Journal Pre-proof

Kalantuboside B induced apoptosis and cytoprotective autophagy in human

melanoma A2058�cells: An in vitro and in vivo study

You-Cheng Hseu, Hsin-Ju Cho, Yugandhar Vudhya Gowrisankar, Varadharajan

Thiyagarajan, Xuan-Zao Chen, Kai-Yuan Lin, Hui-Chi Huang, Hsin-Ling Yang

PII: S0891-5849(19)31278-X
DOI: https://doi.org/10.1016/j.freeradbiomed.2019.08.015
Reference: FRB 14382

To appear in: Free Radical Biology and Medicine

Received Date: 2 August 2019

Revised Date: 17 August 2019
Accepted Date: 17 August 2019

Please cite this article as: Y.-C. Hseu, H.-J. Cho, Y.V. Gowrisankar, V. Thiyagarajan, X.-Z. Chen, K.-
Y. Lin, H.-C. Huang, H.-L. Yang, Kalantuboside B induced apoptosis and cytoprotective autophagy in
human melanoma A2058�cells: An in vitro and in vivo study, Free Radical Biology and Medicine (2019),
doi: https://doi.org/10.1016/j.freeradbiomed.2019.08.015.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier Inc.

 Kalantuboside B (KB)
Activate
Inhibit
Human melanoma A2058 cells

ROS

ERK activation

LC3-II accumulation
AVOs formation
Beclin-1/Bcl-2 dysregulation
Cytochrome c release
PARP cleavage
Caspase-3/-8/-9/-12 activation
Fas activation/Bid cleavage
Bax/Bcl-2 dysregulation

Cytoprotective
Apoptosis
autophagy
Kalantuboside B induced apoptosis and cytoprotective autophagy in human melanoma
A2058 cells: An in vitro and in vivo study

You-Cheng Hseu1,2,3,4, Hsin-Ju Cho5, Yugandhar Vudhya Gowrisankar1, Varadharajan

Thiyagarajan1, Xuan-Zao Chen1, Kai-Yuan Lin6, Hui-Chi Huang7,*, Hsin-Ling Yang5,*

1
Department of Cosmeceutics, College of Biopharmaceutical and Food Sciences, China
Medical University, Taichung 40402, Taiwan
2
Department of Health and Nutrition Biotechnology, Asia University, Taichung 41354,
Taiwan
3
Chinese Medicine Research Center, China Medical University, Taichung 40402, Taiwan
4
Research Center of Chinese Herbal Medicine, China Medical University, Taichung 40402,
Taiwan
5
Institute of Nutrition, China Medical University, Taichung 40402, Taiwan
6
Department of Medical Research, Chi-Mei Medical Center, Tainan 71004, Taiwan
7
Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College
of Chinese Medicine, China Medical University, Taichung 40402, Taiwan

*Corresponding author: Hui-Chi Huang, Associate Professor for Department of Chinese

Pharmaceutical Sciences and Chinese Medicine Resources, College of Chinese Medicine;
Hsin-Ling Yang, Professor for Institute of Nutrition, College of Biopharmaceutical and Food
Sciences, China Medical University, 91 Hsueh-Shih Road, Taichung 40402, Taiwan.
Tel: 886-4-2205-3366 ext 7503; Fax: 886-4-22062891.
e-mail address: hchuang@ mail.cmu.edu.tw; hlyang@mail.cmu.edu.tw.

1
Abbreviations:
KB: Kalantuboside B
MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
PARP: poly (ADP-ribose) polymerase
LC3: microtubule-associated protein light chain-3
AVO: acidic vesicular organelle
3-MA: 3-methyladenine
CQ: chloroquine
ROS: reactive oxygen species
NAC: N-Acetyl-L-cysteine
ERK: extracellular signal-regulated protein kinase
Cdc25C: cell division cycle 25C
PI: propidium iodide
DCFH2-DA: 2’,7’-dihydrofluorescein-diacetate

2
ABSTRACT
Kalantuboside B (KB), a natural bufadienolide derivative extracted from the succulent plant
Kalanchoe tubiflora, is well-known for its cardiotonic, immunomodulatory, and anti-
inflammatory properties. In this study, we tested in vitro and in vivo anti-cancer efficacy with
low concentrations of KB (5–30 ng/mL; 8.7–52.2 nM) on A2058 melanoma cells; and for the
molecular mechanisms that underlie them. KB significantly inhibited the cell viability and
colony formation via arresting the cell cycle at G2/M phase. There was an association with a
decrease in Cyclin A/B1, Cdc25C, and Cdc2 expressions. Further, this treatment indicated the
induction of apoptosis, DNA fragmentation, cytochrome c release, and caspase-3, -8, -9, and -
12 activation, and PARP cleavage, which shows that mitochondrial, death-receptor, and ER-
stress signaling pathways are involved. KB-induced autophagy was apparent from enhanced
LC3-II accumulation, GFP-LC3 puncta, and AVO formation. Surprisingly, KB-mediated cell
death was potentiated by 3-MA and CQ to suggest the role of autophagy as a cytoprotective
mechanism. Moreover, KB-treated A2058 cells enhanced intracellular ROS generation and
antioxidant NAC prevented apoptosis and reversed cytoprotective autophagy. Interestingly,
KB-induced apoptosis (PARP cleavage) and cytoprotective autophagy (LC3-II accumulation)
were mediated by the up-regulation of the ERK signaling pathway. It was also shown that KB
promoted cytoprotective autophagy by a calcium dependent-p53 downregulation pathway. In
vivo data showed that KB suppressed tumor growth significantly in A2058-xenografted nude
mice. A Western blot indicated cell-cycle inhibition (cyclin A reduction), apoptosis induction
(PARP cleavage and Bcl-2 inhibition), and cytoprotective autophagy (LC3-II upregulation
and p53 downregulation) in KB-treated A2058-xenografted mice. Our findings suggested
that KB-induced ROS pathway plays a role in mediating the apoptosis and cytoprotective
autophagy in human melanoma cells. Thus, KB is considered to be a putative anti-tumor
agent.

Keywords: Kalantuboside B, human melanoma, ROS, Apoptosis, cytoprotective autophagy,

p53

3
Introduction
Autophagy and apoptosis are the cellular degradation pathways essential for
organismal homeostasis. Autophagy is a cell-survival pathway conserved in all eukaryotes,
which involves the selective degradation of cellular components, long-lived proteins, protein
aggregates, damaged cytoplasmic organelles, and intracellular pathogens resulting in the
recycling of nutrients and energy generation. Basal levels of autophagy are required for
cellular homeostasis. However, excess autophagy has been implicated in type II cell death [1].
Apoptosis is the best-understood canonical programmed cell death pathway. It is recognized
by distinct morphological characteristics of cells, such as cell shrinkage with nuclear
chromatin condensation and nuclear fragmentation. Apoptosis can be triggered either by
intracellular signals produced in response to cell stresses, such as increased Ca2+
concentration, oxidative damage caused by reactive oxygen species (ROS) [2], and hypoxia
[3] or extrinsic inducers, such as bacterial pathogens, toxins [4], nitric oxide [5], growth
factors [6], and hormones [7]. Autophagy and apoptosis are considered to be the tumor
suppression pathways. Autophagy degrades the oncogenic molecules and prevents the
development of cancers, whereas, apoptosis prevents cancer cell survival. Therefore,
defective or inadequate levels of autophagy or apoptosis lead to cancer. Autophagy and
apoptosis are cross-talked by several molecular nodes; that enables a coordinated regulation
of the degradation of these pathways [8].
Accumulating evidence suggesting that ‘melanoma’, or ‘malignant melanoma’, is the
most dangerous type of skin cancer whose prevalence is increasing worldwide with high
mortality rates [9]. Compared to non-melanoma cancers, malignant melanoma incurs 80% of
deaths in patients [10]. UV exposure to low levels of skin pigment is the primary cause of
melanoma [11]. It is more common in men than in women [12]. Despite the significant
progress in melanoma detection and treatment, the prognosis for malignant melanoma
remains poorly understood [13].
Several studies reported that anticancer drugs stimulates cytoprotective autophagy and
apoptosis as an alternate way of cell-death in different cancer types [14, 15]. These
mechanisms include different molecular and cell signaling pathways [16, 17]. The functional
association between apoptosis and autophagy is very complex and is dependent on the type of
cell that undergoes this phenomenon. Ly et al explained that excessive production of ROS
severely damages the DNA, proteins, and creates an impairment in the mitochondrial
membrane potential (∆Ψm), which leads to apoptosis [18]. Chang et al reported similar

4
observations in triple-negative breast cancer cells (MDA-MB-231) as well [19].
Natural substances account for more than half of the drugs that are used in clinical
trials due to their anti-cancer activity. Among them plant-derived substances are gaining more
importance as effective anticancer agents [20]. A variety of bufadienolide compounds were
isolated from various Kalanchoe species showed strong anti-tumor-promoting activity [21].
Kalanchoe tubiflora (Harvey) Hamet (Crassulaceae) is a succulent plant native to Madagascar
has medical applications in the treatment of abscesses, bruises, coughs, fever, and stomach-
ache [22]. It is also reported to possess anti-leishmanial [23], anti-inflammatory [24], anti-
ulcer [25], anti-microbial [26], cytotoxic [27], insecticidal [28], lymphocyte-suppressive [29],
and EBV-EA inhibitory activities [30]. Further, several bufadienolide compounds were
identified in its flowers [31]. Chemically, bufadienolides are a group of polyhydroxyl C-24
steroids and their glycosides, that contains a six-membered lactone (α-pyrone) ring at the C-
17β position. From the pharmacological point of view, bufadienolides might be a promising
group of steroid hormones with cardioactive properties and anticancer activity.
Depending on various soluble fractions, bufadienolides exhibit different
cytoprotective properties. For example - the water-soluble fraction can induce cell-cycle arrest
in human lung cancer cells and inhibited the tumor growth in A549 xenograft nude mice [32].
Whereas, the butanol fraction showed anti-proliferative activity by targeting the mitotic
apparatus [33]. Recently, Huang et al showed that among the three bufadienolides, the
kalantuboside B (KB) fraction, isolated from this plant was demonstrated to possess potent
anticancer activity against human lung cancer CL1-5 cells [34]. However, from our
knowledge, there was no study that has demonstrated the anti-cancer efficacy of KB in human
melanoma cells. In this study, we tested the therapeutic potentiality of KB, extracted from
Kalanchoe tubiflora, on A2058 human melanoma cells via in vitro and in vivo molecular
approaches and the molecular mechanisms underlying its effect were delineated.

1. Materials and methods

2.1. Reagents
Dulbecco’s Modified Eagle Medium (DMEM), Fetal bovine serum (FBS),
penicillin/streptomycin, and glutamine were purchased from Invitrogen/Gibco BRL (Grand
Island, NY). Antibodies against p53, cytochrome c, Cyclin B1, Cdc2, Bax, Bcl-2, survivin,
ERK1/2, and β-actin were obtained from Santa Cruz Biotechnology Inc. (Heidelberg,
Germany). MTT (3-[4,5-dimethyl-2-yl]-2,5-diphenyl tetrazolium bromide) and BAPTA-AM

5
were obtained from Sigma-Aldrich (St. Louis, MO). Antibodies against Cyclin A, Cdc25C,
caspase-9, caspase-3, LC3B, Beclin-1, AKT, p-AKT, JNK1/2, and p38 were purchased from
Cell Signaling Technology Inc. (Danvers, MA). Whereas, the antibodies against caspase-12
and caspase-8 were procured from Millipore Inc (Temecula, CA) and NeoMarkers Inc.
(Fremont, CA), respectively. Anti-rabbit polyclonal p-ERK1/2, p-JNK1/2, and p-p38 were
procured from Abcam (Cambridge, UK). Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-
[O-methyl]-fluoromethylketone – a very potent irreversible cell permeant caspase inhibitor),
ERK inhibitor (PD98059), and DAPI (4’,6-Diamidino-2-phenylindole dihydrochloride) were
purchased from Calbiochem (La Jolla, CA). All other chemicals, tissue culture, and common
laboratory supplies were purchased from GIBCO BRL (Grand Island, NY, USA) or Merck &
Co., Inc. (Darmstadt, Germany) or Sigma-Aldrich (St. Louis, MO).

2.2. Extraction and characterization of KB

Kalanchoe tubiflora plants were identified by Dr. Yen-Hsueh Tseng [Department of
Forestry, National Chung-Hsing University, Taichung] and collected from the middle
mountains of Nantou County, Taiwan [22]. The whole plants (9.8 kg) were macerated with
ethanol for three times (20 L x 3) and the active compound, kalantuboside B (KB) was
extracted as described by Huang et al [35]. For experimental purpose, KB stock solution was
prepared by dissolving this compound in dimethyl sulfoxide (DMSO) solution (100 µg/mL or
174 µM). (22.8 mg, purity > 96 % by HPLC) (Fig. 1A).

2.3. Cell culture

In this study, our first objective was to determine the ‘half maximal inhibitory
concentration (IC50) of KB on the viability of melanoma cell type. A2058 [a human skin
lymph node derived melanoma cell line] and B16F10 [a murine tumor cell line used as a
model for human skin cancers] were purchased from American Type Culture Collection
(ATCC, Rockville, MD). HaCaT [a spontaneously transformed aneupliod immortal
keratinocyte cell line from adult human skin] were purchased from Cell Line Services (CLS,
Eppelheim, Germany). These cell lines were cultured in DMEM supplemented with 10%
FBS, 3.7 g/L sodium bicarbonate, 2 mM glutamine, 2% non-essential amino acids, and 1%
penicillin/streptomycin and were maintained in an incubator supplied with 5% CO2 at 37°C.

2.4. MTT - Cell viability assay

The IC50 values against KB for these cell types were determined by MTT assay [36].

6
Briefly, A2058 cells were seeded in a 24-well plate and incubated for overnight. On the
following day, cells were treated with different concentrations of KB (5-30 ng/mL, equivalent
to 8.7-52.2 nM) for 24 h. After treatments, culture supernatants were replaced with 100 µL of
0.5 mg/mL MTT and incubated at 37oC for 2 h. MTT formazan crystals were dissolved in 0.5
mL of DMSO and color intensity was measured at 570 nm. Obtained values were then
compared to the MTT standard curve. Cell viability data were represented as the percentage
of viable cells compared to vehicle-treated controls, which we arbitrarily assigned 100%
viability. Each concentration was assayed in triplicates.

2.5. Colony formation assay

Colony formation assay was performed as previously described [37]. A2058 cells
were treated with increasing concentrations of KB (0-30 ng/mL) for 24 h. After incubation,
cells were trypsinized and re-plated in triplicates at a density of 40,000 cells per 35 mm dish
and allowed to incubate with DMEM for five days. After incubation, cells were fixed (10%
neutral formalin, 10min, room temperature) followed by staining with 20% Giemsa solution
(Merck, Darmstadt, Germany). Stained cells were observed for their ability to form colonies.

2.6. Cell-cycle analysis

Propidium iodide (PI) labeling method was used to determine the cellular DNA
content using a previously described procedure [36]. A2058 cells were cultured and the
synchronization of cell-cycle was achieved by double thymidine block. Cells were washed
and incubated with fresh DMEM containing different concentrations of KB (5–30 ng/mL) to
re-stimulate the cells to undergo G1 phase. After 24 h, cells were trypsinized, fixed and re-
suspended in 500 µL PI staining buffer (1% Triton X-100, 0.5 mg/mL RNase A, and 4 µg/mL
PI in PBS) and incubated at room temperature for 30 min. Using a FACScan cytometer (BD
Biosciences, San Jose, CA) equipped with a single argon ion laser (488nm), we detected the
cell cycle progression. Data were analyzed using ModFit software (Verity Software House,
Topsham, ME). During analysis, size gates was established by excluding the cellular debris.
The scattering of light rays towards the forward and right-angle were correlated with cell size
and cytoplasmic complexity, respectively. DNA content was measured using a BD FACS
Calibur system.

7
2.7. TUNEL assay of Apoptotic DNA fragmentation
TUNEL assay was performed to measure DNA fragmentation and apoptosis in A2058
cells using an assay kit (Life Technology, Gaithersburg, MD, USA). Briefly, A2058 cells were
pre-treated with 20 µM Z-VAD-FMK (caspase inhibitor) for 1 h, followed by incubation in
the presence or absence of 20 ng/mL KB for 24 h. Apoptotic cells were collected, fixed, and
mounted on glass slides. 3’-OH ends of fragmented DNA were labeled with biotinylated
Klenow fragment (37oC, 1.5 h). The slides were incubated with horseradish peroxidase (HRP)
conjugated streptavidin, followed by DAB (3,3’-Diaminobenzidine) and hydrogen peroxide.
Fragmented DNA in apoptotic cells was observed under a fluorescence microscope (200 x
magnification) and flow cytometry analysis. Additionally, cells were counterstained with
DAPI for 5 min and the intensity of green fluorescence was quantified using an LS5.0 soft
image (Olympus Imaging America. Inc., PA, USA). The intensity of fluorescence measured
was directly proportional to the number of apoptotic cells. All data were expressed as fold
over control cells that were not treated with KB and had assigned an arbitrary value of 1 [19,
38].

2.8. Annexin V and PI staining

Annexin A5 (or Annexin V) was used as a non-quantitative probe to detect apoptotic
cells that expressed phosphatidylserine (PS) on the cell surface. We measured the rate of
apoptosis in A2058 cells using the Annexin V-fluorescein isothiocyanate (FITC) and PI
double staining technique as described previously [19, 38]. In brief, A2058 cells were treated
with 20 ng/mL KB at different time points (0-24 h) in the presence or absence of 20 µM Z-
VAD-FMK. These cells were trypsinized, washed twice with PBS and then suspended in 0.1
mL of binding buffer. Cells were double-stained using an Annexin V-FITC and PI apoptosis
detection kit (Bio Vision, Mountain View, CA, USA). The resultant green (FITC) and red (PI)
fluorescence emissions for each sample were quantitatively measured using a FACSCalibur
flow cytometer (Becton Dickinson, San Jose, CA, USA) and analyzed with Cell Quest
software. Data obtained were interpreted according to the staining properties of the cells -
(Q1) PI positive but Annexin V-FITC negative: Necrotic cells; (Q2) both PI and Annexin V-
FITC positive: Apoptotic cells; (Q3) both PI and Annexin V-FITC negative: normal live cells
and (Q4) PI negative but Annexin V-FITC positively stained cell: early apoptotic cells.

8
2.9. Cell lysate preparation
Prior to protein estimation determination, A2058 cells (1×106 cells/dish; 10-cm dish)
were washed with PBS containing 0.01 mM sodium vanadate (NaVO4) and solubilized in
lysis buffer (10 mM Tris-HCl, pH 8, 320 mM sucrose, 1% Triton X-100, 5 mM EDTA, 2 mM
DTT, and 1 mM PMSF). Mitochondrial, cytoplasmic, nuclear, and total protein extracts were
prepared using commercially available extraction reagent protocols (Pierce Biotechnology,
Rockford, IL, USA). Solubilized proteins were clarified at 12,000 g for 10 min, at 4oC and the
protein lysate concentrations were measured using bicinchoninic acid (BCA) method. Cell
lysates were stored at -80oC until further use.

2.10. Western blotting

Equal quantities of (50 µg) of solubilized protein samples were loaded and separated
on 8–15% polyacrylamide gradient SDS-PAGE gels. After separation, the proteins were
transferred onto a polyvinylidene difluoride (PVDF) membrane. Non-specific binding was
prevented by incubating the membranes with 5% blotto (5% nonfat dry milk, 1% Tween-20 in
PBS) for 1 h at room temperature. After blocking, membranes were incubated with different
primary antibodies (overnight at 4oC). On the following day, membranes were probed with
either horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mice antibodies for 2
h. Immuno-reactive protein bands were visualized through ImageQuantTM LAS 4000 mini
(Fujifilm) using Super-Signal West Pico Chemiluminescence substrate (Thermo Scientific,
IL). Densitometric analysis was performed using a commercially available quantitative
software (AlphaEase, Genetic Technology Inc. Miami, FL). Data were represented as fold
difference over untreated control. β-actin was used as an internal control.

2.11. GFP-LC3 plasmid transfection and puncta formation

LC3 is a key biomarker used to identify the autophagy in mammalian systems.
Autophagosome formation was detected using green fluorescent protein fused LC3 complex
(GFP-LC3) that is visible in cells and can be observed through a laser scanning confocal
microscope. The protocol was described as previously by Chang et al [37]. A2058 cells were
seeded onto coverslips in each well of a 6-well plate and incubated for overnight. On the
subsequent day, cells were transfected with 2.5 µg of GFP-LC3 using Lipofectamine 2000
reagent (Invitrogen, Carlsbad, CA, USA) and incubated for 24 h to allow for plasmid
expression. After incubation, the medium was replaced with fresh medium containing KB.

9
Cells were then washed twice with PBS, and the expression of GFP-LC3 dot formation was
detected using a laser scanning confocal microscope (200 x magnification).

2.12. Acridine orange (AO) staining

Acridine orange (AO) is a nucleic acid selective cationic membrane permeable
fluorescent dye used to visualize the formation of primary lysosomes and phagolysosomes
(AVOs) that includes the products of phagocytosis of apoptotic cells. A5028 cells were
incubated with 20 ng/mL KB for 24 h, followed by PBS wash and then stained with AO (1
µg/mL in PBS+5% FBS) for 15 min. The formation of AVOs in cells were visualized through
a fluorescence microscope with a red filter (100 x magnification) and quantified by flow
cytometry. The fluorescence emission exhibited by AO is concentration dependent. Red
(lysosomes) or green (cytosol) fluorescence emissions indicate high or low concentrations of
AVOs respectively [37].

2.13. Measurement of intracellular ROS

The effect of time on KB mediated ROS generation in A2058 cells was measured
using a cell-permeable fluorogenic probe 2′,7′-dihydrofluorescein-diacetate (DCFH2-DA).
A2058 cells at 80% cell confluence were incubated in the presence of 20 ng/mL KB for 0, 5,
15, and 30 min. After the incubation, culture supernatants were replaced with fresh medium
containing 10 µM DCFH2-DA and incubated for 30 min at 37°C. The intracellular ROS levels
are proportional to the accumulation of intracellular dichlorofluorescein (DCF) caused by the
oxidation of DCFH2-DA. Fluorescence intensity was measured using an Olympus 1x71
fluorescence microscope (200 × magnification).

2.14. In vivo experiments

In vivo experiments were performed as described previously [37]. 5-7 weeks old,
female athymic BALB/c nude mice were obtained from National Laboratory Animal Center
(NLAC - Taipei, Taiwan), and were maintained in a specially designed pathogen-free animal
facility with 12 h of light and 12 h of night cycle. All mice were given free access to water
and rodent chow (Oriental Yeast Co Ltd., Tokyo, Japan). Animal protocols were approved by
the China Medical University’s Institutional Animal Care and Use Committee (IACUC) and
the experiments were strictly followed the ‘Guidelines for the Care and Use of Laboratory
Animals’ as published by Chinese Society of Animal Science, Taiwan. Efforts were made to
minimize any suffering or discomfort and to reduce the number of animals used.

10
2.15. Xenografting of A2058 cells
Eight nude mice were used in this study. Approximately 0.8 million A2058 tumor cells
suspended in matrix gel (100 µL) were subcutaneously injected to the left hind flanks of each
nude mice for about seven days. Animals were randomly grouped into control (four mice) or
treatment (four mice) sets. The treatment or control group mice received intraperitoneal
injection of 0.4 mg/kg of KB and or 0.1% of DMSO respectively for every 2 days. Body
weight and drug toxicities were monitored for all animals. Caliper measurements of tumor
volumes (length, width, and depth) were calculated every week using the formula - length x
width2 x ½. During the 8th week, all animals were sacrificed and the tumor tissues (the liver,
lung, heart, spleen, and kidney) were weighed and examined by a veterinary pathologist [37].

2.16. Western blot analysis of xenograft tumors

The effect of KB on the expression profiles of different proteins in the excised tumor
tissues were analyzed using western blot technique (refer to the subsection – western blotting
in materials and methods section). The total protein from the excised tumor tissues was
extracted using RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) containing cocktails 1 (1%
of protease inhibitor), 2 (1% Serine/Threonine phosphatase inhibitor) and, 3 (1% Tyrosine
Protein Phosphatase inhibitors). Protein concentrations were determined by the BCA protein
assay method.

2.17. Statistical analysis

All data were expressed as the mean ± standard deviation of three experiments as
indicated. The t-tests or repeated measures of one-way analysis of variance (ANOVA) with
Dunnett’s post-test were used to compare the treatment groups with the control groups. The
criterion for statistical significance was set as *p < 0.05, **p < 0.01, and ***p < 0.001 as
compared to the untreated control cells.

2. Results
3.1. KB suppressed A2058 cell growth and colony formation
The anti-cell proliferative efficacy of KB (Fig. 1A) on human melanoma (A2058),
murine melanoma (B16F10), and human keratinocyte (HaCaT) cells were determined through
the IC50 value. The values obtained were - 13.0, 63.1, and 25.3 ng/mL for A2058, B16F10,
and HaCaT cells respectively. Compared to other cell types, KB showed strong anti-cell

11
proliferation property in A2058 cells (Fig. 1B-D). Besides this, increasing concentrations of
KB caused abrupt morphological changes (Fig. 1E) and suppression of colony formation in
A2058 cells (Fig. 1F & G) [37]. Therefore, the anti-tumor properties of KB were further
investigated using A2058 cells only.

3.2. KB inhibits A2058 cell proliferation through G2/M cell-cycle arrest and apoptosis
Synchronized A2058 cells were treated with different concentrations of KB (0, 5, 10,
20, and 30 ng/mL) for 24 h and subjected them to the flow-cytometry analysis of DNA
staining. As shown in Fig. 2A, KB exposure resulted in progressive and sustained
accumulation of G2/M as well as sub-G1 phase cell populations which signifies that KB
promotes inhibition of cell proliferation by inducing G2/M arrest and apoptosis in A2058
cells. In eukaryotes, cell-cycle progression involves sequential activation of cyclin-dependent
kinases (CDKs), whose activity was dependent on the formation of complexes with regulatory
cyclins [39]. Uhlmann et al reported that cyclin A/B1 and Cdc2 (mitotic-cyclin dependent
kinase) complex formation is key for cells to enter mitosis [40]. Consistent with flow-
cytometry data, western blot data also showed KB mediated dose-dependent reduction in
cyclin A/B1, Cdc25C, and Cdc2 protein expressions that were known to be critically involved
in G2/M phase (Fig. 2B).

3.3. KB induces early apoptosis and potentiates DNA fragmentation in A2058 cells
TUNEL assay was performed to test the ability of KB induced apoptotic cell death in
A2058 cells. As shown in Fig. 3A & B, compared to the control cells, green fluorescence was
upregulated in KB treated cells. However, this upregulation was diminished in the presence of
potent caspase inhibitor Z-VAD-FMK (20 µM). This implies that KB potentiates DNA
fragmentation in A2058 cells. Furthermore, Annexin V-FITC/PI assay results showed that KB
treatment (20 ng/mL for 24 h) significantly upregulated the appearance of Annexin V/PI
labeled cells (red fluorescence) in Q4 (from 12.9±0.5% to 26.6±0.4%) suggesting that there
was an increased early apoptosis in A2058 cells. Conversely, Z-VAD-FMK pretreatment
substantially suppressed this effect (Fig. 3C). This data provided us to infer that KB
effectively inducing early apoptosis in A2058 cells.

3.4. KB induced apoptosis was mediated via the activation of mitochondrial, death
receptor, and ER stress pathways in A2058 cells

12
 We examined the expression patterns of various key protein markers involved in the
KB-induced mitochondria-associated apoptosis in A2058 cells. Cytochrome c has been
reported to be involved in the activation of downstream caspases
that triggers apoptosis [41]. First, we determined the expression levels of cytochrome
c protein from both mitochondrial and cytosolic fractions. Fig. 4A shows that compared to the
untreated cells, KB treatment significantly attenuated the mitochondrial cytochrome c levels
but a proportional increase in cytosolic cytochrome c levels in a time-dependent manner.
Besides cytochrome c, KB treatment induced proteolytic cleavage of procaspase-9 (47 KDa),
procaspase-3 (35 KDa), and PARP (116 KDa) to their active forms (Fig. 4B). This data
provided direct evidence that mitochondrial function was critically impaired in KB-mediated
apoptosis in A2058 cells.
Death receptors triggers the activation of caspase-8 mediated apoptotic cascade after
ligand binding. Several mechanisms have been proposed in linking endoplasmic reticulum
(ER) stress to apoptosis via the activation of ER-associated caspases, especially caspase-12
[42]. Western blot data indicated that KB upregulated the activation of caspase-8 and -12
levels by approximately 2.5 folds in KB treated A2058 cells (Fig. 4C). Cleaved caspase-12
was more prominent than other caspases. However, MTT data showed that Z-VAD-FMK
pretreated and KB exposed cells showed increased cell viability (20% increase) when
compared with KB-alone treatment (50% cell viability) (Fig. 4D). This data allowed us to
interpret that caspase inhibition protected the KB treated cells to undergo caspase-mediated
apoptosis in A2058 cells.

3.5. KB upregulates auto-phagosome formation in A2058 cells

The anti-tumor properties exhibited by KB prompted us to speculate that autophagy
was also playing a key role in A2058 cells. Previous reports suggested that LC3 is a
prominent protein marker that is associated with the cellular autophagy phenomenon [43].
Our western blot data showed that KB dose-dependently upregulated the LC3-I to LC3-II
conversion but survivin levels were downregulated (Fig 5A). Survivin belongs to ‘inhibitor of
apoptosis (IAP)’ protein family that functions to inhibit the caspase activation leading to
negative regulation of apoptosis. Hence, survivin expression was correlated with
tumorigenesis and tumor metastasis [44]. Downregulation of survivin indicated a positive
regulation of KB towards apoptosis or programmed cell death in A2058 cells.
Cytoplasmic p53 is a key ‘tumor suppressor’ marker protein whose inhibition induced
cellular autophagy [18]. As shown in Fig. 5B, KB dose-dependently suppressed p53 protein

13
expression and favors autophagy activity in A2058 cells. This was confirmed by transfecting
the GFP-LC3 plasmid into A2058 cells and the fluorescence emission was measured using
confocal microscopy. Control cells showed diffused and weak LC3 puncta. Whereas, KB
treatment increased the percentage of GFP-LC3 puncta-positive cells as well as average
number of GFP-LC3 puncta per cell. Interestingly, NAC (a pharmacological inhibitor of
ROS) significantly suppressed this effect (Fig. 5C). These results suggested that KB through
the ROS pathway mediating the autophagy in A2058 cells.
One of the characteristic features of autophagy is the formation of acidic vesicular
organelle (AVOs) in cells with increased occurrence of autophagosomes and autolysosomes
[45]. Using the acridine orange (AO) staining method, we tested the KB mediated AVOs
formation in KB treated A2058 cells. Our data showed that KB dose-dependently (5-30
ng/mL, 24 h) increased the AVO formation of (red fluorescence) in A2058 cells (Fig. 5D &
E).

3.6. KB induced autophagy in A2058 cells as a cell survival mechanism

White E et al reported that autophagy plays a double-edged sword role in the
modulation of cancer cell death and survival [46]. Therefore, pharmacological inhibitors
against early autophagy (3-methyladenine – 3MA) and late autophagy (Chloroquine – CQ)
were used to determine whether KB induced autophagy could lead to cell-survival in A2058
cells. Our data showed that KB alone exposed A2058 cells showed 50% cell viability.
However, in the presence of 3-MA and CQ, the cell viability was downregulated to 30% and
20% respectively (Fig. 6A & B). Notably, 3-MA and CQ pretreated cells increased KB
mediated PARP cleavage (apoptosis) inferring enhanced apoptosis pathway (Fig. 6C). This
data signified that KB potentiated autophagy as a cell survival mechanism in A2058 cells.
Singh et al suggested that the proteins in the Bcl-2 family are critical regulators of
mitochondria-mediated apoptotic induction and act either as an activator (Bax) or inhibitor
(Bcl-2) [47]. In this study, KB (20 ng/mL) caused significant inhibition of Bcl-2 and increased
the expression of Bax in a time-dependent manner (Fig. 6D & E). Kang et al suggested that
caspase-mediated cleavage of Beclin-1 promotes cross-talk between apoptosis and autophagy
[48]. Beclin-1 can intervene at every major step in the autophagic pathway via the activation
of specific Beclin-1 binding proteins. These included autophagic inducers and inhibitors. Bcl-
2 was shown to decrease the proautophagic property of Beclin-1, that in turn favors the
autophagy. The association between Beclin-1 and Bcl-2 is a complex phenomenon. In this
study, KB time-dependently increased the Beclin-1 levels, but Bcl-2 expression was totally

14
stopped after 8 h (Fig. 6D & F). These expression patterns suggested that KB plays a key role
in A2058 cells that favors apoptosis and cytoprotective autophagy.

3.7. KB induced apoptosis/autophagy was suppressed in the presence of ROS inhibitor

It has been reported that excessive production of intracellular ROS triggers the cells to
undergo apoptosis and/or autophagy [49, 50]. Fluorescence data showed that there was a
proportional increase in DCF fluorescence with increasing time in KB-treated cells. However,
this effect was significantly suppressed in the presence of NAC (ROS inhibitor) (Fig. 7A &
B). We further tested if KB-induced apoptosis/autophagy is a ROS-dependent pathway. As
shown in Fig. 7C, pretreating cells with NAC suppressed KB-induced cell death (Fig. 7C).
Further, KB induced caspase-3 activation, PARP cleavage (apoptosis) as well as LC3-I/II
accumulation were inhibited in the presence of NAC (Fig. 7D & E). This data clearly
signifies that KB-induced ROS plays a role in apoptosis/autophagy in A2058 cells.

3.8. KB upregulated the ERK signaling pathway in A2058 cells

It has been reported that ERK, AKT, JNK, and p38 pathways play significant roles in
determining the cellular fate against external stimuli [51]. We tested the effect of KB on the
expression and phosphorylation patterns of various signaling proteins in A2058 cells. Our
western blot data showed that KB dose-dependently activated the p-ERK but inhibited the p-
AKT pathway resulted in the induction of apoptosis in A2058 cells (Fig. 8A-C). Interestingly,
KB did not show any effect on JNK and p38 pathways. We further tested the effect of ERK
inhibition on A2058 cell viability as well the expression patterns of apoptosis- and autophagy-
associated proteins. As shown in Fig. 8D, PD98059 pretreatment significantly increased the
PARP cleavage but suppressed the LC3-I/II accumulation in KB-treated cells. Besides this,
cell viability data also suggested that A2058 cells co-treated with both KB and PD98059
showed significant downregulation in the cell viability (30%) compared to the KB alone
treatment A2058 cells (50%) (Fig. 8E). Interestingly, 3-MA and CQ did not affect p-ERK
protein levels in KB-treated cells (Fig. 8F). Therefore, we concluded that the activation of
ERK is pivotal in KB mediated apoptosis and cytoprotective autophagy.

3.9. KB enhanced cytoprotective autophagy via the upregulation of intracellular Ca2+

levels in A2058 cells
Calcium (Ca2+) transfer between the endoplasmic reticulum (ER) and mitochondria
represents a cross-talk leading to Ca2+-dependent apoptosis [52, 53]. We used BAPTA

15
(cytoplasmic calcium chelator) to test whether KB mediated apoptosis/autophagy was a Ca2+-
dependent pathway in A2058 cells. MTT data showed that there was 50% suppression in the
viability of KB alone exposed A2058 cells. However, BAPTA co-treatment further
downregulated the cell viability (10%) (Fig. 9A). Western blot data showed that BAPTA and
KB co-treatment has differential effect on p53 expression, LC3-II accumulation, and PARP
levels in A2058 cells (Fig. 9B-E). These results suggested that KB upregulated the
intracellular Ca2+ levels but downregulated the p53 levels leading to increased cytoprotective
autophagy in A2058 cells.

3.10. KB suppressed A2058 xenograft tumor growth in nude mice

A2058 cells were xenografted into nude mice to evaluate the KB mediated in vivo
tumor growth. Results showed that KB treatment did not affect the weight loss in
experimental mice (Fig. 10A). Compared to the vehicle treatment, KB (0.4 mg/kg)
significantly suppressed the tumor volumes in a time-dependent manner (Fig. 10B-D). This in
vivo data clearly demonstrated the tumor suppression property of KB in nude mice. In
addition, no signs of toxicity were observed in any of the nude mice (body weight and
microscopic examination of individual organs; data not shown).

3.11. Apoptosis and cytoprotective autophagy pathways were involved in KB mediated

suppression of tumor growth in nude mice
Later, we tested the effect of KB on expression profiles of different proteins that are
associated with apoptosis and autophagy in tumor tissues excised from A2058 xenograft mice.
Western blot data showed that KB (0.4 mg/kg) treatment significantly downregulated the
cyclin A, PARP, Bcl-2, p53, and survivin proteins but upregulated the LC3-II expression
(Fig. 11 A-F). This in vivo data allowed us to infer that KB mediated apoptosis and
cytoprotective autophagy mechanisms were involved in the suppression of tumor growth in
nude mice.

4. Discussion
The eukaryotic cell cycle is tightly regulated by cyclin-dependent kinases (CDKs) and
has an association with cyclin proteins [54, 55]. These CDK-cyclin complexes are activated at
synchronized intervals during the cell cycle and can be activated and regulated by external
stimuli, such as: UV-light, thermal disruption, chemicals, and ionizing radiation [56, 57].

16
Exposure to UV radiation is one of the most important risk factors for ‘malignant melanoma’
or ‘skin cancer’ leading to 80% of deaths [58]. Accumulating evidence suggested that natural
substances that arrest cell cycle, or inhibition of cell proliferation, or induction of apoptosis
could be considered as promising interventions in the management of cancers, including
melanoma cancer [59].
In this study, we first tested the effect of KB (Fig. 1A) on cell viability as well as
colony forming abilities in different cell types. Compared to the other cell types, with a low
IC50 value (13 ng/mL), A2058 melanoma cells showed significant decrease in cell
proliferation and anchorage-independent cell growth (Fig. 1B-D). Moreover, KB caused
significant morphological aberrations in A2058 cells that were associated with cell shrinkage
leading to round shape and membrane blebbing emphasizing apoptotic cell death (Fig. 1E-G).
In a recent study, Kalanchoe tubiflora extract was shown to arrest the cell cycle at G2/M
phase and inhibited the proliferation of A549 lung cancer cells [32]. In support to this, our
data also showed that KB downregulated the protein expressions of cyclin A and B with a
concomitant decrease in Cdc2 kinase and increased population of A2058 cells in G2/M phase
(Fig 2A & B). KB was able to decrease the Cdc2 activity through the suppression of Cdc25c,
thus regulating the entry of cells into mitotic phase by maintaining the G2/M transition phase.
These findings suggested that KB has profound effect on A2058 cell progression by inducing
G2/M arrest and inhibition of cyclin A/B through the inhibition of ‘Cdc2/Cdc25C’ in A2058
cells.
Guicciardi and Gores suggested that apoptosis is a way of programmed cell death that
could serve as a potential target in the prevention of proliferating malignant tumor cells [60].
This mechanism could be triggered either by mitochondria (intrinsic) or death receptor
(extrinsic) pathways. The mitochondrial/caspase-mediated signaling cascade is a major
apoptotic pathway that was characterized by mitochondrial outer membrane permeabilization
(MOMP), whose disruption leads to the release of cytochrome c into the cytoplasm, triggering
the caspase cascade and PARP cleavage [61]. In this study, KB treatment induced DNA
fragmentation that further triggered the release of cytochrome c into the cytoplasm and
activated the procaspase-9, -3, and proteolytic cleavage of PARP in A2058 cells. Interestingly,
this effect was significantly suppressed in the presence of caspase inhibitor (Z-VAD-FMK)
signifying the role of caspases involved in DNA fragmentation. On the other hand, the
extrinsic pathway is activated by binding of death receptors such as Fas, FasL, and TRAIl
receptors with cognate extracellular ligands [62]. Activated caspase-8 can promote the
apoptotic signal by direct cleavage of its downstream effector caspase-3 [63]. We observed

17
that KB treatment upregulated the activation of caspase-8 in A2058 cells and strongly
supported the notion that KB-mediated apoptosis was regulated via both mitochondria and
death receptor-mediated pathways (Fig. 3 & 4).
It has been shown that ER stress-inducers were reported as promising anti-cancer
drugs that induce apoptotic cell death via caspase-12 which is independent of mitochondrial
and death receptor pathways [71, 72]. Upon activation, caspase-12 induces the cleavage of
caspase-3 via a cytochrome c independent mechanism [64]. A recent study reported that in
parallel to the caspase-12 activation, ER stress triggers caspase-8 activation, resulting in
cytochrome c/caspase-9 activation through Bid processing [65]. Findings from our study
demonstrated that, in A2058 cells, ER stress plays an important role in KB-induced apoptosis
through the activation of caspase-12 pathway that also signifies that KB affects an additional
apoptotic pathway that was independent of the release of mitochondrial proteins (Fig. 4).
Autophagy is a catabolic pathway that degrades and recycles defective cellular
organelles or long-lived proteins and helps in the maintenance of cellular homeostasis.
Therefore, autophagy could be a promising therapeutic target in anti-cancer therapy [66, 67].
Autophagy acts as a ‘double-edged sword’ and has cell-survival or cell-death mechanisms in
response to therapeutic agents. Significant increase in the appearance of AVOs is a hallmark
event in autophagy that was associated with dramatic accumulation of lipidated LC3 in cells.
As shown in Fig. 5, KB treatment upregulated LC3-I to LC3-II (membrane-bound)
conversion as well as increase in the number of AVOs with more distribution of GFP-LC3-II
puncta in A2058 cells. This explains that auto-phagosomes were recruited with increased
LC3-II protein and KB induced autophagy in A2058 cells. However, KB induced GFP-LC3
puncta formation and LC3-II expressions were significantly inhibited in the presence of NAC
(ROS inhibitor) signifying the fact that KB activated autophagy was a ROS-dependent
pathway.
Accumulating evidence suggests that chemical substances that induce apoptotic cell
death was accompanied by cytoprotective autophagy too [50, 68, 69]. Targeted therapies that
activate the autophagic pathway reportedly contributed to the survival of tumors, and exhibit
resistance to anticancer therapies [14, 70]. To the best of our knowledge, we have shown for
the first time that in the presence of early or late autophagic protein inhibitors (3-MA or CQ)
KB potentiated autophagy as a cell survival mechanism in A2058 cells. Moreover,
pretreatment with these inhibitors eventually enhanced the KB induced apoptosis (PARP
cleavage) (Fig. 6). These findings suggested that KB-induced autophagy was not involved in
A2058 cell death. Perhaps it has played a protective role against the cytotoxic effects of KB.

18
Also, KB mediated Beclin-1 (from 4 h) (Fig. 6D) and cytosolic cytochrome c expressions (16
h) (Fig. 4A) signified the fact that cytoprotective autophagy was followed by the apoptosis
process in A2058 cells.
The interaction between Bax/Bcl-2 (apoptotic proteins) and Beclin-1/Bcl-2
(autophagic proteins) represents a potential point of cross-talk between apoptosis and
autophagic pathways [71, 72]. Kang et al reported that Beclin-1 could not neutralize the anti-
apoptotic activity of Bcl-2. On the other hand, Bcl-2 family proteins decreased pro-autophagic
functions of Beclin-1 [16]. Our study showed that KB down-regulated the Bcl-2 expression
levels but upregulated Beclin-1 levels in A2058 cells. Moreover, there was a time-dependent
upregulation in the ratios of Bax/Bcl-2 and Beclin-1/Bcl-2 suggesting that KB was able to
upregulate A2058 cell death through pro-apoptotic and autophagic signals (Fig. 6F).

Previous studies reported that excessive production of intracellular ROS triggers the
cells to undergo apoptosis and/autophagy [49, 50]. Fluorescence data from our study showed
that KB time-dependently upregulated the generation of ROS in A2058 cells (Fig. 7A & B).
However, pretreatment with an ROS inhibitor (NAC) suppressed the KB induced cell death,
caspase-3 activation, PARP cleavage, LC3-I/II accumulation, and differential cell viability of
A2058 cells (Fig. 7C-E). This data clearly demonstrated the KB-induced ROS role in
apoptosis/cytoprotective autophagy in A2058 cells.
ERK activation has been associated with the induction of autophagy while
accumulating evidence suggested that ERK can be activated by oxidative stress, variety of
oncogenes, growth factors, amino acid deprivation, and certain anticancer agents [67, 73].
Wang et al reported that Cisplatin, a chemotherapy drug, induces apoptosis via ERK
activation [74]. Similarly, other natural compounds like β, β-Dimethylacrylshikonin derived
from Lithospermum erythrorhizon and triptolide derived from Tripterygium wilfordii Hook F
have been shown to induce apoptosis in different cancer cell types via the ERK pathway [75,
76]. Among MAP kinases, ERK has been found to be a downstream effector of ROS in
autophagy induction [77]. In this study, the pharmacological inhibition of ERK pathway
prevented KB induced autophagy and dramatically increased apoptosis in A2058 melanoma
cells (Fig. 8). Furthermore, pretreatment with PD98059 (ERK inhibitor) enhanced the KB
induced cell viability. AKT proteins are one of the key mediators responsible for cell survival
whose overexpression blocks apoptosis [78]. By the same token KB inhibited the
phosphorylation of AKT and resulted in increased apoptosis and/or cytoprotective autophagy
(Fig. 8A & B). Therefore, our findings suggest that ERK activation and AKT inactivation by

19
KB may induce apoptosis and exerts cytoprotective autophagy in A2058 cells.

Regulation of intracellular Ca2+ levels is vital to cell survival and homeostasis. Recent
studies have reported that cytosolic Ca2+ levels regulate the autophagy machinery at different
stages of autophagic flux [79, 80]. An increase in ER stress-induced intracellular calcium
release that in turn activated autophagy via CAMKK2 (calcium/calmodulin-dependent protein
kinase 2) cascade [81]. We found that pretreatment with calcium chelator (BAPTA)
significantly suppressed KB-induced autophagy but potentiated the apoptosis suggesting that
KB triggered intracellular calcium release plays a crucial role in cytoprotective autophagy
(Fig. 9A & B).

p53 has a divergent effect on autophagic regulation. Activated nuclear p53 triggers
autophagy through DRAM upregulation and mTOR inhibition. Whereas, cytoplasmic p53
inhibited autophagy [82]. These observations suggested that baseline p53 inhibited autophagy
and the activation of nuclear or inhibition of cytosolic p53 elicited autophagy [83]. In this
study, pretreatment with BAPTA has a differential effect on p53, LC3-II, and PARP levels in
A2058 cells (Fig. 9C-E). Our findings confirmed that cytoprotective autophagy induction by
the calcium pathway is coupled with p53 downregulation in A2058 cells. This discrepancy in
p53 downregulation and Bax upregulation could be explained from the previous reports.
Bensaad et al, stated that there was an alternate mechanism involved in p53’s role in
autophagy. They found a p53-inducible protein ‘TIGAR phosphatase’ which is involved in the
regulation of intracellular ROS levels in the cell that is inhibiting the autophagy. When
TIGAR phosphatase function is inhibited, increased ROS levels lead to apoptosis and
cytoprotective autophagy induction [84] [85]. Besides this, there was an other report stating
that c-Myc is also reported to be a possible candidate that is known to regulate the expression
of Bcl-2 and Bax, and could induce apoptosis that is independent of p53 pathway [86, 87].
We are speculating similar mechanisms might be involved in KB mediated effect in A2058
cells as well.

Consistent with the in vitro study, our in vivo study also demonstrated that KB
treatment significantly reduced the tumor size in A2058 xenografted nude mice. Western blot
analysis of the excised tumor tissues showed that KB treatment down-regulated the cyclin A,
Bcl-2, survivin, and p53 protein expressions, but upregulated the LC3-I/II levels (Fig. 10 &
11). These findings confirm that KB promotes apoptosis and cytoprotective autophagy in
A2058 human melanoma cancer cells.

20
Conclusion
Melanoma cancer is predominant among Caucasian populations who have a high
incidence rate and genetic mutation [88]. In this study, we demonstrated the anti-cancer
efficacy of KB in A2058 human melanoma cells and the molecular mechanisms underlying
this effect was tested via in vitro and in vivo methods. In A2058 cells, treatment with KB
induced ROS production that to some extent triggered the apoptosis as well as cytoprotective
autophagy. ERK signaling pathway has been shown to play a key role in this effect. Further,
KB promotes cytoprotective autophagy as a calcium-dependent p53 downregulation pathway.
Additionally, KB treatment suppressed tumor growth in A2058 xenograft nude mice through
the induction of apoptosis and cytoprotective autophagy pathways which is evidenced from
the differential protein expression profiles of various autophagy and apoptosis-associated
proteins. Findings from our study implied that KB may serve as a potential novel agent from
an herbal origin in anticancer therapy. Still, more investigations are required to clarify the
possibility of other putative mechanisms involving in KB induced apoptosis and
cytoprotective autophagy. Better insights into KB effects on human melanoma cells will also
help in developing a strategy to prevent cancer progression.

Conflict of interest
All authors declare no conflicts of interest in relation to this study.

Acknowledgments
Ministry of Science and Technology (MOST-106-2320-B-039-054-MY3, MOST-107-2320-
B-039-013-MY3), Asia University, China Medical University (CMU107-ASIA-15,
CMU106-ASIA-19), Taiwan and Chinese Medicine Research Center (CMRC-CHM-8),
China Medical University from the “Featured Areas Research Center Program” within the
framework of the Higher Education Sprout Project by the Ministry of Education (MOE)
funded this study.

21
References

[1] Levine B, Klionsky DJ (2004). Development by self-digestion: molecular mechanisms

and biological functions of autophagy. Dev Cell, 6:463-477.
[2] Annunziato L, Amoroso S, Pannaccione A, Cataldi M, Pignataro G, D'Alessio A, et al.
(2003). Apoptosis induced in neuronal cells by oxidative stress: role played by
caspases and intracellular calcium ions. Toxicol Lett, 139:125-133.
[3] Shimizu S, Eguchi Y, Kamiike W, Itoh Y, Hasegawa J, Yamabe K, et al. (1996).
Induction of apoptosis as well as necrosis by hypoxia and predominant prevention of
apoptosis by Bcl-2 and Bcl-XL. Cancer Res, 56:2161-2166.
[4] Weinrauch Y, Zychlinsky A (1999). The induction of apoptosis by bacterial pathogens.
Annu Rev Microbiol, 53:155-187.
[5] Brune B (2003). Nitric oxide: NO apoptosis or turning it ON? Cell Death Differ,
10:864-869.
[6] Rajah R, Valentinis B, Cohen P (1997). Insulin-like growth factor (IGF)-binding
protein-3 induces apoptosis and mediates the effects of transforming growth factor-
beta1 on programmed cell death through a p53- and IGF-independent mechanism. J
Biol Chem, 272:12181-12188.
[7] Yaoita Y, Nakajima K (1997). Induction of apoptosis and CPP32 expression by thyroid
hormone in a myoblastic cell line derived from tadpole tail. J Biol Chem, 272:5122-
5127.
[8] Lowe SW, Lin AW (2000). Apoptosis in cancer. Carcinogenesis, 21:485-495.
[9] Mortality GBD, Causes of Death C (2016). Global, regional, and national life
expectancy, all-cause mortality, and cause-specific mortality for 249 causes of death,
1980-2015: a systematic analysis for the Global Burden of Disease Study 2015.
Lancet, 388:1459-1544.
[10] Eggermont AM, Spatz A, Robert C (2014). Cutaneous melanoma. Lancet, 383:816-
827.
[11] Kanavy HE, Gerstenblith MR (2011). Ultraviolet radiation and melanoma. Semin
Cutan Med Surg, 30:222-228.
[12] Azoury SC, Lange JR (2014). Epidemiology, risk factors, prevention, and early
detection of melanoma. Surg Clin North Am, 94:945-962, vii.
[13] Chi Z, Li S, Sheng X, Si L, Cui C, Han M, et al. (2011). Clinical presentation,
histology, and prognoses of malignant melanoma in ethnic Chinese: a study of 522

22
 consecutive cases. BMC Cancer, 11:85.
[14] Kondo Y, Kanzawa T, Sawaya R, Kondo S (2005). The role of autophagy in cancer
development and response to therapy. Nature Reviews Cancer, 5:726-734.
[15] Gozuacik D, Kimchi A (2004). Autophagy as a cell death and tumor suppressor
mechanism. Oncogene, 23:2891-2906.
[16] Kang R, Zeh H, Lotze M, Tang D (2011). The Beclin 1 network regulates autophagy
and apoptosis. Cell Death & Differentiation, 18:571-580.
[17] Maiuri MC, Zalckvar E, Kimchi A, Kroemer G (2007). Self-eating and self-killing:
crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol, 8:741-752.
[18] Ly JD, Grubb DR, Lawen A (2003). The mitochondrial membrane potential
(deltapsi(m)) in apoptosis; an update. Apoptosis, 8:115-128.
[19] Chang CT, Korivi M, Huang HC, Thiyagarajan V, Lin KY, Huang PJ, et al. (2017).
Inhibition of ROS production, autophagy or apoptosis signaling reversed the
anticancer properties of Antrodia salmonea in triple-negative breast cancer (MDA-
MB-231) cells. Food Chem Toxicol, 103:1-17.
[20] Newman DJ, Cragg GM (2012). Natural products as sources of new drugs over the 30
years from 1981 to 2010. J Nat Prod, 75:311-335.
[21] Lai ZR, Ho YL, Huang SC, Huang TH, Lai SC, Tsai JC, et al. (2011). Antioxidant,
anti-inflammatory and antiproliferative activities of Kalanchoe gracilis (L.) DC stem.
Am J Chin Med, 39:1275-1290.
[22] Huang HC, Huang GJ, Liaw CC, Yang CS, Yang CP, Kuo CL, et al. (2013). A new
megastigmane from Kalanchoe tubiflora (Harvey) Hamet. Phytochemistry Letters,
6:379-382.
[23] Muzitano MF, Cruz EA, de Almeida AP, Da Silva SA, Kaiser CR, Guette C, et al.
(2006). Quercitrin: an antileishmanial flavonoid glycoside from Kalanchoe pinnata.
Planta Med, 72:81-83.
[24] Costa SS, de Souza Mde L, Ibrahim T, de Melo GO, de Almeida AP, Guette C, et al.
(2006). Kalanchosine dimalate, an anti-inflammatory salt from Kalanchoe brasiliensis.
J Nat Prod, 69:815-818.
[25] Pal S, Nag Chaudhuri AK (1991). Studies on the anti-ulcer activity of a Bryophyllum
pinnatum leaf extract in experimental animals. J Ethnopharmacol, 33:97-102.
[26] Akinpelu DA (2000). Antimicrobial activity of Bryophyllum pinnatum leaves.
Fitoterapia, 71:193-194.
[27] Wu PL, Hsu YL, Wu TS, Bastow KF, Lee KH (2006). Kalanchosides A-C, new

23
 cytotoxic bufadienolides from the aerial parts of Kalanchoe gracilis. Org Lett, 8:5207-
5210.
[28] Supratman U, Fujita T, Akiyama K, Hayashi H (2000). New insecticidal
bufadienolide, bryophyllin C, from Kalanchoe pinnata. Biosci Biotechnol Biochem,
64:1310-1312.
[29] Costa SS, Jossang A, Bodo B, Souza ML, Moraes VL (1994). Patuletin
acetylrhamnosides from Kalanchoe brasiliensis as inhibitors of human lymphocyte
proliferative activity. J Nat Prod, 57:1503-1510.
[30] Supratman U, Fujita T, Akiyama K, Hayashi H, Murakami A, Sakai H, et al. (2001).
Anti-tumor promoting activity of bufadienolides from Kalanchoe pinnata and K.
daigremontiana x tubiflora. Biosci Biotechnol Biochem, 65:947-949.
[31] McKenzie RA, Franke FP, Dunster PJ (1989). The toxicity for cattle of bufadienolide
cardiac glycosides from Bryophyllum tubiflorum flowers. Aust Vet J, 66:374-376.
[32] Hsieh YJ, Huang HS, Leu YL, Peng KC, Chang CJ, Chang MY (2016). Anticancer
activity of Kalanchoe tubiflora extract against human lung cancer cells in vitro and in
vivo. Environ Toxicol, 31:1663-1673.
[33] Hsieh YJ, Yang MY, Leu YL, Chen C, Wan CF, Chang MY, et al. (2012). Kalanchoe
tubiflora extract inhibits cell proliferation by affecting the mitotic apparatus. BMC
Complement Altern Med, 12:149.
[34] Huang HCL, M.S; Chang, W.T., Chen, H.Y., Chen,Y.H., Lin,C.C., Lin,M.K (2018).
Three bufadienolides-induced human lung cancer CL1-5 cell death mainly through
autophagy. Preprints, 2018:2018010134.
[35] Huang HC, Lin MK, Yang HL, Hseu YC, Liaw CC, Tseng YH, et al. (2013).
Cardenolides and bufadienolide glycosides from Kalanchoe tubiflora and evaluation
of cytotoxicity. Planta Med, 79:1362-1369.
[36] Hseu YC, Lee MS, Wu CR, Cho HJ, Lin KY, Lai GH, et al. (2012). The chalcone
flavokawain B induces G2/M cell-cycle arrest and apoptosis in human oral carcinoma
HSC-3 cells through the intracellular ROS generation and downregulation of the
Akt/p38 MAPK signaling pathway. J Agric Food Chem, 60:2385-2397.
[37] Chang CT, Hseu YC, Thiyagarajan V, Lin KY, Way TD, Korivi M, et al. (2017).
Chalcone flavokawain B induces autophagic-cell death via reactive oxygen species-
mediated signaling pathways in human gastric carcinoma and suppresses tumor
growth in nude mice. Arch Toxicol, 91:3341-3364.
[38] Hseu YC, Thiyagarajan V, Ou TT, Yang HL (2018). CoQ0-induced mitochondrial PTP

24
 opening triggers apoptosis via ROS-mediated VDAC1 upregulation in HL-60
leukemia cells and suppresses tumor growth in athymic nude mice/xenografted nude
mice. Arch Toxicol, 92:301-322.
[39] King RW, Deshaies RJ, Peters JM, Kirschner MW (1996). How proteolysis drives the
cell cycle. Science, 274:1652-1659.
[40] Uhlmann F, Bouchoux C, Lopez-Aviles S (2011). A quantitative model for cyclin-
dependent kinase control of the cell cycle: revisited. Philos Trans R Soc Lond B Biol
Sci, 366:3572-3583.
[41] Yang HL, Thiyagarajan V, Liao JW, Chu YL, Chang CT, Huang PJ, et al. (2017).
Toona sinensis Inhibits Murine Leukemia WEHI-3 Cells and Promotes Immune
Response In Vivo. Integr Cancer Ther, 16:308-318.
[42] Shiraishi H, Okamoto H, Yoshimura A, Yoshida H (2006). ER stress-induced
apoptosis and caspase-12 activation occurs downstream of mitochondrial apoptosis
involving Apaf-1. J Cell Sci, 119:3958-3966.
[43] Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, et al. (2000).
LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome
membranes after processing. EMBO J, 19:5720-5728.
[44] Jaiswal PK, Goel A, Mittal RD (2015). Survivin: A molecular biomarker in cancer.
Indian J Med Res, 141:389-397.
[45] Kanematsu S, Uehara N, Miki H, Yoshizawa K, Kawanaka A, Yuri T, et al. (2010).
Autophagy inhibition enhances Sulforaphane-induced apoptosis in human breast
cancer cells. Anticancer Research, 30:3381-3390.
[46] White E, DiPaola RS (2009). The double-edged sword of autophagy modulation in
cancer. Clin Cancer Res, 15:5308-5316.
[47] Singh N (2007). Apoptosis in health and disease and modulation of apoptosis for
therapy: an overview. Indian Journal of Clinical Biochemistry, 22:6-16.
[48] Kang R, Zeh HJ, Lotze MT, Tang D (2011). The Beclin 1 network regulates autophagy
and apoptosis. Cell Death Differ, 18:571-580.
[49] Ly JD, Grubb DR, Lawen A (2003). The mitochondrial membrane potential (∆ψm) in
apoptosis; an update. Apoptosis, 8:115-128.
[50] Park S-H, Kim J-H, Chi GY, Kim G-Y, Chang Y-C, Moon S-K, et al. (2012).
Induction of apoptosis and autophagy by sodium selenite in A549 human lung
carcinoma cells through generation of reactive oxygen species. Toxicology Letters,
212:252-261.

25
[51] Hommes DW, Peppelenbosch MP, van Deventer SJ (2003). Mitogen activated protein
(MAP) kinase signal transduction pathways and novel anti-inflammatory targets. Gut,
52:144-151.
[52] Giorgi C, Bonora M, Sorrentino G, Missiroli S, Poletti F, Suski JM, et al. (2015). p53
at the endoplasmic reticulum regulates apoptosis in a Ca2+-dependent manner. Proc
Natl Acad Sci U S A, 112:1779-1784.
[53] Giorgi C, Baldassari F, Bononi A, Bonora M, De Marchi E, Marchi S, et al. (2012).
Mitochondrial Ca(2+) and apoptosis. Cell Calcium, 52:36-43.
[54] Hartwell LH, Kastan MB (1994). Cell cycle control and cancer. Science, 266:1821-
1828.
[55] Pinto AE, Andre S, Laranjeira C, Soares J (2005). Correlations of cell cycle regulators
(p53, p21, pRb and mdm2) and c-erbB-2 with biological markers of proliferation and
overall survival in breast cancer. Pathology, 37:45-50.
[56] Joaquin M, Gubern A, Posas F (2012). A novel G1 checkpoint mediated by the p57
CDK inhibitor and p38 SAPK promotes cell survival upon stress. Cell Cycle, 11:3339-
3340.
[57] Shackelford RE, Kaufmann WK, Paules RS (1999). Cell cycle control, checkpoint
mechanisms, and genotoxic stress. Environ Health Perspect, 107 Suppl 1:5-24.
[58] Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, et al. (2010).
Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J
Med, 363:711-723.
[59] Hseu YC, Thiyagarajan V, Tsou HT, Lin KY, Chen HJ, Lin CM, et al. (2016). In vitro
and in vivo anti-tumor activity of CoQ0 against melanoma cells: inhibition of
metastasis and induction of cell-cycle arrest and apoptosis through modulation of
Wnt/beta-catenin signaling pathways. Oncotarget, 7:22409-22426.
[60] Guicciardi ME, Gores GJ (2009). Life and death by death receptors. FASEB J,
23:1625-1637.
[61] Xiong S, Mu T, Wang G, Jiang X (2014). Mitochondria-mediated apoptosis in
mammals. Protein & cell, 5:737-749.
[62] Ashkenazi A (2002). Targeting death and decoy receptors of the tumour-necrosis
factor superfamily. Nat Rev Cancer, 2:420-430.
[63] Walczak H, Krammer PH (2000). The CD95 (APO-1/Fas) and the TRAIL (APO-2L)
apoptosis systems. Exp Cell Res, 256:58-66.
[64] Morishima N, Nakanishi K, Takenouchi H, Shibata T, Yasuhiko Y (2002). An

26
 endoplasmic reticulum stress-specific caspase cascade in apoptosis. Cytochrome c-
independent activation of caspase-9 by caspase-12. J Biol Chem, 277:34287-34294.
[65] Jimbo A, Fujita E, Kouroku Y, Ohnishi J, Inohara N, Kuida K, et al. (2003). ER stress
induces caspase-8 activation, stimulating cytochrome c release and caspase-9
activation. Exp Cell Res, 283:156-166.
[66] Sui X, Chen R, Wang Z, Huang Z, Kong N, Zhang M, et al. (2013). Autophagy and
chemotherapy resistance: a promising therapeutic target for cancer treatment. Cell
Death Dis, 4:e838.
[67] Thiyagarajan V, Sivalingam KS, Viswanadha VP, Weng CF (2016). 16-hydroxy-
cleroda-3,13-dien-16,15-olide induced glioma cell autophagy via ROS generation and
activation of p38 MAPK and ERK-1/2. Environ Toxicol Pharmacol, 45:202-211.
[68] Zhang N, Qi Y, Wadham C, Wang L, Warren A, Di W, et al. (2010). FTY720 induces
necrotic cell death and autophagy in ovarian cancer cells: A protective role of
autophagy. Autophagy, 6:1157-1167.
[69] Mai TT, Moon J, Song Y, Viet PQ, Van Phuc P, Lee JM, et al. (2012). Ginsenoside F2
induces apoptosis accompanied by protective autophagy in breast cancer stem cells.
Cancer Letters, 321:144-153.
[70] Janku F, McConkey DJ, Hong DS, Kurzrock R (2011). Autophagy as a target for
anticancer therapy. Nature Reviews Clinical Oncology, 8:528-539.
[71] Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, et al. (2005). Bcl-2
antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell, 122:927-939.
[72] Maiuri MC, Zalckvar E, Kimchi A, Kroemer G (2007). Self-eating and self-killing:
crosstalk between autophagy and apoptosis. Nature Reviews Molecular Cell Biology,
8:741-752.
[73] Cheng P, Alberts I, Li X (2013). The role of ERK1/2 in the regulation of proliferation
and differentiation of astrocytes in developing brain. Int J Dev Neurosci, 31:783-789.
[74] Wang X, Martindale JL, Holbrook NJ (2000). Requirement for ERK activation in
cisplatin-induced apoptosis. J Biol Chem, 275:39435-39443.
[75] Xiu Jin Shen HBW, Xiao Qiong Ma, Jiang Hua Chen (2012). β,β-
Dimethylacrylshikonin Induces Mitochondria Dependent Apoptosis through ERK
Pathway in Human Gastric Cancer SGC-7901 Cells. PLOS ONE, 7:e41773.
[76] Gao H, Zhang Y, Dong L, Qu XY, Tao LN, Zhang YM, et al. (2018). Triptolide
induces autophagy and apoptosis through ERK activation in human breast cancer
MCF-7 cells. Experimental and Therapeutic Medicine, 15:3413-3419.

27
[77] Pattingre S, Bauvy C, Codogno P (2003). Amino acids interfere with the ERK1/2-
dependent control of macroautophagy by controlling the activation of Raf-1 in human
colon cancer HT-29 cells. J Biol Chem, 278:16667-16674.
[78] Franke TF, Kaplan DR, Cantley LC (1997). PI3K: downstream AKTion blocks
apoptosis. Cell, 88:435-437.
[79] Bootman MD, Chehab T, Bultynck G, Parys JB, Rietdorf K (2018). The regulation of
autophagy by calcium signals: Do we have a consensus? Cell Calcium, 70:32-46.
[80] Sun F, Xu X, Wang X, Zhang B (2016). Regulation of autophagy by Ca(2). Tumour
Biol.
[81] Williams JA, Hou Y, Ni HM, Ding WX (2013). Role of intracellular calcium in
proteasome inhibitor-induced endoplasmic reticulum stress, autophagy, and cell death.
Pharm Res, 30:2279-2289.
[82] Mrakovcic M, Frohlich LF (2018). p53-Mediated Molecular Control of Autophagy in
Tumor Cells. Biomolecules, 8.
[83] Tasdemir E, Maiuri MC, Galluzzi L, Vitale I, Djavaheri-Mergny M, D'Amelio M, et
al. (2008). Regulation of autophagy by cytoplasmic p53. Nat Cell Biol, 10:676-687.
[84] Bensaad K, Tsuruta A, Selak MA, Vidal MN, Nakano K, Bartrons R, et al. (2006).
TIGAR, a p53-inducible regulator of glycolysis and apoptosis. Cell, 126:107-120.
[85] Bensaad K, Cheung EC, Vousden KH (2009). Modulation of intracellular ROS levels
by TIGAR controls autophagy. EMBO J, 28:3015-3026.
[86] Eischen CM, Packham G, Nip J, Fee BE, Hiebert SW, Zambetti GP, et al. (2001). Bcl-
2 is an apoptotic target suppressed by both c-Myc and E2F-1. Oncogene, 20:6983-
6993.
[87] Sakamuro D, Eviner V, Elliott KJ, Showe L, White E, Prendergast GC (1995). c-Myc
induces apoptosis in epithelial cells by both p53-dependent and p53-independent
mechanisms. Oncogene, 11:2411-2418.
[88] Leiter U, Garbe C (2008). Epidemiology of melanoma and nonmelanoma skin cancer-
-the role of sunlight. Adv Exp Med Biol, 624:89-103.

28
Figure Legend
Fig 1. Kalantuboside B (KB) inhibits cell proliferation and colony formation in human
melanoma cells. (A) Chemical structure of KB. (B-D) A2058, B16F10, and HaCaT cell-
types were treated with increasing concentrations of KB (0, 5–30 ng/mL; 8.7-34.8 nM) for 24
h, and cell viability was measured using the MTT method. (E) Phase-contrast microscope
images (200 × magnification) showing abrupt morphological changes in A2058 cells that
were exposed to different concentrations of KB (0, 5–30 ng/mL for 24 h). (F-G) Colony
formation ability of A2058 cells on soft agar was tested by treating the cells with either KB
(0, 5–30 ng/mL) or DMSO (vehicle) for every two days and incubated for 5 days at 37°C. p-
iodo nitro-tetrazolium violet (1 mg/mL) was used to stain the colonies (more than fifty cells
per colony). Using a dissecting microscope, stained colonies were counted and the percentage
of colony formation at different concentrations of KB against control A2058 cells were
measured. Colony formation ability of control cells was arbitrarily assigned to 100%. The
data were presented as mean ± SD of three or more independent experiments and the
significant differences were denoted as *p<0.05, **p<0.005 and ***p<0.001.

Fig 2. KB induces G2/M arrest in human melanoma A2058 cells. Cells were treated with 0,
5–30 ng/mL of KB for 24 h. (A) The percentage of cells at different phases of cell-cycle were
determined by flow cytometry. (B) The effect of KB on the expression levels of cell-cycle
regulatory proteins (cyclin A/B1, Cdc25C, and Cdc2) was examined by western blot method
against β-actin as a loading control. The data were presented as mean ± SD of three or more
independent experiments and the significant differences were denoted as *p<0.05.

Fig 3. Induction of apoptosis in A2058 cells by KB treatment. Cells were treated with KB
(20 ng/mL) for 24 h. (A-B) The DNA fragmentation was determined by TUNEL assay. The
green fluorescence indicates TUNEL-positive cells in the microscopic fields (200 ×
magnification) from three separate samples. (C) Annexin V-FITC/PI staining of A2058 cells
was performed to identify early/late apoptosis these cells and the data was analyzed via flow
cytometry. Results in each quadrant were labeled and interpreted as follows: (Q1) Necrosis -
PI positive and Annexin V-FITC-negative cells, (Q2) Apoptosis - PI positive and Annexin V-
FITC-positive cells, (Q3) Normal live cells - PI negative and Annexin V-FITC negative cells,
(Q4) Early apoptosis - PI-negative and Annexin V-FITC-positive cells. The data were
presented as mean ± SD of three or more independent experiments and the significant
differences were denoted as ***p<0.001.
29
Fig 4. KB-induced apoptosis was mediated via the activation of mitochondrial, death
receptor, and ER stress pathways. (A) Effect of time (0-16 h) on expression patterns of the
mitochondrial and cytosolic cytochrome c levels in KB treated A2058 cells. (B) Effect of KB
(5-30 ng/mL) on protein expression patterns of procaspases-9/-3 and PARP (mitochondrial
pathway); (C) caspases-8 (death receptor pathway) and caspase-12 (ER stress pathway) in
A2058 cells. (D) Cell viability was measured by MTT assay. Cells were pretreated with
caspase inhibitor, Z-VAD-FMK (20 µM), for 1 h followed by exposure to KB (20 ng/mL) for
24 h. The data were presented as mean ± SD of three or more independent experiments and
the significant differences were denoted as **p<0.05.

Fig 5. KB triggers autophagy in human melanoma A2058 cells. Cells were exposed to
different concentrations of KB (5-30 ng/mL for 24 h). (A-B) Expression levels of LC3-I,
LC3-II, survivin (A), and p53 (B) were measured by western blotting. (C-D) Cells were
transfected with GFP-LC3 expression vector for 24 h followed by treatment with different
concentrations of KB (5-30 ng/mL) for 24 h. The GFP-LC3 puncta induced by KB were
observed through a confocal microscope. (E) KB (5-30 ng/mL, 24 h) induced AVOs were
detected using acridine orange (AO) stain and were visualized under a red filter fluorescence
microscope (100 × magnification). The percentage of AVOs developed was calculated based
on the results of the fluorescence-activated cell sorting assay. The data were presented as
mean ± SD of three or more independent experiments and the significant differences were
denoted as **p<0.005 and ***p<0.001.

Fig 6. KB enhances autophagy as a cell survival mechanism in A2058 cells. (A-C) Effect
of 3-methyladenine (3-MA) and chloroquine on KB-mediated autophagy, apoptosis, and cell
viability. Cells were pretreated with 3-MA (3-Methyladenine, 5 mM) and CQ (chloroquine,
25 µM) for 1 h followed by exposure to 20 ng/mL of KB for 24 h. (A) Cell viability was
determined by MTT assay. (B-C) Conversions of LC3-I to LC3-II and PARP were examined
by immunoblot. (D-F) Regulation of autophagy by Bax, Beclin-1, Bcl-2, and (dose-
dependent) in KB-treated A2058 cells were detected by western blotting. Cells exposed to 20
ng/mL of KB for 4-24 h. The data were presented as mean ± SD of three or more independent
experiments and the significant differences were denoted as *p<0.05, **p<0.005 and
***p<0.001.

30
Fig 7. KB triggers ROS mediated-apoptosis and autophagy (cytotoxicity) in A2058 cells:
(A-B) Cells were pretreated with 5 mM NAC (ROS inhibitor) for 1 h followed by exposure to
KB (20 ng/mL) at 0, 5, 15, 30 min time points and the production of ROS was measured by
DCFH2-DA method. Data were represented as a fold-over percentage compared to untreated
control cells. (C) Cell viability was measured by MTT assay. Cells were pretreated with ROS
inhibitor, NAC (5mM), for 1 h followed by exposure to KB (20 ng/mL) for 24 h. (D-E) Cells
were pretreated with 5mM NAC for 1 h followed by KB (20 ng/mL for 24 h) and the
expression levels of apoptotic (caspase-3 activation and PARP cleavage) and autophagy
proteins (LC3-I to LC3-II conversion) were measured by western blot method. The data were
presented as mean ± SD of three or more independent experiments and the significant
differences were denoted as *p<0.05, **p<0.005 and ***p<0.001.

Fig 8. KB induces apoptosis and cytoprotective autophagy through upregulated ERK

and downregulated AKT signaling pathways in A2058 cells. (A-C) Cells were incubated
with 20 ng/mL KB for 15-90 min. Total ERK1/2, AKT, JNK1/2, and p38 levels were analyzed
by western blot. The percentage ratios of p-ERK/ERK and p-AKT/AKT in KB-treated A2058
cells were represented. (D-E) Cells were pretreated with inhibitors of ERK (PD98059, 30
µM) for 30 min followed by treatment with KB (20 ng/mL) for 24 h. (D) PARP and LC3I/II
levels were measured by western blot. (E) Cell viability was determined by MTT assay and
the data was represented as fold decrease in KB + PD98059 treated cells compared with KB
alone treated cells. (F) Cells were pretreated with inhibitors of early and late autophagic
protein inhibitors (3-MA, 5 mM and CQ, 25 µM) for 30 min followed by treatment with KB
(20 ng/mL) for 24 h. The expression of p-ERK was measured by western blotting. The data
were presented as mean ± SD of three or more independent experiments and the significant
differences were denoted as *p<0.05, **p<0.005 and ***p<0.001.

Fig 9. Enhanced Calcium-dependent cytoprotective autophagy in KB-treated A2058

cells. Cells were pretreated with BAPTA (5 µM) for 1 h followed by treatment with KB (20
ng/mL) for 24 h. (A) MTT cell viability assay. (B) p53, and LC3-I/II, and PARP protein blot
images. (C-E) The expression patterns of p53, LC3-I/II, and PARP proteins were expressed
as the percentage of expression or fold change. The expression of p-ERK was measured by
western blotting. The data were presented as mean ± SD of three or more independent
experiments and the significant differences were denoted as **p<0.005 and ***p<0.001.

31
Fig 10. Tumor suppression property of KB in A2058 xenografted nude mice. A2058
xenografted nude mice were subjected to either vehicle (control) or KB (0.4 mg/kg,
intraperitoneal injection every 2 days) treatments. (A-B) The body weight and tumor volumes
were measured for 8 weeks. (C-D) On the 8th week, animals were sacrificed, tumors were
excised and their sizes, weights were quantified. The results were presented as the mean ± SD
of three independent assays and significant differences denoted by *p<0.05.

Fig 11. KB inhibited A2058 xenografted tumors in nude mice via apoptosis and
cytoprotective autophagy. A2058 xenografted nude mice were treated with vehicle (control)
or KB (0.4 mg/kg, intraperitoneal injection every 2 days) and the tumors were excised for
further analysis. (A) Western blot analysis of cyclin A, Bcl-2, LC3-I/II, survivin, and p53
protein pattern. (B-F) Fold change in the expression patterns of (B) Cyclin A, (C) Bcl-2, (D)
LC3-I/II, (E) Survivin, and (F) p53 from KB treated were compared with their respective
protein controls in excised A2038 xenografted tumors in nude mice. The results are presented
as the mean ± SD of three independent assays and significant differences denoted by
*p<0.05.

32
 A Fig 1 B C
IC50=13.0 ng/mL IC50=63.1 ng/mL

A2058 cell viability (%)

B16F10 cell viability (%)

100 100
* ** ***
***
** ***
***
50 50

***
Kalantuboside B (KB)

0 0
KB 0 5 10 30 (ng/mL) KB 0 5 10 20 30 (ng/mL)
D F
20
HaCaT cell viability (%)

IC50=25.3 ng/mL KB 0 5 10 20 30 (ng/mL)

100
(A2058)
***
***

50 ***

G
A2058

Colony formation (%)

0 100
KB 0 5 10 20 30 (ng/mL)
80 ***
E ***
KB 0 5 10 20 30 (ng/mL) 60
***
40 ***
(A2058)

20

0
KB 0 5 10 20 30 (ng/mL)
A B
Control Control
0.4 mg/kg 0.4 mg/kg

Tumor volume (mm3)

1200
30
Weight (g)

900
20
600

10 ∗
300

0 0
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
Weeks Weeks

Control 0.4 mg/kg

D 1.2
Tumor weight (g)

Control ∗
0.6
0.4 mg/kg

Fig 10
 A
Control 0.4 mg/kg
Cyclin A (53 KDa)
Bcl-2 (26 KDa)

LC3-I/II (16/14 KDa)

Survivin (16 KDa)

p53 (53 KDa)

β-actin (43 KDa)

B C
2.0
2 2
Cyclin A (fold)

1.5
Bcl-2 (fold)

1 1
1.0

0.5

0 0
0.0

Control 0.4 mg/kg Control 0.4 mg/kg

D E F
6 2 2
Survivin (fold)
LC3-II (fold)

4
p53 (fold)

1 1
2
∗

0 0 0
Control 0.4 mg/kg Control 0.4 mg/kg Control 0.4 mg/kg
Fig 11
A

Apoptotic cells Non-apoptotic cells

KB
(ng/mL) Sub-G1 G1 S G2/M

0 ±0.0
0.9± ±1.5
57.7± ±0.7
16.0± ±1.0
26.3±
***
5 ±0.4***
3.4± ±0.7
47.6± ±0.9
19.9± ±0.7
32.4±
10 ±0.5 ***
8.2± ±0.4
42.2± ±0.2
19.1± ±0.3***
38.6±
20 ±0.5 ***
9.6± ±0.9
40.3± ±0.7
17.8± ±1.5
41.9±
***

30 ±0.7***
15.5± ±0.7
43.2± ±0.5
14.9± ±0.3***
41.9±

B
KB (ng/mL)
0 5 10 20 30
Cyclin A
(53 KDa)
1.00 0.65 0.59 0.39 0.19
Cyclin B1
(62 KDa)
1.00 0.52 0.32 0.30 0.15
Cdc25C
(60 KDa)
1.00 0.74 0.18 0.06 0.05
Cdc2
(34 KDa)
1.00 1.00 0.63 0.30 0.43
β-actin
(43 KDa)

Fig 2
A +Z-VAD-FMK (20 µM)
B
KB 0 20 0 20 (ng/mL) 44
∗∗∗
TUNEL

33

TUNEL (fold)
22
DAPI

11

00
KB 0 20 0 20 (ng/mL)
Merge

+Z-VAD-FMK (20 µM)

C +Z-VAD-FMK (20 µM)

KB 0 20 0 20 (ng/mL)
1.4 ± 0.2 3.7 ± 0.4 0.2 ± 0.0 3.8 ± 0.2 1.0 ± 0.1 3.0 ± 0.3 1.9 ± 0.3 4.3 ± 0.4

82.0 ± 4.5 12.9 ± 0.5 69.4 ± 2.2 26.6 ± 0.4 87.7 ± 6.2 8.4 ± 0.5 76.8 ± 3.6 16.9 ± 0.8

Fig 3
 A KB (20 ng/mL) C KB (ng/mL)
(mitochrondrial) 0 4 8 16 (h) 0 5 10 20 30
Cytochrome c Procaspase-8
(15 KDa) (55 KDa)
(cytosolic) 1.00 0.96 0.67 0.25 Cleaved Caspase-8
(35 KDa)
Cytochrome c 1.00 0.97 0.93 0.92 0.81
(15 KDa) 1.00 1.26 1.77 1.87 2.58
1.00 1.04 1.45 5.70 Procaspase-12
β-actin (55 KDa)
(43 KDa) Cleaved Caspase-12
(39 KDa)
KB (ng/mL) 1.00 1.04 1.02 1.10 1.06
B 1.00 1.13 1.33 1.78 2.35
0 5 10 20 30 β-actin
Procaspase-9 (43 KDa)
(47 KDa)
Cleaved caspase-9 D
( 37 KDa)
1.00 0.89 0.86 0.62 0.58
∗∗
1.00 1.00 1.00 1.13 2.18 100

Cell viability (%)

Procaspase-3
( 32 KDa) 80
Cleaved caspase-3
( 19/17 KDa) 60
1.00 0.96 0.75 0.48 0.36
1.00 1.00 1.00 1.29 1.57
40
PARP
(116 KDa)
Cleaved PARP 20
(89 KDa)
1.00 0.80 0.76 0.72 0.68 0
1.00 0.93 1.27 2.23 2.49 KB 0 20 0 20 (ng/mL)
β-actin Fig 4
(43 KDa) +Z-VAD-FMK (10 µM)
 C +NAC (5 mM)
A KB (ng/mL)
KB 0 20 0 20 (ng/mL)
0 5 10 20 30

(GFP-LC3)
LC3-I/II
(16/14 KDa)
1.00 1.64 2.41 2.67 1.85
Survivin
(16 KDa) 8
1.00 1.03 0.15 0.14 0.12
∗∗

GFP-LC3 puncta/cell
β-actin
(43 KDa)

B KB (ng/mL) 4

0 5 10 20 30
p53
(53 KDa)
1.00 0.47 0.27 0.08 0.07 0
β-actin KB 0 20 0 20 (ng/mL)
(43 KDa)
+NAC (5 mM)
D
E 12
KB 0 5 10 20 30 (ng/mL)
∗∗∗

AVO (fold)
8

∗∗∗
4
∗∗

0
Fig 5
KB 0 5 10 20 30 (ng/mL)
A B C
3-MA (5 mM) CQ (25 µM)
∗
120

∗∗∗
100
100 100 KB 0 20 0 20 0 20 (ng/mL)

Cell viability (%)

PARP
Cell viability (%)

8080 80 (116 KDa)

Cleaved
6060 60 PARP
(89 KDa) 1.0 1.93 1.37 9.18 2.71 8.93
4040 40 LC3-I/II
(16/14 KDa)
2020 20
1.0 2.23 1.10 1.23 6.18 5.92
00 0 β-actin
KB 0 20 0 20 KB 0 20 0 20 (43 KDa)
(ng/mL) (ng/mL)
+3-MA (5 mM) +CQ (25 µM)

D E F
KB (20 ng/mL) 15

0 4 8 16 24 (h)
Bax 12 ∗∗ 1212 ∗∗

Beclin-1/Bcl-2 ratio
Bax/Bcl-2 ratio

(20 KDa)
∗∗
Beclin-1 9 ∗∗ 9
(60 KDa)
Bcl-2 6 6
(26 KDa)
∗∗
LC3-I/II 3 ∗∗ 3 ∗∗
(16/14 KDa)
0 0
β-actin
(43 KDa) 0 4 8 16 24 (h) 0 4 8 16 24 (h)
KB (20 ng/mL) Fig 6 KB (20 ng/mL)
 A KB (20 ng/mL) B - NAC
15 30 (min) 30 + NAC
0 5
∗∗∗

ROS (fold)
─NAC

20 ∗∗∗
∗∗∗
10
+NAC

∗∗ ∗∗ ∗∗
0
0 5 15 30 (min)
D +NAC (5 mM)
C +KB (20 ng/mL)
∗ KB 0 20 0 20 (ng/mL)
100 Caspase-3
Cell viability (%)

(19/17 KDa)
80 E
1.00 15.37 1.04 11.84 +NAC (5 mM)
60 1.00 4.48 1.02 3.61 KB 0 20 0 20 (ng/mL)
β-actin
40 (43 KDa) LC3I/II
PARP (16/14 KDa)
20 (116 KDa) 1.00 1.62 0.78 0.53
Cleaved PARP β-actin
0 (89 KDa)
1.00 0.11 1.53 0.43 (43 KDa)
KB 0 20 0 20
(ng/mL) 1.00 15.63 1.05 9.40
+NAC (5 mM) β-actin
(43 KDa) Fig 7
 A KB (20 ng/mL) B C
0 15 30 45 60 90 (min) 6

p-ERK/ERK ratio fold)

100
∗∗∗

p-AKT/AKT ratio (%)

p-ERK1/2
(42/44 KDa)
4 ∗∗∗ 80 ∗∗ ∗∗ ∗∗
ERK1/2
(42/44 KDa) ∗∗∗ ∗∗∗
60
p-AKT
(60 KDa) ∗∗∗
2 ∗ 40
AKT
(60 KDa) 20
p-JNK1/2 0 0
(46/53 KDa) 0 15 30 45 60 90 (min) 0 15 30 45 60 90 (min)
JNK
(46/53 KDa) KB (20 ng/mL) KB (20 ng/mL)
p-p38 E
(38 KDa) D
p38
(ng/mL) +PD98059 (30 µM) 100 ∗
(38 KDa)

Cell viability (fold)

KB 0 20 0 20
β-actin 80
(43 KDa) LC3-I/II
(16/14 KDa) 60
F 1.00 2.11 0.84 0.75
(ng/mL) 3-MA (5 mM) CQ (25 µM)
1.00 2.75 0.53 0.73
PARP 40
KB 0 20 0 20 0 20 (116 KDa)
Cleaved 20
p-ERK1/2 PARP
(42/44 KDa) (89 KDa) 1.00 0.58 0.94 0.55
0
1.00 3.55 1.79 4.22 1.02 3.22 1.00 1.32 1.03 2.75
β-actin KB 0 20 0 20
1.00 3.21 2.25 3.32 0.72 3.49
(43 KDa) (ng/mL)
β-actin +PD98059 (30 µM)
(43 KDa) Fig 8
 Fig 9 A B +BAPTA (5 µM)
100 ∗∗∗ KB 0 20 0 20 (ng/mL)

Cell viability (fold)

p53
80 (53 KDa)

60 LC3-I/II
(16/14 KDa)
40 PARP
(116 KDa)
Cleaved PARP
20 (89 KDa)
0 β-actin
(43 KDa)
KB 0 20 0 20 (ng/mL)
+BAPTA (5 µM)

C D E
∗∗
3 ∗∗
120 ∗∗∗
2

Cleaved PARP (fold)

p53 expression (%)

100
LC3-II (fold)

2
80

60 1
1
40

20
0 0 0
KB 0 20 0 20 (ng/mL) KB 0 20 0 20 (ng/mL) KB 0 20 0 20 (ng/mL)
+BAPTA (5 µM) +BAPTA (5 µM) +BAPTA (5 µM)
Highlights

Kalantuboside B (KB, 8.7-52.2 nM) shows anti-tumor activity in human melanoma cells

KB inhibited melanoma cell proliferation via G2/M cell-cycle arrest and apoptosis

KB induced autophagy in A2058 cells as a cell-survival mechanism

ROS pathway plays a role in KB-mediated apoptosis and cytoprotective autophagy

KB mediated the suppression of A2058 xenograft tumor growth in athymic nude mice