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박사학위 논문
정상 프리온 단백질 조절을 통한

대장암 세포에서 항암제의 치료효과
향상
The Enhancement of Therapeutic Effects of Anti-cancer Reagents

in Colorectal Cancer via Regulation of Cellular Prion Protein
2022년 02월
순천향대학교 대학원
의 생 명 과 학 과
윤 철 원
정상 프리온 단백질 조절을 통한
대장암 세포에서 항암제의 치료효과
향상
The Enhancement of Therapeutic Effects of Anti-cancer Reagents
in Colorectal Cancer via Regulation of Cellular Prion Protein
지도교수 이 상 훈
이 논문을 박사학위 논문으로 제출함
2021년 12월
의 생 명 과 학 과
윤 철 원
윤철원의 박사 학위논문을 인준함
2021년 12월
위원장 순천향대학교 이 미 영 (인)
위 원 순천향대학교 송 윤 섭 (인)
위 원 순천향대학교 이 상 훈 (인)
위 원 단국대학교 이 준 희 (인)
위 원 충북대학교 이 현 직 (인)
CONTENTS
CONTENTS ······················· Ⅰ
LIST OF TABLES ····················IX
LIST OF FIGURES ···················· X
LIST OF ABBREVIATIONS ··············· XIV
ABSTRACT ······················ XIX
BACKGROUND······················ 1
1. Colorectal Cancer ······················· 1
2. Cellular prion protein in colorectal cancer············ 4
3. The anti-cancer drugs for cancer therapy ··········· 6
4. Extracellular vesicles ····················· 8
5. The relationship between hypoxia and extracellular vesicles in cancer
······························· 10
6. Experimental purpose ····················· 13
I
PART I. The Role of Cellular Prion Protein in Colorectal Cancer
······························· 15
Cellular Prion Protein Enhances Drug Resistance of Colorectal Cancer
Cells via Regulation of a Survival Signal Pathway ······· 15
1. INTRODUCTION ······················· 16
2. MATERIALS AND METHODS ················· 18
2.1. Specimens of patients with colorectal cancer and normal controls 18
2.2. Cell and spheroid culture of human colon cancer cell line and S707 cancer
stem cells ··························· 18
2.3. Preparation of 5FU ······················· 19
2.4. Cell isolation targeting PrPC using magnetic activated cell sorting 20
2.5. RNA Sequencing assay for total RNA of sorted colon cancer cells 20
2.6. Western blot analysis ······················ 21
2.7. Flow cytometry analysis ···················· 21
2.8. Immunofluorescence staining ·················· 22
2.9. siRNA transfection ······················· 23
II
2.10. Kinase assays ························· 23
2.11. Cell cycle analysis ······················· 24
2.12. PI/Annexin V flow cytometric analysis ·············· 25
2.13. Statistical analyses ······················· 25
3. RESULTS··························· 26
3.1. The clinicopathological features in patients with CRC by the expression
of PrPC ···························· 26
3.2. PrPC involved in the CSC properties of CRC ··········· 30
3.3. The change of cancer-related genes in CRC through regulation of
PrPC······························ 32
3.4. Expression of PrPC in drug-resistant CRC cells·········· 34
3.5. PrPC regulates CRC cell survival via the PI3K-AKT axis ····· 36
3.6. PrPC is involved in cancer cell proliferation through the regulation of cell
cycle-associated proteins ··················· 38
3.7. PrPC regulates anti-cancer drug induced stress-associated signaling41
3.8. PrPC inhibits apoptosis of anti-cancer drug resistant CRC cells via
suppression of caspase3 activation and PARP1 cleavage······ 43
III
4. DISCUSSION ························· 45
PART Ⅱ. The Application of Anti-cancer Reagents for Cancer
Therapy ························ 48
A. Melatonin Promotes Apoptosis of Colorectal Cancer Cells via
Superoxide-mediated ER Stress by Inhibiting Cellular Prion
Protein Expression ···················· 48
1. INTRODUCTION ······················· 49
2.1. Preparation of melatonin ···················· 52
2.2. Cells and cell culture ······················ 52
2.3. Cell viability assay ······················· 52
2.7. Statistical analyses ······················· 55
IV
3. RESULTS··························· 56
3.1. Melatonin decreases cell viability and increases production of superoxide
in SNU-C5/WT cells ······················ 56
3.2. Melatonin suppresses the expression of PrPC and PINK1 and increases
the production of mitochondrial superoxide in SNU-C5/WT cells 58
3.3. Melatonin induces ER stress in SNU-C5/WT cells through regulation of
PrPC expression ························ 61
3.4. Inhibition of PrPC expression enhances melatonin-mediated effect of
apoptosis in SNU-C5/WT cells. ················· 63
4. DISCUSSION ························· 65
B. Prion Protein of Extracellular Vesicle Regulates the Progression
of Colorectal Cancer ··················· 68
1. INTRODUCTION ······················· 69
V
stem cells ··························· 71
2.2. Cell culture and characterization of endothelial progenitor cells ·· 72
2.3. Preparation of 5FU and oxaliplatin ················ 72
2.5. Isolation of exosomes······················ 73
2.6. Identification of exosomes via cryo-electron microscopy ····· 74
2.7. Dynamic light scattering analysis ················ 74
2.8. Detection of PrPC concentration via enzyme-linked immunosorbent
assay ····························· 74
2.9. Invasion assay ························· 75
2.10. Wound-healing migration assay ················· 75
2.11. Morpholometric analysis ···················· 75
2.12. Human angiogenesis protein array ················ 76
2.15. BrdU incorporation assay ···················· 77
2.16. Measurements of oxygen consumption rate············ 78
2.17. Tumorigenesis in CRC xenograft mice models ·········· 78
VI
2.18. In vitro vascular permeability assay ··············· 79
2.19. In vivo vascular permeability assay ··············· 79
2.20. Immunofluorescence staining ·················· 80
2.21. Ethics statement ························ 80
2.22. Statistical analysis ······················· 81
3. RESULTS··························· 82
3.1. Hypoxia-induced exosomes isolated from drug-resistant CRC ·· 82
3.2. Hypoxia-induced exosomes isolated from drug-resistant CRC
increasing sphere formation via upregulation of PrPC ······· 85
increasing invasion, migration, and proliferation via upregulation of
PrPC······························ 88
3.4. The characterization of HUVECs and uptake of Hypoxia-stimulated CRC
exosomes ··························· 94
3.5. Hypoxia-stimulated CRC exosomes promoting tumor angiogenesis and
vascular permeability ······················ 97
3.6. Administration of 5FU, anti-PrP antibody, cetuximab inhibiting wild type
CRC progression through suppression of PrPC level ······· 106
3.7. Co-administration of 5FU and anti-PrP antibody inhibiting exosome
VII
treated CRC progression through suppression of PrPC level ··· 109
4. DISCUSSION ························ 122
CONCLUSION ····················· 128
REFERENCES ····················· 133
ABSTRACT IN KOREAN ················ 154
ACKNOWLEDGEMENTS ················ 157
VIII
LIST OF TABLES
Table 1. Clinicopathological features in patients with colorectal
cancer ································· 27
Table 2. Cox regression analysis of the clinicopathological
parameters in colorectal cancer patients ············· 28
IX
LIST OF FIGURES
Figure 1. Identification of PrP C expression in samples of CRC
patients ································ 29
Figure 2. Effect of PrPC of cancer stemness in CRC cells ···· 31
Figure 3. Gene analysis of CRC cells by the PrPC expression ··· 33
Figure 4. The levels of PrPC in SNU-C5/WT, SNU-C5/5FUR, SNU-
C5/OXR and CSC cells ························ 35
Figure 5. The effect of PrP C on cell survival-associated signaling
pathways in CRC cells ························ 37
Figure 6. The effect of PrPC on proliferation in CRC cells ···· 39
Figure 7. The effect of PrPC on cell stress-associated protein in
CRC cells ······························· 42
Figure 8. The effect of PrPC on apoptosis in CRC cells through inhibition of
caspase3 activation and PARP1 cleavage ··············· 44
Figure 9. Melatonin-mediated inhibition of cell viability and
induction of mitochondrial superoxide production in SNU-C5/WT
cells ·································· 57
X
Figure 10. Melatonin mediated inhibition of PrPC and PINK1 in SNU-
C5/WT cells ····························· 59
Figure 11. The effect of PrPC on mitochondrial superoxide production in
SNU-C5/WT cells via treatment with melatonin ··········· 60
Figure 12. The effect of PrPC on melatonin-induced ER stress in
SNU-C5/WT cells ·························· 62
Figure 13. The effect of PrPC on melatonin-enhanced ER stress-
mediated apoptosis in SNU-C5/WT cells ············· 64
Figure 14. Characterization of exosomes secreted by normoxic and hypoxic
drug-resistant CRC cells ······················· 83
Figure 15. Sphere formation of SNU-C5/5FUR, SNU-C5/OXR and
CSC after treatment with exosomes ················ 86
Figure 16. Effect of exosomes secreted by hypoxic drug-resistant
CRC cells on invasion ························ 89
CRC cells on migration ······················· 90
Figure 18. Proliferation capacity of drug-resistant CRC cells after
treatment with exosomes ······················· 91
XI
CRC cells on morphological change ················· 93
Figure 20. Characterization of HUVECs and expression of PrPC after
treatment with exosomes ······················ 95
Figure 21. Uptake of exosomes into HUVECs ············ 96
CRC cells on the motility of HUVECs ··············· 99
CRC cells on angiogenesis ····················· 101
CRC cells on vascular permeability ················· 103
Figure 25. Effect of exosomes secreted by hypoxic drug-resistant CRC
cells on blood vessels ························· 105
Figure 26. Effect of anti-PrP antibody in a wild type CRC xenograft
model ································· 107
Figure 27. Effect of co-administration of anti-PrP antibody and
5FU in a murine xenograft model treated with exosomes secreted
by hypoxic CRC cells ························ 111
XII
Figure 28. Inhibitory effect of anti-PrP antibody with 5FU on proliferation
of CRC cells ······························ 113
Figure 29. Inhibitory effect of anti-PrP antibody with 5FU and
cetuximab on proliferation of CRC cells ·············· 115
Figure 30. Mitochondrial respiration in CRC cells after treatment
with anti-PrP antibody and cetuximab ·············· 117
Figure 31. Effect of co-administration of anti-PrP antibody and
5FU in a murine xenograft model treated with exosomes secreted
by hypoxic CRC cells on proliferation and apoptosis ······ 118
Figure 32. Effect of anti-PrP antibody and 5FU on vascular permeability
in vivo ································· 120
Figure 33. Schematic illustrating the protective mechanism of PrPC on
5FU in 5FU-resistant cancer cells ················· 130
Figure 34. Schematic illustrating the mechanism of melatonin-
mediated anti-cancer effects in SNU-C5/WT cells ······· 131
Figure 35. Schematic illustrating the effects of exosomes secreted by
hypoxic drug-resistant CRC cells on CRC progression and metastasis
···································· 132
XIII
LIST OF ABBREVIATIONS
AKT Protein kinase B
ATF4 Activating transcription factor 4
α-SMA alpha-smooth muscle actin
ATM ATM serine/threonine kinase
ADC Antibody-drug conjugates
BAX Bcl-2-associated X protein
BCL2 B-cell lymphoma 2
BCA Bicinchoninic acid
BrdU 5-bromo-2′-deoxyuridine
CSC Cancer stem cell
CRC Colorectal cancer

CCAAT-enhancerbinding protein
CHOP
homologous protein
CFSE Carboxyfluorescein succinimidyl ester
Cryo-EM Cryo-electron microscopy
CDK Cyclin dependent kinase
DAPI 4’,6-diamidino-2-phenylindole
XIV
Tetramethylindocarbocyanine
DiI
Perchlorate
Dulbecco's Modified Eagle
DMEM/F12
Medium/Nutrient Mixture F-12
Extracellular signal‑regulated protein
ERK1/2
kinase
EBM-2 Endothelial cell growth basal medium-2
EGF Epidermal growth factor
EGFR Epidermal growth factor receptor
EGM-2 Endothelial cell growth medium-2
ELISA Enzyme-linked immunosorbent assay
ENA-78 C-X-C motif chemokine 5
ET-1 Endothelin 1
eIF2α Eukaryotic initiation factor 2-alpha
ER Endoplasmic reticulum
EVs Extracellular vesicles
FBS Fetal bovine serum
FGF Fibroblast growth factor
FFPE Formalin fixed paraffin embedded

Proto-oncogene tyrosine-protein
Fyn
kinase
FITC Fluorescein isothiocyante
XV
Exosomes from SNU-C5/5FUR under
H-5FUR-Exo
hypoxia condition
Exosomes from SNU-C5/OXR under
H-OXR-Exo
hypoxia condition
HIF-1α Hypoxia-inducible factor 1 alpha
HSPA1L Heat shock protein 70 member 1-like
HUVEC Human umbilical vein endothelial cell

Human epidermal growth factor receptor
HER2
2
H&E Hematoxylin and eosin
IRE1 Inositol-requiring enzyme 1
IGF-1 Insulin-like growth factor-1
JNK c-JUN N-terminal kinase
MAPK1 Mitogen-activated protein kinase 1
Melatonin N-acetyl-5-methoxytryptamine
MEK Mitogen activated protein kinases
MACS Magnetic activated cell sorting
MMP11 Matrix metalloproteinase 11

3-(4,5-dimethylthiazol-2-yl)-2,5-
MTT
diphenyltetra-zolium bromide
MDR-1 Multidrug resistance protein 1
MVs Microvesicles
XVI
Exosomes from SNU-C5/5FUR under
N-5FUR-Exo
normal condition
Exosomes from SNU-C5/OXR under
N-OXR-Exo
normal condition
NGS Next generation sequence
OCR Oxygen consumption rate
OX Oxaliplatin
PBS Phosphate-buffered saline
PrPC Cellular prion protein
PARP1 Poly [ADP-ribose] polymerase 1

Platelet endothelial cell adhesion
PECAM-1
molecule-1
Protein kinase R-like endoplasmic
PERK
reticulum kinase
PINK1 PTEN-induced putative kinase 1
PI3K Phosphoinositide 3-kinase
PI Propidium iodide
RNA-seq RNA sequencing
ROS Reactive oxygen species
SNU-C5/5FUR 5FU-resistant SNU-C5 cells
SNU-C5/OXR Oxaliplatin-resistant SNU-C5 cells
SNU-C5/WT Wild type SNU-C5 cells
XVII
Special AT-rich sequence-binding
SATB1
protein-1
SOD Superoxide dismutase
TS Thymidylate synthase
TGFβ Transforming growth factor beta
TP53 Tumor protein P53
VEGF Vascular endothelial growth factor
YAP Yes-associated protein 1
ZO-1 Zonula occludens-1
5FU 5-fluorouracil
XVIII
ABSTRACT
The Enhancement of Therapeutic Effects of Anti-cancer
Reagents in Colorectal Cancer via Regulation of Cellular
Prion Protein
Chul Won Yun
Department of Biomedical Science,
Graduate School of Soonchunhyang University Asan, Korea
(Directed by Professor Sang Hun Lee)
XIX
Colorectal cancer (CRC) is one of the leading causes of cancer-
related death due to its aggressive metastasis in later stages.
Although recent studies have shown the mechanism by which CRC
cells become drug resistant, anti-cancer drug resistance is still a
serious issue for patients with CRC, and novel strategies for
overcoming this drug resistance have not yet been developed. To
solve this problem, several anti-cancer reagents and molecular
targets are tested in this study. Melatonin, an endogenously
secreted indoleamine hormone that is produced in the pineal gland,
is known to possess antitumor effects via various mechanisms
including induction of apoptosis and pro-oxidant effects in various
cancer cells, including CRC. Hypoxia promotes tumor proliferation
and metastasis in CRC. Since the tumor microenvironment is
commonly characterized by hypoxia, its understanding is important
for cancer therapy. Although there is a growing interest in the
tumorigenic role of cellular prion protein (PrPC) in the process of
metastasis, the precise mechanism behind the cellular
communication involving prion proteins remains poorly understood.
Therefore, I hypothesized that 1) PrPC is related to the drug
resistance in CRC cells via regulation of survival signal pathway, and
XX
apoptosis, which is induced by the reactive oxygen species (ROS)-
mediated ER stress; and 2) extracellular vesicles with PrPC
modulated the progression of CRC cells.
To address my hypotheses, I focused on the expression of PrPC
in 5-fluorouracil (5FU)-resistant CRC cells. In specimens from
patients with colorectal cancer, the expression of PrPC was
significantly correlated with metastasis and tumor stages and
affected the tumorigenesis and genetic changes of CRC cells.
Furthermore, in 5FU-resistant CRC cells, PrPC expression is
significantly increased, compared with that in normal CRC cells. In
the presence of 5FU, PrPC increased CRC cell survival and
proliferation by maintaining the activation of the PI3K-AKT
signaling pathway and the expression of cell cycle-associated
proteins, including cyclin E, CDK2, cyclin D1, and CDK4. Moreover,
PrPC inhibited the activation of the stress-associated proteins p38,
JNK, and p53. After treatment of 5FU-resistant CRC cells with 5FU,
moreover, silencing of PrPC triggered apoptosis via the activation of
caspase3.
In addition, I confirmed that melatonin was found to decrease the
expression of PrPC and PTEN-induced putative kinase 1 (PINK1)
XXI
and increase superoxide accumulation in the mitochondria.
Furthermore, PrPC-knockdown potentiated the effects of melatonin
resulting further in significantly reduced expression of PINK1 and
increased superoxide production in CRC. In addition, si-PRNP-
transfected CRC cells treated with melatonin increased the
production of intracellular superoxide and induced endoplasmic
reticulum stress associated protein, and apoptosis.
Next, hypoxic tumor microenvironment increased the PrPC-
expressing exosomes from CRC, and these exosomes regulate the
CRC cell behavior and tumor progression depending on the
expression of PrPC. Hypoxic exosomes from CRC cells promoted
sphere formation, the expression of tumor-inducing genes,
migration, invasion, and tumor growth. Furthermore, these
exosomes increased endothelial permeability, migration, invasion,
and angiogenic cytokine secretion. These effects were associated
with PrPC expression. Application of anti-PrPC antibody with 5FU
significantly suppressed the CRC progression in a murine xenograft
model.
These results indicate that PrPC plays a key role in CRC drug
resistance. Also, melatonin induces mitochondria mediated cellular
XXII
apoptosis in CRC cancer cells via a PrPC-dependent pathway. PrPC
knockdown, combined with melatonin, amplifies the effects of
melatonin, suggesting a novel therapeutic strategy in targeting CRC
cells. Finally, my results indicate that PrP-expressing exosomes
secreted by hypoxic CRC cells are a key factor in the tumorigenic
CRC-to-CRC and CRC-to-endothelial cell communication. Taken
together, these findings suggest that inhibiting PrPC in hypoxic
exosomes during chemotherapy may be an effective therapeutic
strategy in colorectal cancer.
Keywords: colorectal cancer cell, hypoxia, melatonin, 5FU, cellular
prion protein, exosome, drug resistance, antibody therapeutics
XXIII
BACKGROUND
1. Colorectal Cancer
Colorectal cancer (CRC) is the second leading cause of cancer-related
deaths and the third most prevalent malignant tumor worldwide. Early
diagnosis of CRC increases the 5-year survival rate by approximately 64%,
but progression of metastasis decreases the survival rate to 12% (1).
Uncontrolled proliferation of cancer cells is the hallmark of cancer cells and
with this, the ability to metastasize and recur makes it excessively hard to
treat (2). Although the ideal treatment for CRC is surgical control to remove
the tumor and metastases (3), chemotherapy is the typical leading strategy to
control CRC (4). Recent chemotherapy includes fluoropyrimidine-based
single-agent therapy, such as 5-fluorouracil (5FU) and multiple-agent
therapy, including capecitabine, irinotecan, or oxaliplatin (OX) (3). 5FU is a
fluoropyrimidine analogue and is used to treat several cancers, such as
colorectal (4), breast (5), and gastric cancers (6). 5FU acts as an anticancer
agent by inhibiting thymidylate synthase activity, DNA synthesis, and DNA
repair in cancer cells, resulting in cell death (7, 8). Oxaliplatin, a bifunctional
alkylating agent, can inhibit DNA replication and transcription by covalently
binding to DNA, resulting in induction of apoptosis (9).
１
Despite advancements in chemotherapeutic strategies, drug resistance
commonly occurs in many cancer patients receiving cytotoxic chemotherapy
and molecular targeting drugs and significantly restricted the therapeutic
effects by intrinsic or acquired means (10, 11). Also, drug resistance led to
the failure of cancer therapy and increase of cancer recurrence and metastasis
(12). However, the mechanisms inducing drug resistance are complex and not
clear (13, 14). Current findings show that drug resistance may induce the
inhibition of drug transport and targeting signaling, avoided the immune
response and drug-mediated cell death, and induced the drug-tolerant cells.
Intrinsic drug resistance is related to heterogeneity of cancer cells in tumors.
Tumors consist of a heterogeneous population of cancer cells due to a
difference in genetic and functional context (15). Chemotherapeutic reagents
target the actively proliferating cells and main molecular factors of
tumorigenesis. So, non-proliferative or slow cycling cells in tumors via
nutrients limitations, quiescent stem-like cells, and non-presentation of
tumorigenesis factors are shown to resist cancer therapy, and the tumor often
reoccurred. Acquired drug resistance is originated to the treatment of cancer
cells by exposure of anti-cancer drugs. The decrease in drug efficacy usually
occurs because of alterations in cancer cell physiology, such as the mutation
of the target protein, damage to DNA repair, increased anticancer drug efflux,
and the activation of alternative survival signaling pathways (16, 17).
Furthermore, the tumor microenvironment and hypoxia increase drug
resistance in patients with CRC (18), suggesting that novel combination
２
therapies, such as targeted therapy and immune checkpoint inhibitor therapy,
are needed to overcome CRC.
３
2. Cellular prion protein in colorectal cancer
Cellular prion protein (PrPC), a cell surface protein tethered to the
membrane by a glycosylphosphatidylinositol anchor, is highly expressed in a
variety of cells, such as lymphocytes, and tissues, including muscle, heart,
skin, and nervous tissues (19-21). Although studies on PrPC have initially
focused on the nervous system, recent evidence indicates that PrPC regulates
not only self-renewal of stem/progenitor cells and stem cell fate but also
proliferation and resistance to apoptosis in cancer cells (22). Previous studies
indicate that PrPC is associated with cell survival, proliferation, and signal
transduction (23, 24). Also, PrPC promotes anti-oxidant activity against
oxidative stress, which suppresses the formation of ROS generation and
increases interaction with CU2+ (25, 26). Other studies show that PrPC play
important roles in differentiation, neurogenesis, and proliferation of cells (27),
and regulates the expression of extracellular matrix related proteins, which
promotes the adhesiveness of cell (28).
In cancer biology, several studies have indicated that PrPC possess a crucial
role in various physiological functions, such as cell proliferation, apoptosis,
invasion, metastasis, and drug resistance in various cancers (29, 30). PrPC
increases the cell proliferation by activating the PI3K signal pathway and
enhancement of G1/S phase transition via promotion of cell cycle related
proteins in gastric, renal, and colon cancer (31, 32). In CRC, PrPC promotes
４
cancer cell survival by increasing the uptake of glucose (33). The expression
of PrPC is significantly increased in metastatic gastric cancer cells and induces
the invasion and metastasis by upregulating mitogen activated protein kinases
(MEK)/ERK signal pathway, and matrix metalloproteinase 11 (MMP11) (34).
PrPC enhances the migration and invasion of CRC cells by interaction of HOP
and phosphorylation of the extracellular signal‑regulated protein kinase
(ERK1/2) pathway (35). PrPC augments CRC metastasis through the proto-
oncogene tyrosine-protein kinase (Fyn) - SP1 - special AT-rich
sequence-binding protein-1 (SATB1) pathway (36). Also, PrPC increases
the EMT via promotion of ERK2/ mitogen-activated protein kinase 1 (MAPK1)
in colorectal CSC (34). In addition, PrPC regulates the expression of genes
associated with the mesenchymal subtype by modulation of hippo pathway
effectors Yes-associated protein 1 (YAP) and TAZ and transforming growth
factor beta (TGFβ) pathway and promotes metastasis and shows poor
survival (37). Another study has shown that hypoxia increases the expression
of PrPC in CRC cells and that PrPC regulates CSC markers in CRC cells and
tumor progression (24, 38). In particular, tissues of stage III CRC patients
highly expressed PrPC with Oct4-matched expression (24). PrPC induces the
signal pathway by co-localization with epidermal growth factor receptor
(EGFR) and is related to drug resistance, such as cisplatin, by regulation of
KLF4 and p38 (39).
５
3. The anti-cancer drugs for cancer therapy
5FU is widely used in the treatment of several cancers, particularly for CRC.
It is an antimetabolite drug that inhibits thymidylate synthase and is
incorporated into RNA, single DNA, and double DNA helix, leading to cancer
cell death (7). Several clinical studies have shown that 5FU-based
chemotherapies and chemo-radiotherapies increased the survival of patients
with several cancers (40-42). However, despite the benefits of 5FU to cancer
therapy, acquired resistance to 5FU is a major clinical problem. Accumulated
evidence shows that abnormally high activities of thymidylate synthase (TS),
deoxyuridine triphosphatase, B-cell lymphoma 2 (Bcl-2), Bcl-XL, and Mcl-
1 lead to 5FU resistance (43). TS is a key factor for 5FU metabolism, and the
expression of TS is the main regulator of 5FU sensitivity in cancer therapy.
High expression of TS is related to the intrinsic resistance to 5FU and induces
the acquired resistance by affecting TS stability due to the treatment of 5FU
(43). In addition, overexpression of TS induces the oncogenesis via decreased
translation of tumor protein P53 (TP53) (44). Many clinical studies have
demonstrated that patients with low expression of TS are more sensitive to
5FU based therapy in cancer therapy (45-47). However, the detailed
molecular mechanisms for 5FU chemoresistance in CRC require further
investigation.
In addition to genetic factors that contribute to the occurrence of the disease,
６
factors such as diet, physical activity, and physiological homeostasis are
among the important factors associated with occurrence of CRC (48, 49).
Melatonin (N-acetyl-5-methoxytryptamine) is a cytokine secreted by the
pineal gland in the body during sleep that has been known to possess multiple
physiological homeostatic functions and offers numerous benefits associated
with sleep. More specifically, melatonin plays a role in induction of sleep,
regulation of circadian rhythm, immunomodulation, and reduction of oxidation
(50, 51). Interestingly, studies show that melatonin possesses anti-cancer
effects, and in a study related to colorectal cancer, it was shown to induce
senescence and apoptosis of the CRC cells (51, 52). Melatonin promotes the
effects of anti-cancer drugs by decreasing the tumor progression via diverse
signaling pathways including the MAPK, ERK, and AKT pathway (53). The
treatment with melatonin inhibits the cell migration in CRC cells by reducing
the expression of ROCK via modulation of the p38/MAPK signal pathway (54).
The combination of melatonin and 5FU suppresses cell proliferation, migration,
and invasion in CRC cells by caspase/PARP apoptosis pathway and cell cycle
arrest. Also, melatonin and 5FU is inhibited by the phosphorylation of PI3K,
AKT, and enhances the NF-kB translocation to nuclei (55). Cancer cells
treated with melatonin show enhanced production of ROS, resulting in
dysfunctional mitochondria and thus decreased cell viability (56-58). Such
accumulating evidence instigate further investigations into the effects of
melatonin in inducing apoptosis in cancer cells via ROS generation and
mitochondria mediated apoptosis.
７
4. Extracellular vesicles
Extracellular vesicles (EV) are one of the membrane encapsulated vesicles,
which are secreted by diverse cells into the extracellular space (59). EVs are
classified by three main types, such as exosomes, microvesicles, and
apoptotic bodies (60). Exosomes are nano-sized bilayer structures with cup-
shaped morphology and a size between 30-100 nm (61). Microvesicles (MVs)
are classified by one class of EVs, which are shed from the cellular membrane,
including cellular components, with a diameter from 100 to 1000 nm, and MVs
are usually larger than exosomes (62, 63). Apoptotic bodies are released by
dying cells into extracellular space and they are known to range in size
between 50 - 5000 nm in diameter (64). EVs include diverse functional
molecules, such as proteins, DNA, mRNA, miRNA, lipids, and metabolites, as
well as contribute to cell to cell communications (65, 66). Also, EVs are
directly regulated to the biological responses that interact with receptors on
the recipient cells.
Recent studies have identified the important hypoxia-dependent cell-to-
cell communications in the form of tumor-derived EVs. Whereas, identifying
the protein contents of EVs is still progressing, it is known that tumor cells
actively generate, release, and implement EVs to promote the proliferation,
migration, and angiogenesis of cancer (67). The role of tumor-derived EVs
in regulation of angiogenesis and metastasis has been elucidated in breast
８
cancer, multiple myeloma, ovarian cancer, melanoma, glioma, and chronic
myeloid leukemia (68). In addition, cancer-derived exosomes have been
identified as inducers of CSC differentiation and therapy resistance (69-71).
Therefore, it becomes evident that cancer-derived EVs play a major role in
the development and manifestation of the hallmarks of cancer, and further
investigations are necessary to develop an effective anti-cancer therapy.
９
5. The relationship between hypoxia and extracellular vesicles in cancer
Homeostasis for oxygen levels is important for the maintenance of
organisms under physiological and pathophysiological conditions (72).
Hypoxia is a common feature of malignant tumors, which contributes to tumor
angiogenesis, aggressiveness, and metastasis (73, 74). Hypoxia, which is an
environment with restricted oxygen availability, is a characteristic of the
tumor microenvironment during tumorigenesis. Hypoxia plays a pivotal role in
cancer through the alteration of the microenvironment, changes in oncogenes
expression, reprogramming of metabolism, and formation of non-functional
blood vessels, thus, resulting in the induction of metastasis (73, 75). Hypoxia
induces cancer cell proliferation, development of aggressive tumor
phenotypes, CSC phenotypes, and anti-cancer drug resistance (76, 77).
These effects of hypoxia are regulated by hypoxia inducible factor (HIF),
which is a master gene in cancer physiology. It controls the expression of
oncogene transcription factors and relevant target genes as well as cancer cell
metabolism (78, 79). In addition, hypoxia plays a critical, stimulating role in
blood vessel growth and induces tumor growth by angiogenesis in cancer cells
(80). Angiogenesis is associated with the high risk of metastasis, recurrence,
and early patient death (81), and plays important roles in growth, proliferation
and metastasis of CRC (82). Hypoxia is related to stemness and HIF pathway,
and these are pivotal regulators of stem cell differentiation (83). HIF-1α and
１０
HIF-2α play important roles in stemness maintenance and modulate the
genes and transcription factors related to cell-renewal and differentiation
(84). CSC are involved in the initiation and maintenance of tumors, and
associated with cancer recurrence and drug resistance (85). One study
reported that self-renewal and differentiation of CSC is modulated by hypoxia
and its signal pathway (86). Under hypoxic conditions, the expressions of
HIF-1α and multidrug resistance protein 1 (MDR-1), which is multidrug
resistant protein MDR-1/ P-glycoprotein (P-gp), are increased and induce
the drug resistance in CRC cells (87). The inhibition of HIF-1α increases
the sensitivity to anti-cancer drugs and promoted apoptosis of CRC cells via
downregulation of MDR1/P-gp expression (88). However, since HIF protein
regulates various down-stream pathways vital for cell physiology, it is
important to investigate HIF-mediated specific target protein/s for cancer
survival.
Hypoxia is influenced by the production of EVs and the contents and
functions of EVs. The crosstalk between cancer cells and other cells is
regulated by EV, especially exosomes, secreted from hypoxia-stimulated
cancer cells (89). Since hypoxic stress leads to significant alterations in the
molecular content and function of exosomes, hypoxic tumor-derived
exosomes transfer some target genes, including glucose transporter,
epidermal growth factor receptor (EGFR), transfer receptors, and P-gp, to
non-hypoxic cells, resulting in the internalization of receptors and clustering
of oncogene/proto-oncogene-activating receptors (90). Under hypoxia,
１１
melanoma cells secrete more EVs, compared to normoxic cells, and show an
increase in content of specific proteins and miRNA, which is associated with
poor prognosis of patients with melanoma (91). In addition, the change of
exosome contents by hypoxia enhances the communication between cancer
cells and stroma cells (92) and leads to angiogenesis, survival, proliferation,
invasion, metastasis, and drug resistance of cancer cells (90, 93). Thus, novel
approaches for addressing metastatic cancer must be explored to block the
hypoxic exosome-mediated communication between cancer cells and cells
(94-97).
１２
6. Experimental purpose
The relationship with PrPC and drug resistance in colorectal cancer is
examined in diverse studies. My previous studies showed the drug resistant
role of PrPC treated with diverse reagents, such as fucoidan and melatonin in
CRC. PrPC is associated with tumor growth, migration, and proliferation in CRC
cells. The treatment of fucoidan and inhibition of PrPC is shown to reduce cell
proliferation, migration, and induction of apoptosis. Also, during in vivo study,
silencing PrPC and fucoidan decreased the tumor growth and angiogenesis
(98). Another study confirmed that PrPC is related to drug resistance in CRC
cells (99). PrPC demonstrated anti-oxidant effects against the induction of
ROS via treatment of oxaliplatin (OX). The treatment with melatonin inhibited
the PrPC expression and suppressed anti-oxidant enzyme activity, such as
superoxide dismutase and catalase, and induced the ER stress and apoptosis
in OX-resistant CRC cells. Furthermore, melatonin inhibited the colon CSC
via regulation of PrPC-Oct4 axis (24). Co-treatment of 5FU and melatonin
reduced the tumor growth, proliferation, and angiogenesis via a decrease in
CSC abilities by inducing autophagy.
Therefore, in this study, I investigated the effect of PrPC on proliferation
and survival in 5FU-resistant CRC cells. Furthermore, the effects of
melatonin on endoplasmic reticulum stress and apoptosis of colorectal cancer
cells were examined by the regulation of PrPC.
１３
Next, tumor hypoxic conditions increase the PrPC-expressing exosomes
secreted by drug-resistant CRC cells, which controls CRC function and tumor
progression. I aimed to investigate the effect of exosomes derived from
hypoxic 5FU- and OX-resistant CRC cells on tumorigenic potential via PrPC
level. Furthermore, I elucidated a novel therapeutic strategy that involved the
co-administration of 5FU and anti-PrP antibody for clinical application in
patients with CRC.
１４
PART I. The Role of Cellular Prion Protein in Colorectal Cancer
Cellular Prion Protein Enhances Drug Resistance of Colorectal
Cancer Cells via Regulation of a Survival Signal Pathway
Manuscript is published in
Biomol Ther (Seoul). 2018 26(3):313-321.
Cancers (Basel). 2021 13(9):2144.
１５
1. INTRODUCTION
CRC is the third most frequently diagnosed cancer among males and females,
the second leading cause of cancer related death among males, and third
leading cause among females (100). Although surgical techniques,
chemotherapies, and molecular therapies have improved, clinical outcomes
have not kept pace. This lag is mostly because of changes in lifestyle, local
recurrence, distal metastasis, and resistance to chemotherapeutics and
molecularly targeted therapies (17, 101). Among these hurdles, drug
resistance limits therapeutic efficacy, because chemotherapy is one of the
principal modes of cancer therapy (17). In particular, chemotherapy failure
caused by anti-cancer drug resistance induces most cancer related deaths
due to decreases in drug delivery and changes in metabolic enzymes (16).
Therefore, understanding the mechanisms underlying drug resistance plays a
pivotal role in developing reversal strategies and effective cancer therapeutic
combinations.
5FU is widely used in the treatment for several cancers, particularly for
CRC. It is an antimetabolite drug that inhibits thymidylate synthase and is
incorporated into RNA, single DNA, and double DNA helix, leading to cancer
cell death (7). Several clinical studies have shown that 5FU-based
chemotherapies and chemoradiotherapies increased the survival of patients
with several cancers (40-42). However, despite the benefits of 5FU to cancer
１６
therapy, acquired resistance to 5FU is a major clinical problem. Accumulated
evidence shows that abnormally high activities of thymidylate synthase,
deoxyuridine triphosphatase, Bcl-2, Bcl-XL, and Mcl-1 lead to 5FU
resistance (43). However, the detailed molecular mechanisms for 5FU
chemoresistance in CRC require further investigation.
PrPC is highly expressed in a variety of cells, such as lymphocytes, and
tissues, including muscle, heart, skin, and nervous tissues (19). Although
studies on PrPC have initially focused on the nervous system, recent evidence
indicates that PrPC regulates not only self-renewal of stem/progenitor cells
and stem cell fate but also proliferation and resistance to apoptosis in cancer
cells (22). In CRC, PrPC promotes cancer cell survival by increasing uptake of
glucose (33). PrPC augmented CRC metastasis through the Fyn-SP1-SATB1
pathway (36). My previous study reveals that silencing PrPC inhibits colon
cancer cell growth (98). However, there is little evidence of a relationship
between PrPC and anti-cancer chemoresistance in CRC cells.
１７
2. MATERIALS AND METHODS
2.1. Specimens of patients with colorectal cancer and normal controls
This study and the acquisition of clinical samples were approved by the
Ethics Committee of Seoul Hospital, Soonchunhyang University (IRB: SCHUH
2018-04-032-002), and informed consent was obtained from all study
participants. The serum samples of CRC patients (n=18; grade I, n=90; grade
II, n=90; grade III) and normal control (n=45) were obtained from the
Biobanks of Chonbuk National University Hospital, the Ajou University, and
Keimyung University Dongsan Medical Center, South Korea. CRC tissue
specimens (n=288) in a formalin fixed paraffin embedded (FFPE) block were
obtained from Soonchunhyang University. Clinical information was obtained
from reports and histology sections.
stem cells
A human colon cancer cell line (SNU-C5/WT), 5FU-resistant cell (SNU-
C5/5FUR), and oxaliplatin-resistant cell (SNU-C5/OXR) were obtained from
the Chosun University Research Center for Resistant Cells (Gwangju, Korea)
(102). The cells were cultured in RPMI 1640 with 10% fetal bovine serum
(FBS) (Thermo Fisher Scientific, Waltham, MA, USA) at 37℃, 5% CO2
１８
condition in a humidified incubator. S707 human colon CSCs were provided by
Prof. Steven M Lipkin from the Department of Medicine, Weill Cornell College
of Medicine (New York, NY, USA) (103). The cells were cultured in ultra-
low attachment plates as spheres in Dulbecco's Modified Eagle
Medium/Nutrient Mixture F-12 (DMEM/F12) medium with supplements
(Thermo Fisher Scientific) at 37℃, 5% CO2 condition in a humidified incubator.
The CSC spheroids were cultured in ultra-low attachment flasks (Corning,
Corning, NY, USA) and maintained by collection via gentle centrifugation,
dissociation to single cells, and replating. In addition, SNU-C5/5FUR and
SNU-C5/OXR and human CSCs (S707) were cultured in ultra-low attachment
six-well plates for spheroid formation at 37℃, 5% CO2 condition in a
humidified incubator.
2.3. Preparation of 5FU
5FU were purchased from Sigma (St. Louis, MO, USA), and 5FU were
dissolved in DMSO at 10 mM concentration. Stock solution was filter-
sterilized using a 0.22 μm pore filter (Sartorius Biotech GmbH, Gottingen,
Germany). Aliquots of stock solution were stored at 4℃ until use.
１９
2.4. Cell isolation targeting PrPC using magnetic activated cell sorting
Cell isolation by the expression of PrPC was sorted using manual magnetic
activated cell sorting (MACS) according to the manufacturer’s protocol
(Miltenyi Biotec, Bergisch Gladbach, Germany) (104). Cells were incubated
with human CD230 (PrP)-Biotin primary antibody, followed by a wash with
MACS rinsing solution and attached to anti-Biotin MicroBeads secondary
antibody. After another wash, MACS LS column with active magnetic field
were used to sort and isolate the cells, which were used for flow cytometry
analysis, spheroid formation, and RNA sequencing.
2.5. RNA Sequencing assay for total RNA of sorted colon cancer cells
RNA sequencing (RNA-seq) of total RNA was performed at Macrogen
using TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA,
USA). The colon cancer cells, sorted by PrPC expression, were used for the
Illumina Small RNA Sequencing protocol using the NovaSeq 6000 S4 Reagent
Kit. Quality control with FastQC (v0.11.7), read trimming with Trimmomatic
(v0.38), and mapped with HISAT2 (v2.1.0), Bowtie2 (v2.3.4.1), and StringTie
(v1.3.4d) were performed. Gene-set enrichment analysis and functional
annotation were done on differentially expressed genes, using Gene Ontology
database. Morpheus software was used for heatmap analysis.
２０
2.6. Western blot analysis
Total cell protein was extracted by utilizing RIPA lysis buffer (Thermo
Fisher Scientific). Cell lysates were separated by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis and proteins were transferred to
polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The
membranes were blocked with 5% skim milk and incubated with primary
antibodies against PrPC, phospho-phosphatidylinositol-3-kinase (PI3K),
total PI3K, phospho-AKT, total AKT, cyclin dependent kinase (CDK) 2,
CDK4, cyclin D1, cyclin E, phosphor-p38, total p38, phosphor-c-JUN
Nterminal kinase (JNK), total JNK, cleaved caspase-3, cleaved poly [ADP-
ribose] polymerase 1 (PARP1), β-actin (Santa Cruz Biotechnology, Dallas,
TX, USA), phospho-p53, total p53 (Cell Signaling Technology, Danvers, MA,
USA), phospho-ATM serine/threonine kinase (ATM), and total ATM
(Thermo Fisher Scientific). After incubation of membranes with
peroxidaseconjugated goat anti-mouse or anti-rabbit IgG secondary
antibodies (Santa Cruz biotechnology), bands were detected by utilizing
enhanced chemiluminescence reagents (Amersham Biosciences, Uppsala,
Sweden).
2.7. Flow cytometry analysis
Flow cytometry analysis of Oct4, Nanog, and ALDH1A1 was performed to
identify the presence of CSCs (24). A two-color flow cytometry system (BD
２１
FACS Canto II; BD, Franklin Lakes, NJ, USA) was used to examine the
immune-stained cells. By comparing the results with the corresponding
negative controls, the percentage of stained cells was calculated.
SNU-C5/WT and SNU-C5/5FUR cells were subjected to flow cytometry
analysis using anti-PrP antibody (Santa Cruz Biotechnology). Flow cytometry
was performed using a Cyflow Cube 8 (Partec, Münster, Germany). A data
analysis was performed using the FCS Express software package (De Novo
Software).
2.8. Immunofluorescence staining
SNU-C5/WT and SNU-C5/5FUR cells on the cover glass slide were fixed
in 4% paraformaldehyde solution for 10 min. After washing three times in PBS,
cells were blocked with 10% goat serum for 1 h, and then incubated with
primary antibodies against PrPC (Santa Cruz Biotechnology) at 4°C for 24 h.
After washing three times in PBS, cells were incubated with secondary
antibodies conjugated with Alexa-488 (Thermo Fisher Scientific) for 1 h.
Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; Sigma).
Stained slides were imaged using a confocal microscope (ZEISS, Oberkochen,
Germany).
２２
2.9. siRNA transfection
In accordance with the manufacturer’s protocols, LipofectamineTM 2000
reagent (Thermo Fisher Scientific) was used to transfect siRNAs into CRC
cells. The cells were first grown to 70% confluence in culture dishes and
transfected for 48 h with SMART pool siRNAs (100 nM) specific to PrPC
mRNA. The siRNA ordered to block PRNP (The PRNP-siRNA no. 1 sequence:
5’-UCACCGAGACCGACGUUAA-3’, no. 2 sequence: 5’-
GAUCGAGCAUGGUCCUCUU-3’, no. 3 sequence: 5’-
AGAUGUGUAUCACCCAGUA-3’ and no. 4 sequence: 5’-
GACCGUUACUAUCGUGAAA-3’) and a scrambled sequence (The scramble
siRNA no. 1 sequence: 5’-UGGUUUACAUGUCGACUAA-3’, no. 2
sequence: 5’-UGGUUUACAUGUUGUGUGA-3’, no. 3 sequence: 5’-
UGGUUUACAUGUUUUCUGA-3’, and no. 4 sequence: 5’-
UGGUUUACAUGUUUUCCUA-3’) was purchased by Dharmacon (Lafayette,
CO, USA).
2.10. Kinase assays
The cells were lysed using RIPA lysis buffer (Thermo Fisher Scientific).
CDK4 kinase assays were performed using a CKD4 Kinase Assay Kit (Cusabio,
Baltimore, USA). Briefly, 10 mM ATP was added to 1.25 ml of 6 mM substrate
peptide. The mixture was diluted with dH2O to 2.5 ml to yield a 2×
ATP/substrate cocktail. Then 1 ml of 10× kinase buffer was added to 1.5 ml
２３
of dH2O to yield a 2.5 ml 4× reaction buffer and the enzyme was diluted in
reaction buffer to give the reaction cocktail. The reaction cocktail was added
to 12.5 ml/well of prediluted compound of interest (usually approximately 10
mM) and incubated for 5 min at room temperature. ATP/substrate cocktail
was added to 25 ml/well preincubated reaction cocktail/compound. The final
assay conditions for a 50-ml reaction were therefore 25 mM Tris-HCl (pH
7.5), 10 mM MgCl2, 5 mM b-glycerophosphate, 0.1 mM Na3VO4, 2 mM DTT,
200 mM ATP, 1.5 mM peptide, and 50 ng CDK4 kinase. The reaction was
incubated at room temperature for 30 min, then the stop buffer was added.
From each reaction, 25 ml was transferred to a 96-well streptavidin-coated
plate and incubated at room temperature for 1 h. Plates were washed three
times with PBS. Phospho-Rb (Ser780) antibody was added at 100 ml/well
and incubated at room temperature for 2 h. Plates were washed three times
with PBS. HRP-avidin was added to each well, and incubated at 37°C for 1 h.
Plates were washed five times with PBS. TMB substrate and stop solution
were added. The absorbance was read at 450 nm with a microtiter plate reader
(Tecan Group AG, Mänedorf, Switzerland).
2.11. Cell cycle analysis
SNU-C5/5FUR cells were harvested and fixed with 70% ethanol at -20°C
for 2 h. After washing twice with cold PBS, cells were subsequently incubated
with RNase and propidium iodide (PI; Sigma). The cell cycle histograms were
２４
assessed using a Cyflow Cube 8 (Partec). Results were analyzed using the
FCS Express software package (De Novo Software).
2.12. PI/Annexin V flow cytometric analysis
Apoptosis of SNU-C5/5FUR and SNU-C5/OXR cells was evaluated with a
Cyflow Cube 8 (Partec) following staining of the cells with Annexin V-
fluorescein isothiocyante (FITC) and PI (Sigma). Data analysis was
performed using the FCS Express software package (De Novo Software).
2.13. Statistical analyses
Data are expressed as means ± SEM. Statistical significance was assessed
using the Student’s t test, where p< 0.05 was considered significant.
２５
3. RESULTS
3.1. The clinicopathological features in patients with CRC by the expression of
PrPC
Previous study demonstrated that PrPC controls CSC markers in CRC cells
(24). To investigate whether PrPC is involved in CSC properties in CRC, I
assessed the clinicopathological features in patients with CRC depending on
the expression of PrPC (Table 1). Although the expression of PrPC was not
associated with patient age, sex, pT stage, and vascular invasion, the
expression of PrPC was significantly correlated with pN stage, metastasis,
stage, lymphatic invasion, and perineuronal invasion (Table 2). Consistent
with the observed clinicopathological features in patients with CRC, the 5-
year survival of PrPC negative CRC patients was higher than that of PrPC-
positive CRC patients (Figure 1A). In serum samples of PrPC-positive
patients with CRC, the concentration of PrPC was significantly increased in
stage II and III (Figure 1B). In CRC patients with stage III CRC, PrPC was
highly expressed in colon tissues and lymph nodes (Figure 1C). In addition,
PrPC in serum was significantly increased in stage III patients treated with
chemotherapy, compared with that in stage III patients not treated with
chemotherapy (Figure 1D).
２６
Table 1. Clinicopathological features in patients with colorectal cancer.
PrPC Expression Total

Clinicopathological factors p value
Negative (N=133) Positive (N=155) (N=288)
Age, years, mean (SD) 65.3553 (12.2671) 64.0276 (12.8545) 0.365
Gender, N (%) 0.230
M 50 ( 42.1) 66 (56.9) 116
F 83 (48.3) 89 (51.7) 172
pT stage, N (%) 0.424
pT1 and pT2 27 (44.3) 34 (55.7) 61
pT3 and pT4 106 (46.7) 121 (53.3) 227
pN stage, N (%) 0.014
Negative 78 (41.3) 111 (58.7) 189
Positive 55 (55.6) 44 (44.4) 99
Metastasis, N (%) 0.003
Negative 130 (48.5) 138 (51.5) 268
Positive 3 (15.0) 17 (85.0) 20
Stage, N (%) 0.089
and 71 (42.4) 96 (57.5) 167
and 62 (51.2) 59 (48.8) 121
Vascular invasion, N (%) 0.240
Negative 111 (45.1) 135 (54.9) 246
Positive 22 (52.4) 20 (47.6) 42
Lymphatic invasion, N (%) 0.024
Negative 91 (42.5) 123 (57.5) 214
Positive 42 (56.8) 32 (43.2) 74
Perineuronal invasion, N (%) 0.544
Negative 84 (46.2) 98 (53.8) 182
Positive 49 (46.2) 57 (53.8) 106
pT: pathological tumor stage according to the American Joint Committee on Cancer TNM
classification system; pN: pathological lymph node stage according to the American Joint
Committee on Cancer TNM classification system.
２７
Table 2. Cox regression analysis of the clinicopathological parameters in
colorectal cancer patients.
Univariate analysis Multivariate analysis

Clinicopathologic
Variable Hazard ratio Hazard ratio
factors p value p value
(95%/CI) (95%/CI)
Age <60 yr vs. ≥60 yr 1.439 (0.736-2.813) 0.288
Gender Male vs. Female 0.912 (0.481-1.729) 0.778
pT stage Low vs. High 3.065 (0.939-10.011) 0.064
pN stage Negative vs. Positive 1.965 (1.012-3.814) 0.046
Metastasis Negative vs. Positive 2.626 (1.017-6.777) 0.046
Stage Low vs. High 2.698 (1.358-5.361) 0.005 3.568 (1.044-12.200) 0.043
Vascular invasion Negative vs. Positive 1.654 (0.722-3.790) 0.235
Lymphatic invasion Negative vs. Positive 2.826 (1.452-5.500) 0.002 2.566 (1.132-5.769) 0.024
PN. invasion Negative vs. Positive 2.428 (1.246-4.730) 0.009
PrPC expression Negative vs. Positive 3.100 (1.408-6.825) 0.005 3.712 (1.642-8.390) 0.002
２８
Figure 1. Identification of PrPC expression in samples of CRC patients.
(A) Five-year survival of CRC patients depending on PrPC expression
(negative, n=133; positive, n=155). (B) ELISA analysis of PrPC expression
in sera from patients with CRC (normal, 45; stage I, 18; stage II, 90; stage III,
90). (C) Representative immunohistochemistry images of PrPC in tumor
tissues from colorectal cancer patients (n=33). Scale bar = 200 μm. (D)
ELISA analysis of PrPC expression in sera from CRC stage III patients who
were therapy naive (non-chemotherapy, n=70) and patients who were
treated with chemotherapy (Chemo-therapy, n=20). Data are presented as
mean ± SEM. **p<0.01 ((B): ANOVA; (D) unpaired t-test).
２９
3.2. PrPC involved in the CSC properties of CRC
To further explore whether PrPC controls CSC properties in drug-resistant
CRC cells, I investigated the formation of cancer spheres and the expression
of CSC markers, including ALDH1A, Nanog, and Oct4 in 5FU-resistant CRC
cells (SNU-C5/5FUR) and oxaliplatin-resistant CRC cells (SNU-C5/OXR). I
found that the sphere formation capacity and CSC marker expression in each
drug-resistant CRC cell were drastically enhanced in PrP-positive cells
(Figures 2A-D). Moreover, the CSC properties were significantly increased
in PrP-positive CRC stem cells (CSC) (Figures 2E and F).
３０
Figure 2. Effect of PrPC of cancer stemness in CRC cells.
(A-F) Sphere formation assay of PrP-negative and PrP-positive SNU-
C5/5FUR (A and B), SNU-C5/OXR (C and D), and CSC (E and F) in ultra-
low attachment plates for 2 weeks (n=3). CSC markers (ALDH1A, Nanog,
and Oct4) were analyzed using flow cytometry analysis (n=3). Scale bar =
200 μm. Data are represented as the mean ± SEM. *p<0.05, **p<0.01
(unpaired t-test).
３１
3.3. The change of cancer-related genes in CRC through regulation of PrPC
To determine the effect of PrPC on oncogene expression in drug-resistant
CRC cells, I am performed a NGS on PrP-positive and PrP-negative SNU-
C5/5FUR (Figures 3A–E). The transcriptome data showed that tumor
progression-mediated genes, such as CSC markers, metastasis, angiogenesis,
and oncogenes, were overexpressed in PrP-positive SNU-C5/5FUR,
whereas tumor suppressor genes were decreased (Figures 3A–E). These data
suggest that the PrPC expression level is strongly associated with CRC
progression and prognosis through regulation of CSC properties in CRC.
３２
Figure 3. Gene analysis of CRC cells by the PrPC expression.
NGS analysis of PrP-positive and PrP-negative SNU-C5/5FUR (n=3). NGS
analysis of fold changes in PrP-positive vs. PrP-negative cells was
categorized as CSC marker (A), metastasis (B), angiogenesis (C), tumor
suppressor (D), and oncogene (E).
３３
3.4. Expression of PrPC in drug-resistant CRC cells
Several studies have shown that the expression of PrPC is increased in
gastric, breast, and CRC cells (105). In addition, PrPC is associated with
multidrug resistance in gastric and breast cancer cells (106, 107). To explore
whether PrPC is upregulated in drug resistant CRC cells, I analyzed the
expression of PrPC in wild type (SNU-C5/WT), SNU-C5/5FUR, SNU-
C5/OXR and CSC cells by ELISA and western blot assay. In several types of
CRC cells, the expression of PrPC was drastically increased in SNU-C5/5FUR,
SNU-C5/OXR, and CSC, compared with that in SNU-C5/WT cells (Figure
4A). The expression level of PrPC was significantly increased in SNU-
C5/5FUR and SNU-C5/OXR, compared with that in SNU-C5/WT (Figures 4B
and C). In addition, flow cytometry analysis showed that the ratio of PrPC
positive cells was significantly increased in SNU-C5/5FUR (Figure 4D).
Immunofluorescent staining also revealed an increased level of PrPC in SNU-
C5/5FUR (Figure 4E). These results indicate that 5FU/OX-induced drug
resistant CRC cells and CSC express high levels of PrPC.
３４
Figure 4. The levels of PrPC in SNU-C5/WT, SNU-C5/5FUR, SNU-C5/OXR
and CSC cells.
(A) Concentration of the level of PrPC in SNU-C5/WT (WT), SNU-C5/5FUR
(5FUR), SNU-C5/OXR (OXR), and CSC cells (n=3). (B and C) Expression of
PrPC in SNU-C5/WT, SNU-C5/5FUR and SNU-C5/OXR cells (n=3). (D) The
expression of PrPC in SNU-C5/WT and SNU-C5/5FUR cells was analyzed by
flow cytometry (n=3). (E) Immunocytochemistry for PrPC (green) in SNU-
C5/WT and SNU-C5/5FUR cells. Nuclei were stained by DAPI (blue). Scale
bar=100 mm (n=3).
３５
3.5. PrPC regulates CRC cell survival via the PI3K-AKT axis
The phosphoinositide 3-kinase (PI3K)-AKT signaling pathway is a pivotal
regulator and central node in cell survival signaling downstream of growth
factor, cytokines, and several cellular stimuli. To investigate whether PrPC
regulates the PI3K-AKT signaling pathway, I evaluated the phosphorylation
of PI3K and AKT in SNU-C5/WT and SNUC5/ 5FUR. The phosphorylation of
PI3K and AKT were significantly enhanced in SNU-C5/5FUR, compared with
that in SNU-C5/WT (Figure 5A). To investigate whether the expression of
PrPC is associated with regulation of the PI3K-AKT signaling pathway in
5FU-resistant CRC cells, I confirmed activation of PI3K-AKT in the
presence or absence of 5FU (140 mM). Treatment of SNU-C5/WT with 5FU
increased the level of PrPC (Figure 5B). In addition, the level of PrPC was
significantly increased in SNU-C5/5FUR in the presence and absence of 5FU,
compared with in SNU-C5/WT (Figure 5B). The expression of PrPC was
suppressed using PRNP siRNA (si-PRNP) in SNU-C5/5FUR (Figure 5C).
After treatment of SNU-C5/5FUR with 5FU, the suppression of PrPC induced
a decrease in phosphorylation of PI3K and AKT (Figure 5D). These data
suggest that PrPC regulates the PI3K-AKT signaling pathway in 5FU-
resistant CRC cells.
３６
Figure 5. The effect of PrPC on cell survival-associated signaling pathways in
CRC cells.
(A) The level of phospho-PI3K (p-PI3K) and phospho-AKT (p-AKT) in
SNU-C5/WT and SNU-C5/5FUR cells (n=3). (B) The level of PrPC in SNU-
C5/WT cells and SNU-C5/5FUR cells after treatment with 5FU (140 mM)
(n=3). (C) The expression of PrPC after transfection of SNU-C5/5FUR cells
with PRNP siRNA (si-PRNP) (n=3). (D) The level of phosphor-PI3K (p-
PI3K) and phospho-AKT (p-AKT) in SNU-C5/5FUR cells transfected with
si-scr or si-PRNP after treatment with PBS or 5FU (140 mM) for 48 h (n=3).
３７
3.6. PrPC is involved in cancer cell proliferation through the regulation of cell
cycle-associated proteins
To confirm the effect of PrPC on cell proliferation in 5FU resistant CRC cells,
the expression of cell cycle-associated proteins, such as cyclin E, CDK2,
cyclin D1, and CDK4, was analyzed by western blot assay after treatment of
SNUC5/5FUR with 5FU. SNU-C5/5FUR transfected with PRNP siRNA had a
significant decrease in the levels of cyclin E, CDK2, cyclin D1, and CDK4 after
treatment with 5FU (Figure 6A). In addition, SNU-C5/5FUR transfected with
PRNP siRNA showed a significant decrease in CDK4 kinase activity after
treatment with 5FU (Figure 6B). Furthermore, flow cytometry analysis for PI
staining showed that PrPC knockdown SNU-C5/5FUR exhibited a significant
decrease in the ratio of S phase (Figure 6C) after treatment with 5FU. These
findings suggest that PrPC regulates proliferation of 5FU resistant CRC cells
via regulation of cell cycle-associated proteins in the presence of this anti-
cancer drug.
３８
Figure 6. The effect of PrPC on proliferation in CRC cells.
(A) The level of cyclin E, CDK2, cyclin D1, and CDK4 in SNU-C5/5FUR cells
transfected with si-scr or si-PRNP after treatment with PBS or 5FU (140
mM) for 48 h (n=3). (B) The kinase activity of CDK4 in SNU-C5/5FUR cells
transfected with si-scr or si-PRNP after treatment with PBS or 5FU (140
mM) for 48 h (n=3). (C) The percentage of G1, S, and G2/M phase in SNU-
３９
C5/5FUR cells transfected with si-scr or si-PRNP after treatment with PBS
or 5FU was determined by flow cytometry analysis of PI-stained cells (n=3).
４０
3.7. PrPC regulates anti-cancer drug induced stress-associated signaling
The main mechanism of 5FU is the inhibition of DNA synthesis and mRNA
translation (89). To explore whether PrPC regulates stress-associated
proteins in the presence of this drug, I assessed the phosphorylation of
stress-associated proteins, including p38, JNK, p53, and ATM
serine/threonine kinase (ATM), after treatment of SNU-C5/5FUR with 5FU.
SNU-C5/5FUR transfected with PRNP siRNA exhibited a significant increase
in the phosphorylation of p38, JNK, and p53 (Figures 7A-C) after treatment
with 5FU. However, although knockdown of PrPC induced the phosphorylation
of ATM in the PBS-treated group, there was no significant increase in the
phosphorylation of ATM after treatment with 5FU, implying that PrPC is not
involved in ATM signaling in the presence of 5FU (Figure 7D). These results
indicate that PrPC inhibits the activation of stress-associated signaling against
an anti-cancer drug through suppression of p38, JNK, and p53
phosphorylation.
４１
Figure 7. The effect of PrPC on cell stress-associated protein in CRC cells.
The phosphorylation of p38 (A), JNK (B), p53 (C), ATM (D) were determined
by western blot analysis SNU-C5/5FUR cells transfected with si-scr or si-
PRNP after treatment with PBS or 5FU (140 mM) for 48 h (n=3).
４２
3.8. PrPC inhibits apoptosis of anti-cancer drug resistant CRC cells via
suppression of caspase3 activation and PARP1 cleavage
To investigate whether PrPC is involved in apoptosis of 5FU resistant CRC
cells, activation of caspase3 and cleavage of PARP1 were assessed using a
western blot after treatment of SNU-C5/5FUR with 5FU (Figure 8A). The
levels of cleaved caspase3 (C-Caspase3) and cleaved PARP1 (C-PARP1)
were significantly increased in treatment with 5FU, compared with those in
treatment with PBS (Figure 8A). SNU-C5/5FUR transfected with PRNP
siRNA had a significant increase in the levels of C-Caspase3 and C-PARP1
after treatment with 5FU (Figure 8A). In addition, flow cytometry for
PI/Annexin V staining showed that silencing of PrPC significantly increased in
the percentage of early and late apoptotic cells after treatment with 5FU
(Figure 8B). These data indicate that PrPC inhibits apoptosis of CRC cells in
anti-cancer drug conditions via suppression of caspase3 activation and
PARP1 cleavage. In addition, SNU-C5/5FUR transfected with PRNP siRNA
showed a significant decrease in the kinase activity of CDK4 after treatment
with 5FU (Figure 6B).
４３
Figure 8. The effect of PrPC on apoptosis in CRC cells through inhibition of
caspase3 activation and PARP1 cleavage.
(A) The level of cleaved caspase3 (C-Caspase3) and cleaved PARP1 (C-
PARP1) in SNU-C5/5FUR cells transfected with si-scr or si-PRNP after
treatment with PBS or 5FU (140 mM) for 48 h (n=3). (B) Apoptosis was
measured using flow cytometry analysis for PI/Annexin V staining (n=3).
４４
4. DISCUSSION
Previous data have shown a correlation between high PrPC expression and
clinicopathological features in patients with CRC, including metastasis risk,
advanced clinical stage, and survival of CRC patients (38). The expression of
PrPC in tumor tissues from stage III CRC patients is matched with Oct4
expression, indicating that co-expression of PrPC and Oct4 is involved in CRC
metastasis (24). This study revealed that PrP-positive cells were increased
in the properties of a CSC. In RNA-seq data, gene expressions associated
with CSC markers, metastasis, angiogenesis, and oncogenes were
significantly increased in PrP-positive cells, whereas tumor suppressor
genes were reduced. These findings indicated that PrPC plays a pivotal role in
CRC behavior, suggesting that PrPC could be a novel CRC marker for targeted
therapy in CRC patients.
Also, in the present study, I demonstrated that the level of PrPC is increased
in 5FU resistant CRC cells, implying that PrPC increased cell survival in anti-
cancer drug conditions by regulating survival and apoptosis signaling
pathways. After 5FU treatment of SNU-C5/5FUR, a 5FU resistant colorectal
cancer cell line, silencing of PrPC yielded the opposite effects, including
inhibition of proliferation and augmentation of apoptosis. Previous studies
have shown that PrPC contributes to protection from oxidative stress,
regulation of copper metabolism, cell cycle control, and cell adhesion (31,
４５
108-110). In addition, PrPC promotes pro-proliferative and anti-apoptotic
effects in stem cells, such as hematopoietic stem cells, neural stem cells,
mesenchymal stem cells, and human embryonic stem cells (22). In cancer cell
biology, PrPC accelerates cell proliferation, invasion, metastasis, and
resistance to cytotoxic drugs (29, 31, 34, 36). My results revealed for the
first time that 5FU resistant CRC cells express high levels of PrPC. In gastric
cancer cells, PrPC expression enhanced drug resistance via an increase in
Bcl-2 expression (111). Moreover, treatment of HCT116 CRC cells with
PrP-specific antibodies inhibited cancer cell growth and promoted the effects
of anticancer drugs such as 5FU, cisplatin, and doxorubicin (112). I also
confirmed that the level of PrPC expression was significantly increased in
oxaliplatin-resistant SNU-C5 cells, compared with wild-type cells. These
findings suggest that PrPC could be a key protein for resistance to anticancer
drugs in CRC cells.
The PI3K signaling pathway is involved in various cellular processes,
including cell survival, metabolism, inflammation, motility, and cancer
progression (113). In particular, the PI3K-AKT signal axis regulates survival
and proliferation in cancer cells (114). In human lung cancer cells, AKT
amplification plays a pivotal role in acquiring cisplatinum resistance (115). My
results indicated that the phosphorylation of PI3K and AKT are significantly
increased in 5FU resistant CRC cells, and inhibition of PrPC expression
reduced the activation of PI3K and AKT in the presence of 5FU. Activation of
AKT also promotes cell proliferation through multiple downstream targets for
４６
cell cycle regulation (116). I also revealed that downregulation of PrPC
inhibited proliferation via suppression of cell cycle-associated proteins, such
as cyclin E, CDK2, cyclin D1, and CDK4 after 5FU treatment of 5FU resistant
cells. PrPC accelerated the G1/S transition via the PI3K-AKT-cyclin D1 axis
(31). These findings suggest that PrPC plays pivotal roles in cell survival and
proliferation in drug resistant CRC cells via regulation of the PI3K-AKT
signaling pathway.
Activation of PI3K-AKT pathway by PrPC expression promotes anti-
apoptotic effect (117). Conversely, downregulation of PrPC promotes
caspase3 activation by reducing AKT activation (118). In hypoxic conditions,
increased PrPC inhibits TRAIL-mediated apoptosis (119). My previous study
revealed that silencing of PrPC increased apoptosis of CRC cells. To explore
the relationship between PrPC and anti-cancer drug resistance, I showed that
treatment with 5FU induced the activation of stress-associated proteins, such
as p38, JNK, and p53, and suppression of PrPC triggered additional activation
of these proteins. This additional activation implies that PrPC inhibits the
activation of stress-associated proteins in drug resistant CRC cells.
Furthermore, PrPC suppressed the activation of caspase3 after 5FU treatment
of drug-resistant CRC cells. These results suggest that the level of PrPC plays
an important role in the development of chemoresistance by CRC cells.
４７
PART II. The Application of Anti-cancer Reagents for Cancer Therapy
Melatonin Promotes Apoptosis of Colorectal Cancer Cells via
Superoxide-mediated ER Stress by Inhibiting Cellular Prion Protein
Expression
Manuscript is published in Anticancer Res. 2018 38:3951-3960.
４８
1. INTR0ODUCTION
CRC is one of the most commonly diagnosed cancers in the world, placing it
as the most common cause of mortality (120). Uncontrolled proliferation of
cancer cells is the hallmark of cancer cells and along with this, the ability to
metastasis and recurrence makes it excessively hard to treat (2). In response,
patients go on extensive chemotherapies inducing apoptosis of target cancer
cells in the body, as well as surgery to remove the cancer tumors (101).
There has been a proliferation of research in drugs used to control CRC and
the progression of the disease. For example, OX, a bifunctional alkylating
agent, can inhibit DNA replication and transcription by covalently binding to
DNA, resulting in induction of apoptosis (9). However, drug resistance of
cancer cells is a major limitation in chemotherapy (17). Although the mutation
of anticancer drug target protein, defective DNA damage repair, enhanced
anticancer drug efflux, and alternative compensating signaling pathways are
potential mechanisms of drug resistance, their toxicity could lead to numerous
side-effects, even favoring growth of cancer cells by killing the patients’
immune system. Thus, natural compounds have been known to be highly
desirable clinically due to their minimal side-effects which are more prevalent
in chemotherapies and other drugs. Not surprisingly, there exist studies that
show natural compounds with anti-cancer effects to be a promising strategy
for cancer prevention and therapy (98, 121-123). More studies on novel
４９
compounds and therapeutic mechanisms associated with the compounds are
desirable for developing efficient therapeutic strategies targeting CRC.
In addition to genetic factors that contribute to the occurrence of the disease,
factors such as diet, physical activity, and physiological homeostasis are
among the important factors associated with occurrence of CRC (48, 49).
Melatonin is a cytokine secreted by the pineal gland in the body during sleep
that has been known to possess multiple physiological homeostatic functions
and offer numerous benefits associated with sleep. More specifically,
melatonin plays a role in induction of sleep, regulation of circadian rhythm,
immunomodulation, reduction of oxidation (50, 51). Interestingly, studies
show that melatonin possesses anti-cancer effects, and in a study related to
colorectal cancer, it was shown to induce senescence and apoptosis of the
CRC cells (51, 52). Cancer cells treated with melatonin show enhanced
production of ROS, resulting in dysfunctional mitochondria and thus decreased
cell viability (56-58). Such accumulating evidence instigate further
investigations on the effects of melatonin in inducing apoptosis in cancer cells
via ROS generation and mitochondria mediated apoptosis.
A previous study suggested that melatonin exerts its effects through PrPC-
dependent pathway (99). PrPC, a cell surface protein tethered to the
membrane by glycosylphosphatidylinositol anchor, is highly expressed in cells
of various tissues including nervous, muscle and heart tissue (19-21).
Although mutated prion proteins are most widely known for their
５０
neurodegenerative properties, several studies have indicated that cellular
prion proteins possess a crucial role in various physiological functions, such
as cell proliferation, apoptosis, invasion, metastasis and drug resistance in
various cancers (29, 30). My previous studies demonstrated that PrPC is
related to cancer proliferation, and that knockdown of PrPC inhibits colorectal
cancer cell growth (98) and induces tumor cell death (124). PrPC levels in
cancer are associated with ROS-mediated endoplasmic reticulum (ER) stress,
thereby destroying mitochondria and ER to induce cell death (125). PTEN-
induced putative kinase 1 (PINK1), a protein located to the mitochondrial
outer membrane, maintains mitochondria homeostasis through mitochondria
quality control pathway (126). In damaged mitochondria, PINK1 recruits
parkin and autophagy related proteins and results in degradation of damaged
mitochondria through autophagy (127). In a recent study, it was shown that
PINK1 functions as a regulator of cell cycle progression and has tumor
promoting properties (128).
５１
2.1. Preparation of melatonin
Melatonin was purchased from Sigma, and was dissolved in 100% ethanol,
filter-sterilized through a 0.45 μm pore filter (Sartorius Biotech GmbH)
Aliquots of stock solution were stored at 4℃ until use.
2.2. Cells and cell culture
The human colorectal cancer cell line (SNUC5/WT) was obtained from the
Chosun University Research Center for Resistant Cells (Gwangju, Republic of
Korea). The cells were maintained in RPMI 1640 supplemented with 10% FBS,
l-glutamine, and antibiotics (Biological Industries, Beit Haemek, Israel) at
37˚C with 5% CO2 in a humidified incubator.
2.3. Cell viability assay
The exponentially growing colon cancer cells were incubated in 96-well
plates with each drugs (0-1 mM Melatonin) for 24 h. The cell viability was
determined by a modification of the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetra-zolium bromide (MTT) assay, in which the tetrazolium salt (3-
(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-
５２
sulfophenyl)-2-tetrazolium was converted to formazan by mitochondrial
dehydrogenase in the viable cells. The formazan was quantified by measuring
the absorbance at 570 nm using a microplate reader (BMG Labtech).
Total cell protein was extracted by utilizing RIPA lysis buffer (Thermo
Fisher Scientific). Cell lysates were separated by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis and proteins were transferred to
polyvinylidene fluoride membranes (Millipore). The membranes were blocked
with 5% skim milk and incubated with primary antibodies against PrPC,
phospho-protein kinase R-like endoplasmic reticulum kinase (PERK), PERK,
phospho-eukaryotic initiation factor 2-alpha (eIF2α), eIF2α. activating
transcription factor 4 (ATF4), phospho-c-JUN N-terminal kinase (JNK),
total JNK, phospho-p38, Bcl-2-associated X protein (BAX), cleaved
Caspase-3, cleaved poly [ADP-ribose] polymerase 1 (PARP1), β-Actin
(Santa Cruz Biotechnology), PTEN-induced putative kinase 1 (PINK1),
CCAAT-enhancerbinding protein homologous protein (CHOP) (Novus
Biologicals, Littleton, CO, USA). After incubation of membranes were
incubated with peroxidase conjugated goat anti-mouse or anti-rabbit IgG
secondary antibodies (Thermo Fisher Scientific). Protein bands were
visualized by utilizing enhanced chemiluminescence reagents (Amersham
Biosciences).
５３
UGGUUUACAUGUUUUCCUA-3’) was purchased by Dharmacon.
To measure mitochondrial superoxide, SNU-C5/WT cells were stained with
MitoSOX red (Thermo Fisher Scientific) for 30 min at 37˚C and washed with
PBS three times. To confirm apoptosis, the cells were stained with Annexin
V-FITC and PI (Sigma). Each sample was quantitively analyzed using CyFlow
Cube 8 (Sysmex Partec). Data analysis was performed using the FCS Express
５４
software package (De Novo Software).
2.7. Statistical analyses.
Results are expressed as the mean ± SEM and analyzed by ANOVA. In some
experiments, this was followed by a comparison of the treatment mean with
the control using a Bonferroni-Dunn test. Data were considered to be
significantly different at values of p<0.05.
５５
3. RESULTS
3.1. Melatonin decreases cell viability and increases production of superoxide
in SNU-C5/WT cells
To evaluate the effect of melatonin on SNU-C5/WT cells, cell viability was
measured by utilizing the MTT assay after treatment of SNU-C5/WT cells
with melatonin (0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1 mM) at various periods
of time (0, 6, 12, and 24 h). Melatonin treatment was shown to reduce the cell
viability of SNU-C5/WT cells in dose- and time-dependent manner (Figures
9A and B). In order to assess superoxide production in SNU-C5/WT cells
treated with Melatonin, the production of mitochondrial superoxide was
examined by flow cytometric analysis (Figures 9C and D). The level of
mitochondrial superoxide was significantly increased after treatment with
melatonin (1 mM). These results suggest that melatonin inhibits cell viability
of SNU-C5/WT cells and increases superoxide production.
５６
Figure 9. Melatonin-mediated inhibition of cell viability and induction of
mitochondrial superoxide production in SNU-C5/WT cells.
Cell viability was measured using the MTT assay. (A) SNU-C5/WT cells
were treated with melatonin (0-1 mM) for 24 h (n=3). (B) SNU-C5/WT
colon cancer cells were treated with melatonin (1 mM) for various periods of
time (0, 6, 12, and 24 h). Values represent the means ± SEM. *p<0.05 vs.
control and **p<0.01 vs. control. (C) SNU-C5/WT colon cancer cells were
treated with melatonin (1 mM) for 24 h and superoxide production was
assayed using flow cytometric analysis of MitoSOX red staining (n=3). (D)
The quantitation of the percentage of mitochondrial superoxide levels. Values
represent means ± SEM. **p<0.01 vs. control.
５７
3.2. Melatonin suppresses the expression of PrPC and PINK1 and increases the
production of mitochondrial superoxide in SNU-C5/WT cells
In order to assess the effect of melatonin on the expression of PrPC and
PINK1 in SNU-C5/WT, the cells were treated with various concentrations of
melatonin (0, 0.2, 0.6, and 1 mM) at various time periods (0, 6, 12, and 24 h).
Western blot analysis was performed to evaluate the expression of PrPC and
PINK1. The results show that PrPC and PINK1 expression are significantly
reduced upon melatonin (1 mM) treatment for 24 h (Figures 10A-D).
Next, to measure the expression of PrPC and PINK1, SNUC5/WT cells were
transfected with si-PRNP following treatment with melatonin (1 mM). In si-
PRNP transfected cells, melatonin treatment significantly reduced the
expression of PrPC and PINK1 (Figures 11A and B). In order to evaluate the
effect of melatonin on the production of superoxide in SNUC5/WT flow
cytometric analysis was performed. MitoSOX red staining showed that
knockdown of PrPC induced significant production of mitochondrial superoxide
upon melatonin treatment compared to control (Figure 11C). These results
demonstrate that the knockdown of PrPC significantly potentiated the effects
of melatonin.
５８
Figure 10. Melatonin mediated inhibition of PrPC and PINK1 in SNU-C5/WT
cells.
SNU-C5/WT cells were treated with different concentrations of melatonin.
PrPC (A) and PTEN-induced putative kinase 1 (PINK1) (B) was determined
by western blot analysis (n=3). (C) SNU-C5/WT cells were treated with
melatonin for different periods of time. PrPC (C) and PINK1 (D) were
determined by western blot analysis (n=3).
５９
Figure 11. The effect of PrPC on mitochondrial superoxide production in SNU-
C5/WT cells via treatment with melatonin.
The expression levels of PrPC (A) and PINK1 (B) in SNU-C5/WT cells
transfected with si-PRNP or si-scr following treatment with melatonin (1
mM) for 24 h (n=3). (C) Mitochondrial superoxide was measured using flow
cytometry analysis for MitoSOX red staining (n=3).
６０
3.3. Melatonin induces ER stress in SNU-C5/WT cells through regulation of
PrPC expression
To assess the effect of melatonin-induced ER stress in si-PRNP
transfected SNU-C5/WT cells, the expression and activation of ER-stress
associated proteins, such as PERK, eIF2α, ATF4, IRE1α, JNK, and p38 was
examined by western blot analysis (Figures 12A-F). si-PRNP transfected
SNU-C5/WT treated with melatonin (1 mM) showed significantly higher
levels of expression of the ER stress marker (ATF4) and of the
phosphorylation of ER stress regulators (PERK, eIF2α, IRE1α, JNK and p38)
compared to control. These findings indicate that melatonin induces ER stress
and knockdown of PrPC enhances melatonin mediated induction of ER stress.
６１
Figure 12. The effect of PrPC on melatonin-induced ER stress in SNU-C5/WT
colorectal cancer cells.
The expression levels of PERK (A), eIF2α (B), ATF4 (C), IRE1α (D), JNK
(E), and p38 (F) were determined by western blot analysis in SNU-C5/WT
cells transfected with si-PRNP or si-scr following treatment with melatonin
(1 mM) for 24 h (n=3).
６２
3.4. Inhibition of PrPC expression enhances melatonin-mediated effect of
apoptosis in SNU-C5/WT cells
In order to investigate melatonin-mediated effect on apoptosis in si-PRNP
transfected SNU-C5/WT cells, ER stress marker (CHOP) and apoptosis
associated proteins, including BAX, cleaved caspase3 and cleaved PARP1
were determined by western blot analysis. Under melatonin-mediated
induction of ER stress, knockdown of PrPC significantly enhanced CHOP, Bax,
cleaved caspase3 and cleaved PARP1, compared to untreated cells (Figures
13A-D). Melatonin significantly increased the percentage of early and late
apoptotic cells to 24.47% compared to control (2.79%). In addition, si-PRNP
transfected SNU-C5/WT treated with melatonin show significant increase in
early and late apoptotic cells to 42.81% (Figure 13E). These findings indicate
that combination of si-PRNP and melatonin induces apoptosis of SNU-C5/WT
via ER stress induction.
６３
Figure 13. The effect of PrPC on melatonin-enhanced ER stress-mediated
apoptosis in SNU-C5/WT cells.
The expression levels of ER stress marker, CHOP (A) and apoptosis
associated proteins, including BAX (B), cleaved Caspase3 (C), and cleaved
PARP1 (D), were determined by western blot analysis in SNU-C5/WT cells
transfected with si-PRNP or si-scr following treatment with melatonin (1
mM) for 24 h (n=3). (E) Apoptosis of cells was measured utilizing PI/annexin
V staining and flow cytometric analysis (n=3).
６４
4. DISCUSSION
In this study, the effects of melatonin, a hormone secreted by the pineal
gland, on the viability of cancer cells was investigated. Melatonin treatment of
SNU-C5/WT induced superoxide levels and decreased cell viability.
Moreover, melatonin inhibited PrPC expression and subsequently reduced
PINK1 expression ultimately destroying mitochondrial homeostasis. In
combination with melatonin, knockdown of PrPC induced further inhibition of
PINK1 expression and production of superoxide, melatonin-mediated ER
stress, and ultimately cellular apoptosis. Together, melatonin treatment and
PrPC knockdown led to increased apoptosis of SNU-C5/WT via PrPC-
dependent mitochondria/ER signaling. My study suggests melatonin as a novel
anti-cancer agent.
Melatonin, a hormone that is most widely known for its sleep inducing
abilities has the potential to regulate CRC cell growth by inducing apoptosis.
Several studies demonstrated that melatonin causes cancer cell death via
mitochondrial reactive oxygen species production (56, 57) and inhibits tumor
growth (122, 129). In addition, another study suggested that melatonin
regulates Endothelin 1 (ET-1) levels and suppresses tumor progression in
CRC (130), suggesting that melatonin has a clear potential in cancer therapy.
In accordance with my previous study, SNU-C5/WT cells, when treated with
melatonin, show decreased cell viability and increased mitochondrial
６５
dysfunction in dose and time dependent manner. Many cancer cells show
elevated ROS production compared to normal cells, because of oncogenic
mutation, increased metabolic activity, and cancer microenvironment.
Specifically, superoxide has been known as one of the most potent
mitochondrial deregulators and excess superoxide is associated with
apoptosis in many cancer cells (131-135). My results also suggested that
melatonin caused an increase in superoxide production in cancer cells, leading
to the apoptotic potentials of the cells after the treatment.
Previous studies suggested that PrPC plays a role in the protection of cells
against oxidative stress by clearing up toxic superoxide dismutase (SOD)
activities (108). In addition, studies have shown that when the gene for PrPC,
PRNP, is silenced, the activities of antioxidant enzymes including catalase and
glutathione reductase are reduced (136, 137). Thus, in this study the effects
of melatonin in the induction of ROS and mitochondrial dysfunction through
PrPC dependent pathway was examined. It was found that PINK1 expression
is decreased even further when PrPC was silenced via si-PRNP transfection
compared to treatment with melatonin alone, and that knockdown of PrPC
increased superoxide production in CRC cells even further than melatonin
treatment alone. These results are supported by previous studies showing
that melatonin-induced ROS production of CRC cells lines (56, 57), and this
effect was mediated by decrease of PINK1 expression (138). Thus, my
results suggest that melatonin plays a critical role in superoxide production
and PINK1 expression in SNU-C5/WT cells via PrPC dependent pathway.
６６
Further study is required to investigate the precise mechanism of the effects
of melatonin-mediated PINK and PrPC expression in CRC cells.
A previous study has suggested that PrPC function may be related to anti-
oxidant activities against ROS production in cancer (139). Other studies on
PrPC and cancer indicated that PrPC expression levels associate with ROS, ER
stress (125) and induction of tumor cell death (98, 124). The results of this
study support the hypothesis that ER stress is involved in melatonin/PrPC,
mediated apoptosis of cancer cells. My results from western blotting analysis
suggest that melatonin increased ROS-mediated ER stress-associated
proteins resulting in alterations in the levels of oxidative stress-related
proteins, including p-PERK, p-eIF2α, ATF4, p-IRE1α, p-JNK, p-P38, and
CHOP and subsequent apoptosis associated proteins, such as BAX, C-
Caspase3, and C-PARP1, finally leading to cancer cell death (140, 141). In
addition, my western blot analysis results show that silencing of PrPC
magnifies the effects of melatonin on ER and apoptosis, highlighting the
importance of PrPC in melatonin mediated cancer therapy. These results
signify the importance of combination of melatonin and silencing PrPC in CRC
cell treatment which regulates superoxide production and ER stress. However,
further mechanistic studies are required to investigate whether combination
of melatonin and anticancer drugs have synergistic effects in CRC cancer.
６７
PART II. The Application of Anti-cancer Reagents for Cancer Therapy
Prion Protein of Extracellular Vesicle Regulates the Progression of
Colorectal Cancer
Manuscript is published in Cancers (Basel). 2021 13(9):2144.
６８
1. INTRODUCTION
CRC is the second leading cause of cancer-related deaths and the third most
prevalent malignant tumor worldwide. Early diagnosis of CRC increases the
5-year survival rate by approximately 64%, but progression of metastasis
decreases the survival rate to 12% (1). Although the ideal treatment for CRC
is surgical control to remove tumor and metastases (142), chemotherapy is
the typical leading strategy to control CRC (3). Recent chemotherapy includes
fluoropyrimidine-based single-agent therapy, such as 5FU, and multiple-
agent therapy, including capecitabine, irinotecan, or OX (3). Despite the
advancements in chemotherapeutic strategies, drug resistance restricts the
chemotherapeutic effect by increasing the DNA repair process and drug-
releasing metabolism (143, 144). Furthermore, the tumor microenvironment,
hypoxia, increases drug resistance in patients with CRC (18), suggesting that
novel combination therapies, such as targeted therapy and immune checkpoint
inhibitor therapy, are needed to overcome CRC (3, 144).
Hypoxia is a common feature of malignant tumors, which contributes to
tumor angiogenesis, aggressiveness, and metastasis (73, 74). In particular,
the crosstalk between cancer cells and cells is regulated by extracellular
vesicles, especially exosomes, secreted from hypoxia-stimulated cancer
cells (89). Since hypoxic stress leads to significant alterations in the
molecular content and function of exosomes, hypoxic tumor-derived
６９
exosomes transfer some target genes, including glucose transporter,
epidermal growth factor receptor (EGFR), transfer receptors, and P-gp, to
non-hypoxic cells, resulting in the internalization of receptors and clustering
of oncogene/proto-oncogene-activating receptors (90). These hypoxia-
induced exosomes promote tumor angiogenesis, invasion, metastasis, and
tumor immune system. Thus, novel approaches for addressing metastatic
cancer must be explored to block the hypoxic exosome-mediated
communication between cancer cells and cells (90, 94-97).
PrPC is a cell surface glycoprotein and misfolding of PrPC is associated with
neurodegenerative diseases, including transmissible spongiform
encephalopathy and prion diseases (145). In tumor biology, studies have
indicated that PrPC plays an important role in cancer proliferation, invasion,
metastasis, apoptosis, and drug resistance (29, 146). My recent studies have
shown that hypoxia increases the expression of PrPC in CRC cells and that
PrPC regulates CSC markers in CRC cells and tumor progression (24, 38). In
particular, tissues of stage III CRC patients highly expressed PrPC with Oct4-
matched expression (24).
７０
stem cells
A human colon cancer cell line (SNU-C5/WT), 5FU-resistant cell (SNU-
C5/5FUR), and oxaliplatin-resistant cell (SNU-C5/OXR) were obtained from
the Chosun University Research Center for Resistant Cells (102). The cells
were cultured in RPMI 1640 with 10% FBS (Thermo Fisher Scientific) at 37℃,
5% CO2 condition in a humidified incubator. S707 human colon CSCs were
provided by Prof. Steven M Lipkin from the Department of Medicine, Weill
Cornell College of Medicine (103). The cells were cultured in ultra-low
attachment plates as spheres in Dulbecco's Modified Eagle Medium/Nutrient
Mixture F-12 (DMEM/F12) medium with supplements (Thermo Fisher
Scientific) at 37℃, 5% CO2 condition in a humidified incubator. The CSC
spheroids were cultured in ultra-low attachment flasks (Corning) and
maintained by collection via gentle centrifugation, dissociation to single cells,
and replating. In addition, SNU-C5/5FUR and SNU-C5/OXR and human CSCs
(S707) were cultured in ultra-low attachment six-well plates for spheroid
formation at 37℃, 5% CO2 condition in a humidified incubator. SNU-C5/5FUR
and SNU-C5/OXR and CSCs were treated with exosomes from 1) normoxic,
2) hypoxic, and 3) PRNP siRNA treated conditions. Spheroids measured with
a visual inverted microscope (Olympus, Tokyo, Japan).
７１
2.2. Cell culture and characterization of endothelial progenitor cells
Human umbilical vein endothelial cells (HUVECs) (purchased from Thermo
Fisher Scientific) were cultured in complete endothelial cell growth medium-
2 (EGM-2) medium (Lonza, Walkersville, MD, USA), and characterized with
flow cytometry analysis (147) using positive HUVEC marker (anti-human
CD31 or platelet endothelial cell adhesion molecule-1 (PECAM-1); and
negative HUVEC markers (anti-human CD45, and anti-human CD11b)
purchased from BD Pharmingen, San Diego, CA, USA.
2.3. Preparation of 5FU and oxaliplatin
5FU and oxaliplatin were purchased from Sigma, and 5FU and oxaliplatin
were dissolved in DMSO at 10 mM concentration. Stock solution was filter-
sterilized using a 0.22 μm pore filter (Sartorius Biotech GmbH). Aliquots of
stock solution were stored at 4℃ until use.
Flow cytometry with CD81 and CD63 was used to confirm exosome markers
(148). A two-color flow cytometry system was used to investigate the
immune-stained exosomes. The percentage of stained exosomes was
calculated by comparing the negative controls. Each sample was quantitatively
７２
analyzed using a CyFlow Cube 8 (Sysmex Partec). A data analysis was
performed using the FCS Express software package (De Novo Software).
To analyze the cell cycle, cells were incubated with RNase and stained with
PI (Thermo Fisher Scientific). To confirm the cell proliferation, the cells were
washed once in phosphate-buffered saline (PBS) and incubated with 10 μM
Carboxyfluorescein succinimidyl ester (CFSE) (Molecular Probes, Eugene,
Oregon, USA) for 10 min at 37℃. The CSFE-labeled cells were washed twice
in PBS and analyzed using a flow cytometer (CyFlow Cube 8) (24).
2.5. Isolation of exosomes
Conditioned media were obtained from CRC cells (3 × 106) after 48 h of
incubation with serum-free media. Exosomes were isolated from conditioned
media (60 ml per group) of normoxic, hypoxic, or si-PRNP pretreated
hypoxic CRC cells using an exosome isolation kit (Rosetta Exosome, Seoul,
Korea) (149). First, conditioned media was precleared by differential
centrifugation and concentrated using a centrifugal filter (Sigma-Aldrich).
And then, extracellular vesicle enrichment is obtained by using Solution A, B,
and C. Finally, purified exosomes is collected by the usage of spin-based
size-exclusion column. The protein concentration of exosomes is measured
by the bicinchoninic acid (BCA) assay. For cell treatment, 2 μg of exosomes
based on protein measurement using BCA assay were added to 2 × 105 cells.
７３
2.6. Identification of exosomes via cryo-electron microscopy
For cryo-electron microscopy (Cryo-EM), 5 μL of exosomes were loaded
on 300-mesh EM carbon grids with a hydrophilic surface and frozen using
Vitrobot (Thermo Fisher Scientific) in liquid nitrogen (150). The grids were
observed and analyzed using Talos L120C cryo-EM (Thermo Fisher
Scientific), and images were recorded at 13,000 magnification.
2.7. Dynamic light scattering analysis
The size of exosomes derived from colon cancer cells was measured using
the ELSZ-1000 analyzer (Otsuka electronics, Kobe, Japan) (151). Briefly,
exosomes (10 μL) were diluted to 1:100 in PBS. The solution was measured
by performing the zeta-potential and particle size analysis to confirm the
presence of exosomes.
2.8. Detection of PrPC concentration via enzyme-linked immunosorbent assay
Concentrations of PrPC in serum sample (100 μL) or isolated exosomes
(50 μg) were analyzed using enzyme linked immunosorbent assay (ELISA)
with commercial kit manufactured by Lifespan Biosciences, Seattle, WA, USA.
PrPC were quantified by its absorbance at 450 nm using a microplate reader
(BMG Labtech).
７４
2.9. Invasion assay
Matrigel-coated transwell cell culture chambers (8-μm pore size; Sigma-
Aldrich) and serum-free RPMI 1640 or endothelial cell growth basal
medium-2 (EBM-2) medium were used to assess the invasion of SNU-
C5/5FUR, SNU-C5/OXR, and HUVECs. The cells were first treated with
SNU-C5/5FUR or SNU-C5/OXR exosomes derived from different conditions
for: hypoxic, normoxic, and/or transfected with si-PRNP, and incubated for
72 h at 37℃ then invasion assay was performed. Cells were stained with 2%
crystal violet, and invasive cells were quantified and photographed using a
light microscope.
2.10. Wound-healing migration assay
Cells were added in 60 mm cell culture plates and grown at 90% confluency
in 4 ml of culture medium (98). A scratch was made in the cell layer by using
sterile pipette tip and cultured for 24 h at 37℃. The images were obtained
using a microscope (Eclipse TE300, Nikon, Tokyo, Japan).
2.11. Morpholometric analysis
Morphological changes in colon cancer cell lines were examined by phase-
contrast microscopy (Nikon, Tokyo, Japan). The cells were cultured in 24-
well plates (7,000 cells/well). Cell images were obtained using phase-
７５
contrast microscopy. The average cell size was calculated from at least three
different visual fields in three independent dishes using ImageJ software.
2.12. Human angiogenesis protein array
Commercially available human angiogenesis antibody array (Abcam,
Cambridge, UK) (152) was used to measure the expression of angiogenesis
proteins in HUVECs treated with exosomes, Approximately 200 μg of total
lysates protein was analyzed following the protocol.
７６
UGGUUUACAUGUUUUCCUA-3’) was purchased by Dharmacon.
Total protein was extracted using RIPA lysis buffer (Thermo Fisher
Scientific). Cell lysates (20 μg) were separated by 10% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, and proteins were transferred
onto polyvinylidene fluoride membranes for detection. After washing with
TBST (10 mM Tris-HCl [pH 7.6], 150 mM NaCl, and 0.05% Tween-20), the
membranes were blocked with 5% skim milk for 1 h and then incubated with
primary antibodies specific to PrPC, CD81, CD63, and β-actin, followed by
washing and another incubation peroxidase-conjugated secondary antibodies.
Visualization of the band was done with chemiluminescence.
2.15. BrdU incorporation assay
The cell proliferation parameters were assessed using a cell proliferation
5-bromo-2′-deoxyuridine (BrdU) ELISA colorimetric kit (Roche,
Mannheim, Germany). To perform ELISA, 100 μg/mL BrdU was added to
CRC cells and incubated at 37℃ for 3 h. Subsequently, 1 M H2SO4 was added
to stop the reaction. The light absorbance at 450 nm of the samples was
measured by using an ELISA reader (BMG labtech).
７７
2.16. Measurements of oxygen consumption rate
The mitochondrial oxygen consumption rate (OCR) was measured using an
XF96e Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA,
USA) following the instructions. Sequential injections of oligomycin, carbonyl
cyanide m-chlorophenylhydrazone, rotenone, and antimycin A provide
information for mitochondrial basal respiration, maximal respiration, ATP
turnover, and spare respiratory capacity respectively. Final results were
presented as the percentage of change compared with the control.
2.17. Tumorigenesis in CRC xenograft mice models
Mice xenograft model of CRC using BALB/c nude mouse was created with
subcutaneous injection of SNU-C5/WT cells (5 × 106). When the tumors
reached a volume of 10 mm3, 5FU (Sigma-Aldrich) and anti-PrP antibody
(Santa Cruz Biotechnology) were administered together. After 28 days of
drug administration, the mice were euthanized for histology. Two
perpendicular tumor dimensions (a = length, b = width) were measured with
a Vernier caliper and the volume (V; mm3) was calculated with the formula V
= (a × b2)/2 (153). The tumor specimens were fixed in 4% formaldehyde,
embedded in paraffin, sliced into 4 μm-thick sections, and stained for
immunohistochemistry-based analysis.
７８
2.18. In vitro vascular permeability assay
The permeability of treated HMVEC monolayers grown on transwell filters
(0.4 μm pore size; BD Biosciences) was assessed by the passage of
rhodamine B isothiocyanate-dextran (average MW ~70,000; Sigma). Briefly,
rhodamine-dextran was added to the top well at 20 mg/ml, and the appearance
of fluorescence in the bottom well was monitored by measuring 40 μl medium
aliquots in a time course using a victor3 multiple microplate reader (Perkin
Elmer, Waltham, Massachusetts) at 544 nm excitation and 590 nm emission.
2.19. In vivo vascular permeability assay
For the in vivo vascular permeability assay, the mice were treated with
experimental exosomes for 21 days (154). 100 mg/kg rhodamine-dextran
(Sigma-Aldrich; average MW ~70,000) were administered intravenously via
tail vein. Transcardiac perfusion was performed 3 hours post injection to
remove the excess dye. Tissues specimens were either embedded in Tissue-
Plus OCT Compound (Thermo Fisher Scientific) and cryo-sectioned for
fluorescent microscopy or fixed for hematoxylin and eosin (H&E) staining for
histological analysis.
７９
2.20. Immunofluorescence staining
For histological analysis, human colon cancer tissue or mouse tumorigenesis
model cancer tissue was fixed in 4% paraformaldehyde (Affymetrix, Santa
Clara, CA, USA) and embedded in paraffin. Paraffin-embeded sections of
tissue samples were incubated with the primary anti-bodies against CD31,
zonula occludens-1 (ZO-1), KI67, and cleaved caspase-3 (Santa Cruz
Biotechnology) depending on the experimental conditions. Alexa488-
conjugated or Alexa594-conjugated secondary antibodies (Thermo Fisher
Scientific) were used. DAPI (Vector Laboratories, Burlingame, CA, USA) was
used to mark cell nuclei. Confocal microscopy (Leica, Wetzlar, Hesse,
Germany) images were taken for subsequent analysis.
2.21. Ethics statement
All animal studies were approved by the Institutional Animal Care and Use
Committee of Soonchunhyang University and fulfilled in accordance with the
National Research Council Guidelines for the Care and Use of Laboratory
Animals. This study used male Balb/C nude mice (8–10 weeks old;
Biogenomics, Seoul, Korea). All animals were maintained in a pathogen-free
facility under a 12-h light/dark cycle at 25℃ with free access to water and
laboratory chow.
８０
2.22. Statistical analysis
Results were expressed as the mean ± SEM, and two-tailed Student’s t
test or one- or two-way analysis of variance was used to compute the
significance between the groups. Comparisons of three or more groups were
performed using Dunnett’s or Tukey’s posthoc test. A p value of < 0.05
was considered significant.
８１
3. RESULTS
During the rapid development of tumors, hypoxia stimulates the
hypersecretion of membrane-bound vesicle known as exosomes that can
induce angiogenesis, metastasis, and immunosuppression to drive tumor
progression (155, 156). To assess the effect of hypoxia on the component of
exosomes in drug-resistant CRC and to identify the key molecules in
regulating drug-resistant CRC properties, I am isolated the exosomes from
SNU-C5/5FUR under normal (N-5FUR-Exo) and hypoxic conditions (H-
5FUR-Exo) and characterized them (Figures 14A–C). N-5FUR-Exo and H-
5FUR-Exo expressed the exosome markers CD81 and CD63 (Figure 14D).
My previous studies have shown that Oct4 and PrPC are highly expressed
simultaneously in patients with CRC, and hypoxia significantly increased the
level of PrPC (24, 38), and this was confirmed as significant PrPC upregulation
in N-5FUR-Exo (Figure 14D) and even higher expression H-5FUR-Exo
(Figures 14D and E).
８２
８３
Figure 14. Characterization of exosomes secreted by normoxic and hypoxic
drug-resistant CRC cells.
(A) Representative cryo-electron microscopy analysis of exosomes isolated
from SNU-C5/5FUR under normoxic and hypoxic conditions. Scale bar = 200
nm (n=3). (B) Representative flow cytometry histogram of exosome markers,
CD81 and CD63, on exosomes under normoxic and hypoxic conditions for 48
h (n=3). (C) Size distribution analysis by dynamic light scattering (n=3). (D)
Expression of PrPC, CD81, and CD63 in N-5FUR-Exo, H-5FUR-Exo, H-
5FUR-Exo + si-PRNP, and H-5FUR-Exo + si-scr (n=3). (E) ELISA
analysis of PrPC expression in N-5FUR-Exo, H-5FUR-Exo, H-5FUR-Exo
+ si-PRNP, and H-5FUR-Exo + si-scr (n=3). Data are represented as the
mean ± SEM. *p<0.05, **p<0.01 (ANOVA).
８４
3.2. Hypoxia-induced exosomes isolated from drug-resistant CRC increasing
sphere formation via upregulation of PrPC
To determine whether PrPC in exosomes regulates the function of drug-
resistant CRC cells, I assessed the sphere formation in SNU-C5/5FUR and
SNU-C5/OXR after treatment with exosomes isolated from each cell under
normoxic or hypoxic conditions (Figure 15A). The capacity of sphere
formation in SNU-C5/5FUR and SNU-C5/OXR was significantly increased
following treatment with H-5FUR-Exo or H-OXR-Exo (Figure 15B). To
further confirm the effect of exosomes on tumor function in CRC, I initially
assessed the capacity of sphere formation in CSC after treatment with CSC-
derived exosomes (Figure 15C). The number of spheres in CSC were
significantly increased after treatment with H-CSC-Exo, compared with that
without treatment and in N-CSC-Exo (Figure 15D). In particular, the
inhibition of PrPC significantly decreased the number of spheres in CSC
treated with H-CSC-Exo (Figures 15C and D), suggesting that PrPC in cancer
cell-derived exosomes regulates the ability of sphere formation in CRC.
８５
８６
Figure 15. Sphere formation of SNU-C5/5FUR, SNU-C5/OXR and CSC after
treatment with exosomes.
(A) Sphere formation assay of SNU-C5/5FUR and SNU-C5/OXR treated with
exosomes isolated from SNU-C5/5FUR and SNU-C5/OXR under normoxic
and hypoxic conditions. Scale bar = 200 μm (n=3). (B) Quantification of the
number of spheres is shown as a bar graph. (C) Sphere formation assay of
CSC treated with exosomes isolated from CSC under normoxic and hypoxic
conditions. Scale bar = 200 μm (n=3). (D) The graph shows the number of
CSC spheres after treatment with exosomes. Data are presented as mean ±
SEM. **p<0.01 vs. non-treatment; ##p<0.01 vs. non-treatment (ANOVA).
８７
3.3. Hypoxia-induced exosomes isolated from drug-resistant CRC increasing
invasion, migration, and proliferation via upregulation of PrPC
In addition, to determine whether PrPC in exosomes regulates the function
of drug-resistant CRC cells, I confirmed the invasion and migration in SNU-
C5/5FUR and SNU-C5/OXR after treatment with exosomes isolated from
each cell under normoxic or hypoxic conditions (Figures 16 and 17). The
invasion and migration capacities of drug-resistant CRC cells were
significantly increased after treatment with H-5FUR-Exo or H-OXR-Exo
(Figures 16A and B, 17A and B). The proliferation capacity of CRC cells also
significantly increased after treatment with hypoxia-stimulated exosomes
(Figures 18A–F). Furthermore, the elongated mesenchymal-like morphology
was significantly induced after treatment with H-5FUR-Exo or H-OXR-Exo
(Figures 19A and B). However, the knockdown of PRNP blocked the effect of
hypoxia-stimulated exosomes (Figures 16-19). These findings indicated
that increased levels of PrPC in hypoxia-stimulated exosomes enhance CRC
cell functions, such as sphere formation, invasion, migration, and proliferation.
８８
Figure 16. Effect of exosomes secreted by hypoxic drug-resistant CRC cells
on invasion.
(A) Representative invasion analysis is shown as scale bar = 200 μm (n=3).
(B) The average number of invasive cells is shown as a bar graph. Data are
presented as mean ± SEM. *p<0.05, **p<0.01 vs. non-treatment; #p<0.05,
##p<0.01 (ANOVA).
８９
on migration.
(A) Representative wound-healing analysis is shown as scale bar = 200 μm
(n=3). (B) The average wound closure is shown as a bar graph. Data are
presented as mean ± SEM. *p<0.05, **p<0.01 vs. non-treatment; #p<0.05,
##p<0.01 (ANOVA).
９０
９１
Figure 18. Proliferation capacity of drug-resistant CRC cells after treatment
with exosomes.
CFSE analysis of SNU-C5/5FUR (A), SNU-C5/OXR (B), and CSC (C) after
treatment with exosomes secreted by each cell under normoxic and hypoxic
conditions (n=3). BrdU incorporation analysis in SNU-C5/5FUR (D), SNU-
C5/OXR (E), and CSC (F) after treatment with exosomes secreted by each
cell under normoxic and hypoxic conditions (n=3). Data are presented as
mean ± SEM. *p<0.05, **p<0.01 vs. non-treatment; #p<0.05, ##p<0.01
(ANOVA).
９２
on morphological change.
(A) Representative morphological change analysis of SNU-C5/5FUR and
SNU-C5/OXR treated with exosomes isolated from SNU-C5/5FUR and
SNU-C5/OXR under normoxic and hypoxic conditions. Scale bar = 50 μm
(n=3). (B) The average number of morphologically changed cells is shown as
a bar graph (n=3). Data are presented as mean ± SEM. *p<0.05 vs. non-
treatment; #p<0.05 (ANOVA).
９３
3.4. The characterization of HUVECs and uptake of Hypoxia-stimulated CRC
exosomes
To investigate whether hypoxia-stimulated CRC exosomes induce tumor
angiogenesis, I am characterized HUVECs (Figures 20A and B) and treated
Dil-labeled 5FUR-exosomes with HUVECs. Fluorescence microscopic
images showed that Dil-labeled 5FUR exosomes were taken up by HUVECs
(Figure 21A). In HUVECs containing Dil-labeled 5FUR exosomes, H-5FUR-
Exo significantly increased the level of PrPC compared with N-5FUR-Exo
and si-PRNP + H-5FUR-Exo (Figure 20C), suggesting that PrPC in
exosomes isolated from CRC cells is transferred into endothelial cells.
９４
Figure 20. Characterization of HUVECs and expression of PrPC after treatment
with exosomes.
(A) Representative flow cytometry histogram of HUVECs positive markers,
CD31, and negative markers CD45 and CD11b (n=3). (B) Representative flow
cytometry histogram of the uptake of tetramethylindocarbocyanine
perchlorate (DiI)-labeled exosomes in HUVECs (n=3). (C) ELISA analysis
of PrPC expression in HUVECs after treatment with exosomes secreted by
normoxic and hypoxic SNU-C5/5FUR (n=3). Data are presented as mean ±
SEM. **p<0.01 vs. non-treatment; ##p<0.01 (ANOVA).
９５
Figure 21. Uptake of exosomes into HUVECs.
(A) Representative immunofluorescence analysis of intracellular uptake of
DiI-labeled exosomes (red) to HUVECs. Scale bar = 50 μm (n=3).
９６
3.5. Hypoxia-stimulated CRC exosomes promoting tumor angiogenesis and
vascular permeability
To assess the effect of hypoxia-stimulated exosomes on HUVEC migration
and invasion, the migration and invasion capacities of HUVECs after treatment
with exosomes were assessed. The migration and invasion capacities of
HUVECs were significantly augmented after treatment with H-5FUR-Exo
compared with that in other groups (Figures 22A–D). The silencing of PRNP
in H-5FUR-Exo significantly inhibited the migration and invasion capacities
(Figures 22A–D). In particular, the expression of angiogenic cytokine, C-X-
C motif chemokine 5 (ENA-78), in HUVECs treated with exosomes
significantly increased after treatment with H-5FUR-Exo (Figures 23A and
B). Moreover, permeability assay showed that treatment of HUVECs with H-
5FUR-Exo significantly increased the permeability of HUVECs (Figure 24A).
To further reveal whether exosomes secreted by hypoxia-stimulated drug-
resistant CRC cells attenuated the endothelial barrier in vivo, I am injected
exosomes isolated from SNU-C5/5FUR to mouse models and analyzed the
permeability of blood vessels (Figure 24B). In the liver and lungs, injection
with H-5FUR-Exo increased the permeability of blood vessels (Figure 24B).
Furthermore, the endothelial tight junction was significantly decreased after
injection of H-5FUR-Exo by decreasing in the level of ZO-1 (Figure 25A).
However, knockdown of PRNP blocked the effect of H-5FUR-Exo on
angiogenesis and permeability of endothelial cells in vitro and in vivo (Figures
22–25). These data indicated that exosomes secreted by hypoxia-stimulated
９７
drug-resistant CRC cells increase tumor angiogenesis and the permeability
of endothelial cells via PrPC.
９８
on the motility of HUVECs.
(A) Representative wound healing assay of HUVECs treated with exosomes
isolated from SNU-C5/5FUR under normoxic and hypoxic conditions. Scale
bar = 200 μm (n=3). (B) The average number of wound closures is shown
９９
as a bar graph. Data are presented as mean ± SEM. **p<0.01 vs. Prior to
migration. #p<0.05, ##p<0.01 (ANOVA). (C) Representative invasion
analysis of HUVECs treated with exosomes isolated from SNU-C5/5FUR
under normoxic and hypoxic conditions. Scale bar = 200 μm (n=3). (D) The
average number of invasive cells is shown as a bar graph. Data are presented
as mean ± SEM. *p<0.05, **p<0.01 vs. non-treatment. ##p<0.01 (ANOVA).
１００
on angiogenesis.
(A) Representative immunoblot analysis of angiogenesis related proteins in
HUVECs treated with PBS, N-5FUR-Exo, H-5FUR-Exo, and si-PRNP +
１０１
H-5FUR-Exo (red square: angiogenin, yellow square: EGF, green square:
ENA-78, and blue square: bFGF). (B) Average pixel density of immunoblot
are shown as the bar graph (n=2). Data are presented as mean ± SEM.
*p<0.05, **p<0.01 vs. PBS (ANOVA).
１０２
on vascular permeability.
(A) The permeability of treated HUVEC monolayers grown on 0.4 mm filters
was measured by the appearance of rhodamine-dextran, which was added to
the top well at the beginning of the experiment, in the bottom well during a 1
h time course. The absorbance at 590 nm at each time point is indicated (n=3).
Data are presented as mean ± SEM. **p<0.01 (ANOVA). (B) Representative
１０３
images of vascular permeability in vivo on liver and lung treated with
intravenously injected exosomes detected by the appearance of intravenously
injected rhodamine-dextran (red) (n=3). Scale bar = 50 μm.
１０４
on blood vessels.
(A) Representative immunofluorescence analysis of ZO-1 (green) and CD31
(red) expression in liver and lung treated with intravenously injected
exosomes (n=3). Scale bar = 50 μm.
１０５
3.6. Administration of 5FU, anti-PrP antibody, cetuximab inhibiting CRC
progression through suppression of PrPC level
To confirm the effect of 5FU, anti-PrP antibody, or cetuximab on CRC
progression, I initially assessed the tumor size after treatment with 5FU, anti-
PrP antibody (0.05 or 0.5 mg/kg), or cetuximab (0.5 mg/kg) twice a week in
a SNU-C5/WT xenograft model and investigated the level of PrPC in serum
(Figures 26A–D). In a wild-type CRC xenograft model, treatment with 5FU,
anti-PrP antibody (0.05 or 0.5 mg/kg), or cetuximab significantly decreased
the tumor size (Figures 26B and C). The level of PrPC in serum was
significantly reduced after treatment with 5FU, anti-PrP antibody (0.05 or
0.5 mg/kg), or cetuximab, compared with that treated with PBS (Figure 26D).
１０６
Figure 26. Effect of anti-PrP antibody in a wild type CRC xenograft model.
(A) Schematic illustration of SNU-C5/WT cells in vivo subcutaneously
transplantation and injection with PBS, 5FU, anti-PrP antibody (0.05 mg or
0.5 mg), and cetuximab (0.5 mg). (B) Photographs of tumor growth in a
１０７
murine xenograft mouse model. (C) Quantification of tumor size in each group
(n=10). (D) ELISA analysis of PrPC expression in sera isolated from a murine
xenograft model (n=10). Data are presented as mean ± SEM. *p<0.05,
**p<0.01 (ANOVA).
１０８
3.7. Co-administration of 5FU and anti-PrP antibody inhibiting CRC
progression through suppression of PrPC level
To further determine whether hypoxia-induced exosomes isolated from
drug-resistant CRC affect CRC progression and co-administration of 5FU and
anti-PrP antibody suppresses CRC progression in SNU-C5/WT cells
pretreated with an exosomes xenograft model (Figure 27A), I assessed the
tumor size in SNU-C5/WT cells pretreated with exosomes xenograft model
after treatment with H-5FUR-Exo + PBS, H-5FUR-Exo + 5FU, H-5FUR-
Exo + 5FU + anti-PrP antibody, or H-5FUR-Exo + anti-PrP antibody
(Figure 27B). I also assessed the tumor size in a SNU-C5/WT xenograft
model, as a negative control, after treatment with PBS (no Exo + PBS) and
5FU (no Exo + 5FU) (Figure 27B). In a SNU-C5/WT pretreated with H-
5FUR-Exo xenograft model, co-treatment with 5FU and anti-PrP antibody
(H-5FUR-Exo + 5FU + Anti-PrP) significantly decreased the tumor size
(Figure 27C). The concentration of PrPC in serum was also significantly
reduced after co-treatment with 5FU and anti-PrP antibody, compared with
that in cells treated with PBS (H-5FUR-Exo), 5FU (H-5FUR-Exo + 5FU),
or anti-PrP antibody (H-5FUR-Exo + anti-PrP) (Figure 27D). In the
context of tumor proliferation in vitro, co-treatment of SNU-C5/WT with
anti-PrP antibody and 5FU significantly decreased the S phase of the cell
cycle (Figures 28A and B). In particular, the S phase after co-treatment with
anti-PrP antibody and 5FU was decreased in a 5FU dose-dependent manner
(Figure 28C). In comparison with the anti-proliferative effect of the anti-PrP
１０９
antibody and cetuximab, treatment with a high concentration of cetuximab (10
μg/mL) decreased the S phase of the cell cycle of SNU-C5/WT, com-pared
with that treated with 5FU, anti-PrP antibody, or low concentration of
cetuximab (1 μg/mL) (Figure 29A). Co-treatment with cetuximab, an anti-
PrP antibody, and 5FU showed the most suppressive effect on CRC
proliferation (Figure 29B). Furthermore, treatment of SNU-C5/WT with
anti-PrP antibody or cetuximab significantly decreased mitochondrial
oxidative phosphorylation, including mitochondrial basal respiration, ATP
turnover, and spare respiratory capacity, compared with the non-treatment
group (Figures 30A–E). In a SNU-C5/WT pretreated with H-5FUR-Exo
xenograft model, co-administration of 5FU and anti-PrP antibody
significantly reduced the expression of Ki-67 in tumor tissues (Figures 31A
and B). Conversely, the level of apoptotic marker cleaved caspase3 in tumor
tissues was significantly increased after co-treatment with 5FU and anti-PrP
(Figures 31C and D). In the H-5FUR-Exo-treated tumor tissues,
immunofluorescence staining of ZO-1 showed that the tight junctions of tumor
blood vessels decreased after co-administration of 5FU and anti-PrP
antibody (Figure 32A). These findings suggest that co-administration of 5FU
and anti-PrP antibody suppresses CRC progression by blocking PrPC.
１１０
１１１
Figure 27. Effect of co-administration of anti-PrP antibody and 5FU in a
murine xenograft model treated with exosomes secreted by hypoxic CRC cells.
(A) Schematic illustration of SNU-C5/WT cells pretreated with/without H-
5FUR-Exo in vivo subcutaneous transplantation and injection with PBS, 5FU,
5FU + anti-PrP antibody, and anti-PrP antibody. (B) Photographs of tumor
growth in a murine xenograft mouse model. (C) Quantification of tumor size
in each group (n=6). (D) ELISA analysis of PrPC expression in sera isolated
from a murine xenograft model (n=6).
１１２
Figure 28. Inhibitory effect of anti-PrP antibody with 5FU on proliferation of
CRC cells.
(A) Cell cycle analysis in SNU-C5/WT cells after treatment with 5FU, anti-
１１３
PrP antibody, or 5FU + anti-PrP antibody (n=3). (B) The graph shows S
phase levels of SNU-C5/WT after treatment with 5FU, anti-PrP antibody, or
5FU + anti-PrP antibody. (C) Cell cycle analysis in SNU-C5/5FUR after
treatment with 5FU (10, 50, and 100 μM), anti-PrP antibody, and anti-PrP
antibody + 5FU (10, 50, and 100 μM) (n=3). Data are presented as mean
± SEM. **p<0.01 vs. control; #p<0.05, ##p<0.01 (ANOVA).
１１４
１１５
Figure 29. Inhibitory effect of anti-PrP antibody with 5FU and cetuximab on
proliferation of CRC cells.
(A) Cell cycle analysis in SNU-C5/WT cells after treatment with 5FU, anti-
PrP antibody, or cetuximab (1 and 10 μg/mL) (n=3). (B) Cell cycle analysis
in SNU-C5/WT cells after treatment with 5FU, 5FU + anti-PrP antibody,
cetuximab + 5FU, cetuximab + anti-PrP antibody, and cetuximab + anti-PrP
+ 5FU (n=3).
１１６
Figure 30. Mitochondrial respiration in CRC cells after treatment with anti-PrP
antibody and cetuximab.
(A) OCR in SNU-C5/WT after treatment with anti-PrP antibody and
cetuximab (n=10). (B–E) Mitochondrial basal respiration (B), maximal
respiration (C), ATP turnover (D), and spare respiratory capacity (E) in
SNU-C5/WT after treatment with anti-PrP antibody and cetuximab (n=10).
Data are presented as mean ± SEM. *p<0.05, **p<0.01 vs. non-treatment
(ANOVA).
１１７
Figure 31. Effect of co-administration of anti-PrP antibody and 5FU in a
murine xenograft model treated with exosomes secreted by hypoxic CRC cells
on proliferation and apoptosis.
(A) Representative immunofluorescence staining analysis of Ki-67 (green)
in colorectal cancer tissues (n=3). Scale bar = 50 μm. (B) The graph shows
Ki-67-positive cells in tumor tissues (C) Representative
１１８
immunofluorescence staining analysis of cleaved caspase3 (red) in colorectal
cancer tissues (n=3). Scale bar = 50 μm. (D) The graph shows cleaved
caspase3-positive cells in tumor tissues. Data are presented as mean ± SEM.
*p<0.05, **p<0.01 (ANOVA).
１１９
１２０
Figure 32. Effect of anti-PrP antibody and 5FU on vascular permeability in vivo.
(A) In a mouse pre-injected with or without exosomes isolated from hypoxic
SNU-C5/5FUR, mice were injected with PBS, 5FU, 5FUR + anti-PrP
antibody, or anti-PrP antibody. Vascular permeability was assessed by
immunofluorescence staining of ZO-1 (green) and CD31 (red). Scale bar =
50 μm (n=3).
１２１
4. DISCUSSION
It has been shown that PrPC regulates proliferation, drug resistance,
metastasis, and CSC properties in various type of cancers including pancreatic,
breast, and colon cancers (157). Although exosomal PrPC is known to inhibit
amyloid beta-mediated neurotoxicity in Alzheimer’s disease (158), and
facilitate intercellular prion transmission in prion disease (159), studies on
the effect of exosomal PrPC on the tumor progression are limited. In this study,
I demonstrated that exosomal PrPC promotes proliferation, invasion and
migration of cancer cells. I found that exosomal PrPC inhibits migration and
invasion of vascular endothelial cells and increases permeability of blood
vessels. Therefore, PrP antibody treatment can reduce permeability of blood
vessels and inhibit metastatic CRC development by neutralizing exosomal PrP.
In addition, I also demonstrated that co-administration of anti-PrP antibody
with chemotherpy can improve cancer treatment efficacy.
This study showed that hypoxia induced the expression of PrPC in exosomes
secreted by drug-resistant CRC cells and that PrPC-expressing exosomes
promote CSC properties and tumor progression. PrPC is a highly ubiquitous
glycoprotein that affects the process of tumor progression, such as
proliferation, migration, invasion, metastasis, chemoresistance, and apoptosis
as well as stemness of cancer cells (22, 146, 160). Previous studies indicated
that PrPC promotes tumor metastasis, epithelial–mesenchymal transition, and
１２２
glucose metabolism through the regulation of Fyn, cytoskeletal regulatory
proteins (33, 36). PrPC also regulates multi-drug resistance via interaction
with CD44 (141, 157). Under hypoxic conditions, PrPC induces tumor
progression in CRC by targeting the heat shock protein 70 member 1-like
(HSPA1L)/HIF-1α/GP78 signal axis (38).
Hypoxia is a pathophysiological tumor microenvironment, which affects the
cell cycle, morphological conformation, energy metabolism, differentiation,
apoptosis, and autophagy (94, 161, 162). Under hypoxic conditions, cancer
cells regulate a wide range of gene expression in conjunction with the major
components of the hypoxia signaling pathway through expression of HIFs (74,
90). In patients with pancreatic tumors, the level of HIF-1α expression was
significantly increased (163). Among HIFs, the overexpression of HIF-1α
in response to hypoxia promotes tumor blood vessel formation,
aggressiveness, metastasis, and drug resistance. Hypoxia and HIF-1α
contribute to abnormal blood vessels, which are newly formed by
discontinuous endothelium and the blockage of lymphatic drainage, resulting
in the production of vascular hyperpermeability and increased permeation
(164, 165).
Recently, several studies have revealed that the crosstalk between tumor
cells and the microenvironment is a key factor for tumor progression through
extracellular vesicles and exosomes secreted by hypoxic cancer cells (70,
71). Hypoxia-induced exosomes released from cancer cells contain plasma
１２３
membrane receptors, including glucose transporter, EGFR, P-glycoprotein,
and multidrug resistance protein 1; angiogenic proteins, such as VEGF, FGF,
and angiogenin; and various noncoding RNAs, including miRNAs and lncRNAs
(162, 165, 166).
My study observed that targeted genes associated with CSC markers,
metastasis, angiogenesis, and oncogenes were significantly increased in
exosomes secreted from hypoxic 5FU-resistant CRC cells. In particular, the
expression of these genes in exosomes was regulated by the expression of
PrPC. These exosomes enhanced the cancer sphere formation, invasion,
migration, and proliferation in drug-resistant and CSCs in CRC. Notably,
exosomes secreted by hypoxic drug-resistant CRC cells are incorporated into
HUVECs and increase the migration, invasion, permeability, and production of
angiogenic cytokines.
In vivo study, these exosomes augmented the vascular hyperpermeability
through inhibition of tight junction protein ZO-1 expression. These effects
were blocked by silencing of PRNP. For stabilization of PrPC, My previous
study showed that HIF-1α-induced HSPA1L downregulated the expression
of GP78, a ubiquitinase for PrPC, resulting in the stabilization of PrPC under
hypoxic conditions (38). In lung cancer, exosomes secreted by hypoxic
cancer increased the expression of miR-23a, which inhibited the expression
of ZO-1, resulting in the induction of vascular permeability and cancer trans-
endothelial migration (95). Exosomes secreted by metastatic breast cancer
１２４
also destroyed endothelial ZO-1 expression and integrity through the
upregulation of miR-105 (154). This evidence indicates that exosomes
secreted by hypoxic tumor cells play important roles in the reduction in
endothelial integrity and tumor progression, suggesting that targeting of PrP-
expressing exosomes secreted by hypoxic tumors might be a novel strategy
for patients with CRC.
Clinically, antibody therapy for tumors provides the possibility for treating
patients with cancer in a targeted fashion by decreasing severe side effects,
compared with conventional chemotherapy (167). For the development of
advanced and highly therapeutically efficient antibody therapy in cancer
biology, discovery of specific molecular biomarkers in a wide range of solid
malignancies is a key process for beneficial therapeutic outcomes (167).
According to the unmet medical needs of patients with tumors, tumor
therapeutic antibodies, including trastuzumab (anti-human epidermal growth
factor receptor 2 [HER2] therapy) and cetuximab (anti-EGFR therapy), were
developed and clinically approved as treatments for a solid carcinoma and a
broad range of cancers, respectively (168, 169).
Cetuximab is a chimeric human mouse anti-EGFR monoclonal antibody and
it is used in combination with chemotherapy or as a single agent in metastatic
colon cancer and metastatic squamous cell head and neck cancer (170). EGFR
is overexpressed in most epithelial cell carcinomas such as colorectal cancer,
breast cancer, and lung cancer, and it is known that activation of EGFR
１２５
promotes cancer proliferation, angiogenesis, metastasis and inhibits apoptosis
(171). Cetuximab selectively binds to EGFR and competitively inhibits the
binding of EGF and other ligands, preventing its activation, eventually
inhibiting the growth of cancer cells and production of MMP and EGF and
inducing apoptosis (172).
However, intrinsic phenotypic variation and adaptive phenotypic
modifications in tumor cells can induce repeated exposure to sub-optimal
doses of the biotherapeutic, resulting in acquired resistance to monoclonal
antibody therapy (167, 173, 174). Antibody–drug conjugates (ADC) are
another option for treating tumors as a novel antibody-based therapeutic.
ADC consist of targeted antibody and anti-cancer drugs covalently attached
to the antibody, resulting in ADC reaching the tumor and killing the tumor.
Although ADC have the potential for this concept, the clinical outcomes are
limited because some chemical drugs can be released from the tumor and
diffuse into the surrounding cells (167, 175175).
In this study, I identified a novel CRC target, PrPC, and assessed the effect
of anti-PrP antibody on CRC by co-administration of 5FU. In vitro, anti-PrP
significantly inhibited proliferation and mitochondrial respiration. In exosome
non-treated murine xenograft model, the administration of anti-PrP antibody
significantly decreased the tumor size and serum PrPC concentration. This
effect was similar to the effect of cetuximab. In an H-5FUR-Exo-treated
xenograft model, co-administration of anti-PrP antibody and 5FU
１２６
significantly reduced the tumor size, PrPC expression, and tumor cell
proliferation. Furthermore, co-administration drastically increased the
number of apoptotic CRC cells in tumor tissues. These findings indicate that
co-administration of anti-PrP antibody and 5FU suppresses CRC tumor
progression, suggesting that anti-PrP antibody-based therapy could be a
novel and powerful strategy for clinical application in patients with CRC.
１２７
CONCLUSION
My findings reveal for the first time that 5FU-resistant colorectal cancer
cells express high levels of PrPC, and that PrPC facilitates anti-cancer drug
resistance by regulating survival, proliferation, and apoptosis signaling
pathways (Figure 33). Further studies are required to understand how the
expression of PrPC is induced during the acquisition of anti-cancer drug
resistance. This study may help clarify the physiological function of PrPC in
drug resistant colorectal cancer cells. The strategy of targeting PrPC may
enable novel molecular target combination chemotherapies. Furthermore, my
findings reveal that melatonin inhibits cell viability via excessive production
of superoxide and increased PrPC expression. In addition, silencing PrPC
enhances melatonin mediated accumulation of superoxide and activates ROS
mediated ER stress. Finally, apoptosis of CRC cancer cells is activated (Figure
34). This study demonstrated that the combination of melatonin and silencing
PrPC in SNU-C5/WT cells results in increased anticancer effects. These
findings indicate that understanding the effects of melatonin and PrPC on
colorectal cancer may help design a novel therapeutic strategy in cancer
treatment.
Next, these results indicate that exosomes secreted by hypoxic drug-
resistant CRC cells are key regulators of CRC progression (Figure 35). PrPC
is a pivotal messenger for regulating CRC behavior through the secretion of
１２８
exosomes by hypoxic tumors. Furthermore, my results suggest the possibility
of clinical application of anti-PrP antibody with anti-cancer drugs. Although
my results indicated that the administration of anti-PrP antibody did not have
an effect on several organs and tissues in a pre-clinical study, its side effects
and safety should be investigated prior to the application of this antibody in
patients with CRC. In conclusion, the co-administration of anti-PrP antibody
and anti-cancer drugs might be a potential therapeutic strategy for patients
with CRC through inhibition of exosomal PrPC expression and suppression of
CRC progression.
Taken together, these findings suggest that targeting PrPC might be a
potential therapeutic strategy for CRC therapy and induction of synergistic
effects with diverse anti-cancer reagents and could be an effective diagnostic
factor.
１２９
Figure 33. Schematic illustrating the protective mechanism of PrPC on 5FU in
5FU-resistant cancer cells.
In 5FU-resistant cancer cells, the expression level of PrPC is increased. In
the presence of 5FU, PrPC promotes cell survival, proliferation via activation
of the PI3K-AKT signaling pathway, and blockage of stress- and apoptosis-
mediated protein activation and reduction of apoptosis.
１３０
Figure 34. Schematic illustrating the mechanism of melatonin-mediated anti-
cancer effects in SNU-C5/WT CRC cells.
In colorectal cancer cells, melatonin induces the production of superoxide and
decreases the levels of PrPC. Silencing PrPC expression enhanced the
anticancer effects of melatonin via the production of mitochondrial superoxide,
activation of ER stress- and apoptosis- mediated proteins.
１３１
Figure 35. Schematic illustrating the effects of exosomes secreted by hypoxic
drug-resistant CRC cells on CRC progression and metastasis.
PrPC are overexpressed in cancers and related to cancer proliferation,
invasion, metastasis, and drug resistance. PrPC-expressing exosomes
induced the microenvironment of metastasis via increase of endothelial
permeability and angiogenic cytokine secretion. The treatment of anti-PrPC
and 5FU decreased the tumor progression.
１３２
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１５３
ABSTRACT IN KOREAN
정상 프리온 단백질 조절을 통한 대장암 세포에서의 항암제의
치료효과 향상
윤 철 원
순천향대학교 대학원 의생명과학과
(지도교수 : 이 상 훈)
대장암은 질병의 후기 진행단계에서 공격적인 전이로 인한 암 관련 죽음의
주요원인 중 하나이다. 최근 연구들을 통해 대장암세포의 약물내성 획득에 관한
기작을 확인했지만, 약물내성을 극복하기 위한 새로운 치료법은 아직도 개발되지
않았다. 여전히 항암제 내성은 대장암 환자의 심각한 문제이고, 이러한 문제를 해
결하기 위해 다양한 항암 인자와 분자적 타겟에 대한 연구가 진행되고 있다. 송과
선에서 만들어져 내재적으로 분비되는 인돌아민 호르몬인 멜라토닌은 대장암을
포함한 다양한 암에서 산화성 스트레스 및 세포사멸을 유도하는 다양한 기작을
통해 항암효과를 보이는 것으로 알려져 있다. 저산소증은 대장암에서 종양의 증식
과 전이를 촉진시킨다. 종양 미세환경은 일반적으로 저산소증으로 특징 지어지기
때문에, 이에 대한 이해는 암 치료에 있어서 중요하다. 또한, 암 전이에 대한 프
１５４
리온 단백질의 역할에 대한 관심이 증가하고 있지만, 프리온 단백질을 통한 세포
내 상호작용을 통한 정확한 기작은 잘 알려져 있지 않다. 따라서 본인은 1) 프리
온 단백질이 세포 생존신호경로 조절을 통해 대장암의 약물내성 및 멜라토닌을
통한 활성산소가 매개된 세포사멸에 관여하고, 2) 프리온 단백질을 포함한 세포외
소포체를 통한 대장암세포의 종양진행을 조절할 것이라 가정하였다.
5FU 내성 대장암 세포에서 프리온 단백질의 발현에 따른 대장암의 변화 양
상을 확인하였다. 대장암 환자로부터 얻은 검체에서 세포 프리온 단백질의 발현은
대장암의 전이 및 종양의 단계와 유의미한 상관관계를 보여주었고 대장암 세포의
종양형성 및 유전학적 변화에 영향을 주었다. 또한, 항암제 내성 대장암세포에서
프리온 단백질의 발현이 상당히 증가됨을 확인하였고, 5FU 처리 후 프리온 단백
질은 PI3K-AKT 신호경로 및 세포 주기 관련 단백질의 활성화 유지를 통해 대
장암 세포의 생존과 증식을 촉진시키며 스트레스 관련 단백질의 활성화를 억제하
였다. 프리온 단백질의 발현을 억제한 후 5FU의 처리는 caspase-3의 활성화를
통해 세포사멸을 증가시켰다.
대장암에서 멜라토닌 처리는 프리온 단백질과 PINK1의 발현을 감소시키고
미토콘드리아에 과산화물의 축적을 증가시켰다. 프리온 단백질의 억제는 멜라토닌
의 효과를 강화시키고 PINK1의 발현을 상당히 감소시켜 대장암세포에 과산화물
의 축적을 증가시켜 ER stress 유도를 통한 세포사멸을 증가시켰다. 다음으로, 저
산소 종양 미세환경은 대장암에서 프리온 단백질을 발현하는 엑소좀을 증가시키
고, 이러한 엑소좀은 프리온 단백질 발현에 따라 대장암 세포의 행동과 종양의 진
행을 조절한다. 저산소 상태에서 나온 대장암 세포 엑소좀은 종양의 구형 형성,
종양 유도 유전자 발현, 이동성 및 종양의 성장을 촉진시켰다. 추가로, 엑소좀은
１５５
혈관 내피 세포의 침투성, 이동성과 혈관 형성 사이토카인의 분비를 증가시켰다.
이러한 효과는 프리온 단백질 발현과 관련되어 있고, 마우스 동물 모델에서 5FU
와 항 프리온 단백질 항체와의 병합처리는 대장암의 진행을 상당히 억제하였다.
결론적으로 프리온 단백질이 통해 대장암세포에서 약물 내성을 획득하는데
중요한 역할을 하고 있다는 것을 제시한다. 또한, 항암치료제로서 멜라토닌은 프
리온 단백질과 미토콘드리아의 항상성 조절에 관여하는 PINK1 단백질의 조절을
통해 대장암세포의 미토콘드리아 활성산소종의 생성을 증가시키고, ER stress를
통한 세포사멸을 유도하였고, 프리온 단백질의 siRNA를 통한 억제는 멜라토닌의
효과를 강화시켜 대장암 치료를 위한 새로운 치료방법이 될 수 있음을 확인하였
다. 추가로, 저산소 상태에서 대장암세포에서 분비되는 프리온 단백질을 발현하는
엑소좀이 대장암간/대장암과 내피세포의 세포 신호전달에 중요한 인자임을 확인
하였다. 마지막으로 프리온 단백질의 억제를 통하여 항암화학 치료 시 대장암의
치료효율을 향상시킬 수 있음을 보여준다.
주제어 : 대장암, 저산소 조건, 멜라토닌, 5FU, 정상 프리온 단백질, 엑소좀, 항암
제 내성, 항체 치료
１５６
ACKNOWLEDGEMENTS
학위기간 중에 많은 어려움들이 있었지만 박사학위를 마칠 수 있었던 것은
많은 분들의 도움과, 격려로 무사히 학위과정을 마칠 수 있었습니다. 우선 박사
학위를 마칠 수 있도록 이끌어주신 이상훈 교수님께 감사의 뜻을 전하고
싶습니다. 학위 과정 동안 진행한 연구를 전폭적으로 지원해 주시고 다양한
관점에서 연구의 방향을 이끌어 주셔서 감사 드립니다. 또한, 한 명의 연구자로서
교수님의 모습을 본보기 삼아 성장하고 훌륭한 연구자가 되고 초심을 잃지
않도록 하겠습니다. 석사, 박사 학위과정 동안 지도해 주셔서 감사합니다.
대학원 학위과정 동안 저에게 힘이 되어주고 격려와 믿음으로 기다려 주신
부모님께 진심으로 감사의 말씀을 드립니다. 대학원 진학이라는 저의 결정을
존중해주시고 여러 방면으로 도움을 주셔서 이렇게 무사히 학위과정을 마칠 수
있었다고 생각합니다. 앞으로 새로운 환경에서 새로이 시작될 학위과정 이후의
삶에서도 이러한 고마움을 생각하며 더 발전하도록 하겠습니다.
학위논문의 심사과정에서 아낌없는 충고, 조언과 격려를 해주신 순천향대학교
의료생명공학과 이미영 교수님, 순천향대학교 비뇨기과 송윤섭 교수님,
단국대학교 치과대학 이준희 교수님, 그리고 충북대학교 수의학과 이현직
교수님께도 감사의 인사를 드립니다. 부족한 저를 격려해 주시고 진심 어린
조언을 통해 저의 학위논문의 주제와 내용이 더 풍성해 질 수 있었다고
생각합니다. 교수님들의 조언을 통해 좀 더 발전하고 노력하는 연구자가 될 수
있도록 노력하겠습니다.
１５７
마지막으로 저의 학위과정 동안 만났던 모든 분들께 진심으로 감사의 말씀을
드립니다. 연구실에서 많은 일이 있었지만, 학위과정 동안 서로 의지할 수 있었던
한용석, 윤여민, 이가은에게 감사하고, 연구실 생활을 하는 동안 다양한 조언을
해주신 고경윤 박사님, 예지혜 박사님, 박희정 박사님께 감사의 인사를 드립니다.
짧은 감사의 글이지만 저에게 도움을 주신 분들께 다시 한번 감사의 인사를
드리며 글을 마치겠습니다.
１５８

정상 프리온 단백질 조절을 통한 대장암 세포에서 항암제의 치료효과 향상

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정상 프리온 단백질 조절을 통한

The Enhancement of Therapeutic Effects of Anti-cancer Reagents

이 논문을 박사학위 논문으로 제출함

위원장 순천향대학교 이 미 영 (인)

LIST OF TABLES ····················IX

LIST OF FIGURES ···················· X

LIST OF ABBREVIATIONS ··············· XIV

ABSTRACT ······················ XIX

1. Colorectal Cancer ······················· 1

2. Cellular prion protein in colorectal cancer············ 4

3. The anti-cancer drugs for cancer therapy ··········· 6

4. Extracellular vesicles ····················· 8

5. The relationship between hypoxia and extracellular vesicles in cancer

6. Experimental purpose ····················· 13

Cellular Prion Protein Enhances Drug Resistance of Colorectal Cancer

Cells via Regulation of a Survival Signal Pathway ······· 15

2. MATERIALS AND METHODS ················· 18

2.1. Specimens of patients with colorectal cancer and normal controls 18

stem cells ··························· 18

2.3. Preparation of 5FU ······················· 19

2.6. Western blot analysis ······················ 21

2.7. Flow cytometry analysis ···················· 21

2.8. Immunofluorescence staining ·················· 22

2.9. siRNA transfection ······················· 23

2.11. Cell cycle analysis ······················· 24

2.12. PI/Annexin V flow cytometric analysis ·············· 25

2.13. Statistical analyses ······················· 25

3.1. The clinicopathological features in patients with CRC by the expression

3.2. PrPC involved in the CSC properties of CRC ··········· 30

3.3. The change of cancer-related genes in CRC through regulation of

3.4. Expression of PrPC in drug-resistant CRC cells·········· 34

cycle-associated proteins ··················· 38

3.7. PrPC regulates anti-cancer drug induced stress-associated signaling41

suppression of caspase3 activation and PARP1 cleavage······ 43

PART Ⅱ. The Application of Anti-cancer Reagents for Cancer

A. Melatonin Promotes Apoptosis of Colorectal Cancer Cells via

Superoxide-mediated ER Stress by Inhibiting Cellular Prion

Protein Expression ···················· 48

2. MATERIALS AND METHODS ················· 52

2.1. Preparation of melatonin ···················· 52

2.2. Cells and cell culture ······················ 52

2.3. Cell viability assay ······················· 52

2.4. Western blot analysis ······················ 53

2.5. siRNA transfection ······················· 54

2.6. Flow cytometry analysis ···················· 54

2.7. Statistical analyses ······················· 55

3.1. Melatonin decreases cell viability and increases production of superoxide

in SNU-C5/WT cells ······················ 56

the production of mitochondrial superoxide in SNU-C5/WT cells 58