Biomedicine & Pharmacotherapy 112 (2019) 108602

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Original article

Ribociclib, a selective cyclin D kinase 4/6 inhibitor, inhibits proliferation T

and induces apoptosis of human cervical cancer in vitro and in vivo
Yudi Xionga,b, Tianqi Lia,b, Ganiou Assania,b, Huan Linga,b, Qian Zhoua,b, Yangyang Zenga,b,
Fuxiang Zhoua,b, Yunfeng Zhoua,b,
⁎

a
Hubei Cancer Clinical Study Center, Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital, Wuhan University, Wuhan, China
b
Department of Radiation Oncology and Medical Oncology, Zhongnan Hospital, Wuhan University, Wuhan, China

ARTICLE INFO ABSTRACT

Keywords: Cervical cancer remains one of the main factors leading to tumor-related death worldwide. Many strategies of
Cervical cancer cancer treatment such as chemotherapy are developed and used nowadays. However, for the cancer che-
CDK4 motherapy resistance, reduction of the limitation of cancer chemotherapy efficacy is one of the aims of several
CDK6 oncology teams. Moreover, the cyclin-dependent kinase 4/6-cyclin D-retinoblastoma protein-E2F pathway is an
Ribociclib
important mechanism for cell cycle control and its dysregulation is one of the key factors for cancers devel-
Cell cycle
Cell apoptosis
opment including cervical cancer. Ribociclib is one of the selective CDK4/6 inhibitors and is a new therapeutic
approach showing promise as a good strategy of therapy in many human cancers. However, there are not the
studies regarding the investigation of effects of Ribociclib in cervical cancer yet. In the present study, by western
blotting and immunofluorescence assay, we found respectively that CDK4, CDK6 and cyclin D1 are highly ex-
pressed and are mostly localized in the nucleus with some localized in the cytoplasm of cervical cancer cell lines.
Moreover, Ribociclib induced cell cycle arrest in G0-G1 phase and cell apoptosis, and inhibited C33A cell
proliferation in dose – dependent manner following by decreased expression of certain related genes such as
CDK4, CDK6, E2F1, P-Rb, and increased Bax expression. In C33A xenografts, Ribociclib inhibited tumor growth
associated with decreased expressions of CDK4, CDK6, cyclin D1, Rb and Ki-67, and also significantly increased
tumor cell apoptosis. However, we didn’t find side effect of Ribociclib concerning heart, liver and kidney per-
turbation and any Ribociclib anti-tumor effects on HeLa in vitro and in vivo which may be due to Hela cell
infection by HPV. Based on our findings, the Rb-E2F pathway can be considered as an important factor in human
cervical cancer pathogenesis and as a mechanism of Ribociclib, a potential strategy of treatment for the im-
provement of new therapeutic measures for the treatment of HPV-negative cervical cancer which application for
HPV-positive cervical cancer is desired in further study.

1. Introduction
Cervical cancer is the fourth most common cancer in women and
represents the leading cause of death among women in the world [1].
More than 85% of the global burden occurs in developing countries
where it accounts for 12% of all female cancers. Cervical cancer re-
presents the 2nd common cancer in less developed regions and the 11th
in more developed regions [1]. It was estimated that 528.000 new cases
occurred in 2012 and its incident cases were increased by 9% (95% UI,
2%–17%) between 2006–2016 [2].Based on these data, cervical cancer
can be considered as an important public issue and need strong stra-
tegies of treatment for its curative treatment as for its preventive
treatment.
Many strategies of curative treatment of cervical cancer such as
surgery which including pelvic lymphadenectomy and radical hyster-
ectomy in early stages, chemotherapy, and radiation therapy in more
advanced stages are developed and used in nowadays. Radical hyster-
ectomy and radiotherapy were considered as a treatment for localized
disease, while concurrent radiochemotherapy remains the main cor-
nerstone intervention for advanced cancer [3]. However,

Abbreviations: CDK, cyclin-dependent kinase; Rb, retinoblastoma; HPV, human papillomavirus; FDA, the Food and Drug Administration; HR, hormone receptor;
HER2, human epidermal growth factor receptor 2; HE, Hematoxylin-eosin staining
⁎
Corresponding author at: Department of Radiation Oncology & Medical Oncology, Zhongnan Hospital, Wuhan University, 169 Donghu Road, Wuhan, 430071,
China.
E-mail address: yfzhouwhu@163.com (Y. Zhou).

https://doi.org/10.1016/j.biopha.2019.108602
Received 31 October 2018; Received in revised form 17 January 2019; Accepted 18 January 2019
0753-3322/ © 2019 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Y. Xiong, et al.
radioresistance and chemotherapy resistance represents the limitation
of the effectiveness for the use of radiotherapy and/or chemotherapy in
clinical treatment for patients with cervical cancer [4] which suggests
that the availability of effective therapeutic measure is limited and need
the alternative treatment strategies for the patients with cervical
cancer. Although target therapy became a research hotspot in recent
years for the treatment of cervical cancer [5,6].However, the survival
rate of cervical cancer did not significantly increase or increased with
adverse events.
Moreover, the families of cyclin-dependent-kinase (CDK) are the key
regulator of cell cycle progression, which represents one of the hall-
marks of cancer [7]. As one of the essential signaling pathways involved
in cell cycle progression, the cyclin-dependent kinase (CDK) 4/6-cyclin
D-retinoblastoma protein (Rb)-E2F1 pathway (CDK4/6-cyclin D-Rb-
E2F1 pathway) is frequently found to be over-activated in cancer [8]
where Cyclin D is a catalyst for CDK4 and CDK6 and interacts with
CDK4/CDK6 to form the Cyclin D-CDK4/6 complexes [9,10]. CDK4 or
CDK6 are kinds of the serine/threonine (Ser/Thr) protein kinases im-
plicated in cell cycle progression through the G1-S phase [11]. Cyclin D
including cyclin D1, D2 and D3 combine with CDK4 or CDK6, and
phosphorylates the tumor suppressor protein Rb [12]. This phosphor-
ylation in turn, via the activation of the transcription factor E2F1, leads
to the transition from the G1 phase to the S-phase and finally, cell
proliferation [13,14].Amplification of cyclin D1 or CDK4 genes, or
overexpression of their protein products has been found in cervical
cancer and overexpression of cyclin D1 was an independent poor
prognostic marker in cervical carcinoma suggesting that target CDK4/6-
cyclin D-Rb-E2F1 pathway by inhibiting CDK4 or cyclinD1 would be a
good treatment strategy for cervical cancer patients [15].
Ribociclib(LEE011) is one of the small-molecule selective inhibitors
of CDK 4/6, developed to target the ATP-binding site of CDK4/6 by
inhibiting CDK4 and CDK6, by restoring the activation of Rb which
leads to cell cycle arrest in G1/S phase. The Food and Drug
Administration (FDA) have approved the CDK 4/6
inhibitors,Palbociclib(PD0332991), Ribociclib, and Abemaciclib
(LY2835219) as initial endocrine-based therapy for the treatment of
postmenopausal women with hormone receptor (HR)-positive, human
epidermal growth factor receptor 2 (HER2)-negative advanced or me-
tastatic breast cancer in combination with an aromatase inhibitor, le-
trozole [16–20]. Ribociclib anti-cancer effect has been reported with
tolerable safety [21,22]. Moreover, CDK 4/6 inhibitors have been de-
monstrated to suppress tumor growth in preclinical models with neu-
roblastoma [23], liposarcoma [24], ovarian cancer [25], thyroid cancer
[26], synovial sarcoma [27], non-small cell lung cancer [28], gastric
cancer [29] and other malignant tumors. However, the potential me-
chanism of targeting CDK4/6 by using the specific inhibitor Ribociclib
as a putative therapeutic strategy needs to be elucidated in cervical
cancer. Our study aimed to evaluate the anticancer effects of Ribociclib
on cervical cancer in vitro and in vivo where we could discover the
mechanisms of CDK 4/6 selective inhibitor-mediated reduction of
cancer growth.
2. Materials and methods
2.1. Cell lines and cell culture
The human cervical cancer cell lines C33A and HeLa were obtained
from the Shanghai Life Science Institute Cell Library (Shanghai, China)
and were maintained in the Key Laboratory of Tumor Biological
Behavior of Hubei Province. The cell lines were cultured in DMEM
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Biomedicine & Pharmacotherapy 112 (2019) 108602
(Hyclone, USA) containing 10% fetal bovine serum (Wisent, Canada),
100 U/mL penicillin and 100 μg/ml streptomycin. All cells were in-
cubated in 5% CO2 at 37 °C.
2.2. Immunofluorescence assay
The immunofluorescence assay was used to determine CDK4, CDK6
and cyclin D1 background expression of cervical cancer cells, C33A,
and HeLa cells were grown in 12-well plates for 12 h and fixed with 4%
paraformaldehyde for 15 min following by the permeabilization with
0.2% Triton X-100 in PBS for 20 min and blocked with bovine serum
albumin(BSA) for 30 min at room temperature. The cells were then
incubated with the CDK4 primary antibody (1:100
dilution,#A0366,ABclonal,USA),CDK6 primary antibody (1:100
dilution,#A1545,ABclonal,USA) and cyclin D1 primary antibody
(1:100 dilution,#A10757,ABclonal,USA) at 4 °C overnight. Then wa-
shed with 1% BSA in PBS following by its incubation with Alexa Fluor
594 (Red) conjugated donkey anti-rabbit antibody (1:200 dilution,
#SA00006-8, Proteintech, China) for 2 h. The coverslips were mounted
in VECTASHIELD mounting medium with DAPI (#H-1200, Vector,
USA). Finally, cells were imaged on a Nikon Eclipse Ti-U fluorescence
microscope (Diagnostic Instuments Inc., USA) equipped with a digital
camera.
2.3. Pharmacologic treatment
10 mg of LEE011 powder (M.W.:434.54 g/mol, HY-15777,MCE) was
solubilized in 2.3013 ml of DMSO and 10 mM stock solution was
made,2 mg of LEE011 powder was solubilized into 20 mM stock solu-
tion. Finally, 0–20 μM of LEE011 was produced from stock solutions by
using serum-free media. C33 A and HeLa were incubated under the
treatment of various concentrations of LEE011.
For animal experiments, animals bearing engrafted tumors of 100-
200 mm3 are randomized to oral treatment with 200 mg/kg of LEE011
in 0.5% methylcellulose or vehicle daily for 21 days.
2.4. Morphological observation assay
The effect of LEE011 on cell proliferation and viability were as-
sessed by morphologic changes. C33A and HeLa cells were seeded into
6-well plates at a density of 30%–40% per well, and incubated for 48 h
in various concentrations (0, 5, 10, 15, 20 μM) of LEE011.At the end of
cell treatment, C33A and HeLa cells were observed after 2 days of
LEE011 exposure. Cells were finally imaged on a Nikon Eclipse Ti-U
fluorescence microscope (Diagnostic Instuments Inc., USA) equipped
with a digital camera.
2.5. Western blotting
Protein lysates were extracted from C33A and HeLa cells with RIPA
lysis buffer (#P0013B,Beyotime biotechnology, China) supplemented
with 1XPMSF (#ST506,Beyotime biotechnology, China) and phospha-
tase inhibitor tablets (#4906837001,Roche,USA). The concentrations
of the protein lysates were quantified by BCA assay (#23227, Thermo,
USA), and then incubate the samples in boiled water for 10 min.These
samples were separated by 10% SDS-PAGE and then transferred to
PVDF membranes. After blocking with 5% skim milk in TBST for 2 h,the
membranes were incubated with primary antibodies human RB (1:1000
dilution,#25628-1-AP, Proteintech, China);pRBSer780 (1:1000
dilution,#9307,Cell Signaling Technology, USA);E2F1 (1:1000
Y. Xiong, et al.
dilution,#12171-1-AP, Proteintech, China);Cyclin E1 (1:1000
dilution,#A14225,ABclonal,USA);CDK2 (1:1000 dilution,#2546,Cell
Signaling Technology, USA);CDK4(1:1000 dilution,#A0366,ABclonal,
USA);CDK6(1:1000dilution,#A1545,ABclonal,USA);Cyclin D1 (1:5000
dilution,#60186-1-lg, Proteintech, China);BCL-2 (1:2000 dilu-
tion,#12789-1-AP, Proteintech, China);BAX (1:5000 dilution,#50599-
2-lg, Proteintech, China);P16 (1:1000 dilution,#10883-1-AP,
Proteintech, China);GAPDH (1:5000,#60004-1-Ig,Proteintech,China)
at 4 °C overnight. At the end of time of the primary antibodies in-
cubation, the membranes were washed with TBST for 5 times, 10 min
per time of washing following by the incubation with Horseradish
peroxidase-conjugated to the secondary antibody diluted at 1:10000 for
2 h and specific bands were visualized by ECL (Advansta, USA).The
bands were detected using the ChemiDoc XRS + system (Bio-Rad,
Hercules, CA, USA) and quantification for the interested proteins was
performed using ImageJ program.
2.6. Cell cycle analysis
C33A and HeLa cells were cultured under the treatment of LEE011
(10 μM) or with DMSO control for 48 h, harvested, and fixed in cold
70% ethanol. Nuclear staining for cell cycle analysis was performed by
using propidium iodide (#C1052, Beyotime, China) following the
manufacturer’s instructions. Then, the samples were analyzed by flow
cytometry (FACS AriaIII, BD, USA).The above procedures were per-
formed three times under the same condition.
2.7. Apoptosis assay
Cell apoptosis in C33A and HeLa cells was analyzed by flow cyto-
metry after 48 h of LEE011 (10 μM) treatment. For cell apoptosis ana-
lysis, the cells were collected and FITC-conjugated annexin V/propi-
dium iodide method was used to perform the experiments (Annexin V-
FITC/PI Apoptosis Kit, Bestbio, China).We also evaluated the apoptosis
by using TUNEL assay kit.
2.8. In vivo tumor growth
For cell lines xenograft experiments, 5*106 cells were suspended in
100 ml PBS, and subcutaneously injected into male nude mice (aged
4–6 weeks). Two to four weeks later, mice bearing engrafted tumors of
100–200 mm3 were randomized to oral treatment with 200 mg/kg
LEE011 in 0.5% methylcellulose (n = 5) or vehicle(n = 5) daily for a
total of 21 days. The body weight of mice and tumor sizes were mon-
itored for 3 weeks. The perpendicular tumor diameters were measured
with calipers, and tumor volumes (V) were calculated according to the
formula of the rotational ellipsoid: V = A * (B2/2), (A = longer dia-
meter, B = smaller diameter).The animal experiments were approved
by the Institutional Animal Care and Use Committee of Wuhan
University and performed by following Institutional Guidelines and
Protocols. All efforts including euthanasia were made to reduce suf-
fering. At the end of the study, animals were collected eyeball blood
after euthanasia and sacrificed by cervical dislocation, the heart, liver
and kidney tissues were used for hematoxylin-eosin staining analysis,
and tumor tissues were excised for immunohistochemical analysis.
2.9. Blood biochemical analysis
After collecting the eyeball bloods, different biochemical indicators
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Biomedicine & Pharmacotherapy 112 (2019) 108602
(ALT,AST,ALB,ALP,BUN,CR,LDH-L,CK) were detected from these blood
samples by using different kits (Changchun Huili Biotech Co., Ltd.,
China) according to the manufacturer’s instructions. Then, the samples
were analyzed by an automatic biochemical analyzer (Chemray240,
Rayto, China).
2.10. Hematoxylin-eosin staining of tissues
The tissues sections were mounted on glass slides and treated with
hematoxylin-eosin staining solution (#G1005, Servicebio, China) based
on the manufacturer’s instructions, which were stained with hematox-
ylin for 5 min after routine de-waxing and washing. The color was se-
parated and the sections were changed from back to blue, dyed with
eosin for 5 min, dehydrated in increasing concentrations of alcohol,
hyalinized with dimethylbenzene, followed by sealing with neutral gum
for observation under the microscope.
2.11. Immunohistochemistry of tumor xenografts
Immunohistochemical staining was performed with primary anti-
bodies on 4 mm-thick formalin fixed paraffin-embedded (FFPE) tissue
sections. The tissue sections were deparaffinized in a graded series of
xylene and rehydrated in a graded series of ethanol. Antigens were
retrieved in citrate buffer (PH 9.0) using heat-induced epitope retrieval
method. To inactivate endogenous peroxidases, the tissue sections were
incubated in 3% hydrogen peroxide for 25 min at room temperature
and then blocked for 30 min with 3% bovine serum albumin (BSA). The
primary antibodies used were CDK4 (1:100 dilution, #A0366,
ABclonal, USA);
CDK6 (1:100dilution, #A1545,ABclonal,USA);CyclinD1(1:100
dilution,#A10757,
ABclonal, USA); RB (1:100 dilution, #10048-2-lg, Proteintech,
China); Detection was performed using the automatic digital slide
scanning and analysis system (Aperio VERSA 8, LEICA, Germany).
Tissue slides were visualized at x200 or x400 using a microscope. The
quantification for the interested proteins was performed using Image-
pro plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).
2.12. Statistical analysis
The Data were analyzed statistically by using Student's t- test. A P
value of < 0.05 was considered statistically significant. Statistical
analyses were carried out with IBM SPSS Statistics v20.0 software.
3. Results
3.1. CDK4/6, and cyclin D1 are highly expressed in cervical cancer
It was demonstrated by western blotting that the human cervical
cancer cell lines exhibited high levels of CDK4, CDK6 and cyclin D1
expression (Fig. 1B). We quantified CDK4, CDK6 and cyclin D1 relative
expressions compared with GAPDH in C33A and HeLa (Fig. 1C).
Moreover, immunofluorescence assay was performed in order to assess
the subcellular localization of CDK4, CDK6 and cyclin D1 in C33A and
HeLa where the red staining represents the CDK4, CDK6 and cyclin D1
protein and the blue staining represents the nucleolus. As shown, CDK4,
CDK6 and cyclin D1 protein are mostly localized in the nucleus of
cervical cancer cells, but with some localized in the cytoplasm (Fig. 1A).
Y. Xiong, et al. Biomedicine & Pharmacotherapy 112 (2019) 108602

Fig. 1. CDK4, CDK6 and Cyclin D1 are highly expressed in cervical cancer.
A. Expression of CDK4, CDK6 and cyclin D1 in C33A and HeLa cells was assessed by immunofluorescence. Cells were imaged on a fluorescence microscope equipped
with a digital camera after incubation with Alexa Fluor 594 (Red) conjugated donkey anti-rabbit antibody or DAPI (blue), the magnification is 200X(scale bar,
100 μm). B. Expression levels of CDK4, CDK6 and cyclin D1 in human cervical cancer cell lines C33A and HeLa were detected by Western blotting. C. Relative
expressions of CDK4, CDK6 and cyclin D1 compared with GAPDH in C33A and HeLa.

3.2. LEE011 inhibits cell proliferation and promotes apoptosis by targeting
the Rb-E2F1 pathway in C33A
Morphologic changes were observed after 2 days of LEE011 ex-
posure. When treated with increased doses of LEE011 (0, 5, 10, 15,
20 μM) for 48 h, C33A cells show pronounced morphologic signs of
toxicity, including abnormal shape and appearance, cellular lysis, and
destruction (Fig. 2A). C33A cells have started to show observable signs
of toxicity in the form of cells reduction and abnormal cells morphology
when treated with 10 μM LEE011, and cellular death becomes more
pronounced when the drug concentration increased up to 20 μM.
However, HeLa cells show little changes in morphology and little cel-
lular death (Fig. 2A). Furthermore, western blotting showed that
treatment of C33A cells with LEE011 significantly reduces the expres-
sion of CDK4,CDK6,E2F1,P-Rb,P16,cyclin E1,CDK2 in a dose-dependent
manner, whereas the expression of Rb remains unchanged (Fig. 2B). A
dose-dependent increase of the expression of the pro-apoptopic protein
Bax and decrease of the expression of the anti-apoptotic protein Bcl-2
were also observed after treating with LEE011 in dose dependent
manner (Fig. 2B).But the expressions of these proteins did not have a
significant change in HeLa cells (Fig. 2B).
3.3. LEE011 induces cell cycle G1 arrest and cell apoptosis in C33A
We assessed the effect of LEE011 on cell cycle and cell apoptosis by
treating C33A and HeLa cells with high concentrations of LEE011. After
the treatment with LEE011 (10 μM),C33 A and HeLa cells were in-
cubated for 48 h. LEE001 induces a significant G1 cell-cycle arrest ac-
companied by a reduction in the fraction of cells in S phase and G2/M
phase in C33 A cells (P < 0.05), compared with the control (Fig. 3A
and B). Moreover, after treatment of C33A cells with 10 mM of LEE011,
G0/G1 portion increased with 19.23%, S portion decreased with 9.68%,

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Y. Xiong, et al. Biomedicine & Pharmacotherapy 112 (2019) 108602

Fig. 2. LEE011 inhibits cell proliferation and promotes apoptosis by targeting the Rb-E2F1 pathway in C33A but not HeLa.
A. C33A and HeLa cells were treated with increasing concentrations of LEE011, and the morphologic changes of cells were observed by microscopy after 48 h of
LEE011 treatment (the magnification is 200X, scale bar, 100 μm).
B. The expression of proteins in Rb-E2F1 pathway and apoptosis in C33A cells were detected by western blotting after 2 days of LEE011 treatment.
C. The expression of proteins in Rb-E2F1 pathway and apoptosis in HeLa cells were detected by western blotting after 2 days of LEE011 treatment.

and G2/M portion decreased with 10.18% were observed. However,
HeLa cells treated with LEE011 did not have a significant result on cell
cycle arrest (Fig. 3C and D).
Otherwise, LEE011-induced apoptosis was measured by AnnexinV-
FITC/PI Apoptosis Kit and flow cytometric analysis (Fig. 4). C33A cells
were cultured on 10 μM of LEE011 for 48 h. LEE011 treatment led to
apoptosis 2.24 times (31.7%) more than control (14.13%) in C33A
(P < 0.05) (Fig. 4A and B), while LEE011 treatment did not sig-
nificantly increase the apoptotic index compared with control, in HeLa
cells (Fig. 4C and D).
These results indicate that cell treatment with LEE011 could only
induce cell cycle and cell apoptosis in C33 A with no significant influ-
ence on HeLa cell lines cycle and apoptosis process.
3.4. LEE011 effectively suppresses in vivo tumor growth in xenograft models
For performing the in vivo test of the effectiveness of LEE011, we
conducted the C33A and HeLa xenografts. The xenograft models were
treated with 200 mg/kg of LEE011 in 0.5% methylcellulose or vehicle
daily during a total of 21 days.LEE011 effectively hindered tumor

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Y. Xiong, et al.
Fig. 3. LEE011 induces cell cycle G1 arrest in C33 A but not HeLa.
A.B. After treatment of C33A cell lines with LEE011 (10 μM) during 48 h, G0/G1 portion increased with 19.23% (P < 0.05), S portion decreased with 9.68%
(P < 0.05), and G2/M portion decreased with10.18% (P < 0.05). C.D. After treatment of HeLa cell lines with LEE011 (10 μM) during 48 h, there are no significant
changes in the cell cycle process.
growth in C33A xenograft models. The result shown that there are the
reduction of kinetic of the tumor volume growth in C33A xenografts
treated with LEE011 compared with the untreated control group,
(Fig. 5A–C). Moreover, LEE011 had no significant effect on body weight
in C33 A xenografts (Fig. 5D). For HeLa xenografts, LEE011 did not
induce suppression of tumor growth (Fig. 5A–C), but caused the mice
lost weight (Fig. 5D).In another hand, we confirmed the suppression of
the CDK4/6-cyclin D-Rb-E2F1 pathway by immunohistochemical
staining for CDK4,CDK6,cyclin D1, Rb and assessed the anti-pro-
liferative effect by the proliferation marker Ki-67 immunostaining.
Furthermore, we also performed TUNEL assay to detect apoptotic cells
on the xenografts (Fig. 5E and F).We found that the expression of
CDK4,CDK6,cyclin D1 and Rb were reduced, and the proportion of
apoptotic cells was increased in LEE011 treatment group compared
with the control group in C33 A xenografts. However, we did not found
the significant effects of LEE011 on the HeLa xenografts.
Furthermore, to evaluate the safety of LEE011 on the xenograft
models, we detected different biochemical indicators (ALT, AST, ALB,
ALP, BUN, CR, LDH-L, CK) as a heart, liver and kidney enzymatic
markers by using relative different kits (Fig. 5I) follow by the ob-
servation of the morphologic of heart, liver and kidney (Fig. 5G,H).No
obvious change of the morphologic and enzymatic marker of liver,
heart and kidney were found, suggesting that the use of LEE011 for
treatment did not have obvious adverse effects on xenograft mice
models.
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Biomedicine & Pharmacotherapy 112 (2019) 108602
4. Discussion
With recent development and FDA approval of highly specific
CDK4/6 inhibitors (Palbociclib, Ribociclib and Abemaciclib) for ad-
vanced metastatic breast cancer, there has been more interest in clinical
trials with these drugs in various tumors (https://clinicaltrials.gov/
).But we have not found the clinical trials concerning the effect of
Ribociclib on cervical cancer yet. In order to find and confirm that the
therapies with CDK4/6 inhibitors may be beneficial for cervical cancer
treatment, we investigated the effects of Ribociclib, as one of the target
therapies on cervical cancer. We found that the human cervical cancer
cell lines exhibited high levels of CDK4, CDK6 and cyclin D1 expression
by western blotting, and the proteins were found to be localized in the
nucleus by immunofluorescence staining. Moreover, it has been found
that cyclin D1 accumulates in the nucleus and triggers the active cyclin
D1-CDK4 complex formation in proliferating cells during G1 phase
[30]. Another study has analyzed the expression of cyclin D1 and CDK4
gene in 120 cervical carcinoma tissue samples and demonstrated that
the patients with overexpression of cyclin D1 and CDK4 gene had a
poor prognosis [15].This suggests that cervical cancer is Rb-E2F
pathway-activated cancer and may be susceptible to Ribociclib.
The treatment of C33A with 10 μM of Ribociclib for 48 h sig-
nificantly reduced the expression of the CDK4, CDK6, E2F1, P-Rb, P16,
cyclin E1 and CDK2, whereas the expression of Rb remains unchanged.
These data suggest that Ribociclib inhibited the formation of CDK4/6-
Y. Xiong, et al. Biomedicine & Pharmacotherapy 112 (2019) 108602

Fig. 4. LEE011 induces cell apoptosis in C33A but not HeLa.

A.B. After treatment of C33A cell lines with LEE011 (10 μM) during 48 h, the apoptotic index increased 2.24 times more (31.7%) than control (14.13%) in C33A
(P < 0.05). Alive cells are shown in the lower left part of the panel (Q3); Early apoptotic cells are shown in the lower right part of the panel (Q4); Late apoptotic cells
are shown in the higher right part of the panel (Q2); Necrotic cells are shown in Q1. Apoptotic cells were exhibited as annexin V + cells. C.D. In HeLa cells, treatment
with 10 μM of LEE011 resulted in apoptosis augmentation with no significant changes compared with control. Alive cells are shown in the lower left part of the panel
(Q8); Early apoptotic cells are shown in the lower right part of the panel (Q7); Late apoptotic cells are shown in the higher right part of the panel (Q6); Necrotic cells
are shown in Q5. Apoptotic cells were exhibited as annexin V + cells.

cyclin D complexes, CDK2-cyclin E complexes and phosphorylation of
Rb. Ribociclib was supposed to inactivate the transcription factor E2F1
by Rb which lead to G1 cell cycle arrest, and hold back cell prolifera-
tion. Our study consistently showed that Ribociclib can suppress
phosphorylation of Rb and also causes degradation of CDK4/6 and
E2F1. This finding correlated with the report of Lee et al. where it found
in thyroid cancer cell lines that Ribociclib decreased Rb after its de-
phosphorylation and E2F expression via E2F target gene FOXMI, Cyclin
A1, Myc [26]. Moreover, Ribociclib changes the CDK4/6-cyclin D-Rb-
E2F1 pathway and CDK2-cyclin E-Rb-E2F1 pathway which control G1-S
phase cell cycle progression. Moreover, CDK4/6-cyclin D complexes
and CDK2-cyclin E complexes are responsible for successively phos-
phorylating Rb, thereby alleviating its inhibition on E2F1 and allowing
the activation of genes necessary for promoting S phase entry and DNA
synthesis [31]. It seems that the anti-tumor effects of Ribociclib were
attributed to the restoration of the antimitogenic function of Rb which
inactivation is one of the most frequent and early recognized molecular
hallmarks of cancer.
Furthermore, it has reported that Rb-E2F pathway is implicated in
cancer cell apoptosis [32]. Bax and Bcl-2 are two members of Bcl-2
family and are known to control apoptosis [33]. Under dose-dependent
treatment, Ribociclib increased the expression of Bax, and decreased
the expression of Bcl-2. Suggesting that Ribociclib could influence
cervical cancer cell lines apoptosis. This finding confirmed the finding
of Lee et al. mentioned that thyroid cancer cell lines treated with Ri-
bociclib lead to apoptosis 2.8 times more (4.2%) than control (1.5%)
[26]. In another hand, by performing flow cytometry analysis to assess
the cell cycle and apoptosis in C33A treated with Ribociclib. In order to
confirm apoptosis capacity of Ribociclib assessed by Bax and Bcl2 ex-
pression level and confirm cell cycle as a mechanism of anti-pro-
liferative effect of Ribociclib on cervical cancer cell, we found that
Ribociclib induced cell apoptosis and cell cycle arrest as it has been
reported by Lee et al. in human thyroid cancer cells [26]. However,
there are no significant effects of Ribociclib on Hela cancer cell line. We
also found that Ribociclib inhibited cell proliferation at in-vitro and in-
vivo level in C33A cells, with no significant anti-tumor effect in HeLa
cells at in vitro and in vivo level which suggested that the effect of Ri-
bociclib in cervical cancer cell line could be tumor-type dependent ef-
fect or drug concentration of treatment dependent effect. We suppose
that the difference results between the two treated cancer cell lines
could be due to the different genetic backgrounds of these two cell
lines.C33 A is an HPV-negative cell line and HeLa is an HPV-positive
cell line. The expression of human papillomavirus(HPV) oncoprotein
has changed the regulatory mechanism of the cell cycle. Certain human

7
Y. Xiong, et al. Biomedicine & Pharmacotherapy 112 (2019) 108602

Fig. 5. The effects of LEE011 treatment on tumor growth in vivo.

A. After euthanasia, tumors were extracted from both of control group and LEE011 treated group. The sizes of tumors extracted from LEE011 group were smaller than
the control group in C33A xenograft models. However, the sizes of tumors in the control group and LEE011 treated group had no difference in HeLa Xenografts. B–C.
Treated with 200 mg/kg, LEE011 significantly inhibited tumor growth in C33A xenograft models (P < 0.05). Tumor growth had no change in HeLa xenograft
models.D.LEE011 had no effect on body weight in C33A xenografts, but caused the HeLa models lost weight. E. The antiproliferative effect of LEE011 was confirmed
by Ki- 67 staining in C33A xenografts, and suppression of the RB-E2F1 pathway was confirmed by immunohistochemical staining for CDK4, CDK6, cyclin D1 and Rb.
LEE011-induced apoptosis was detected by using TUNEL assay (scale bar, 100 μm). F. The index of the antiproliferative effect, the RB-E2F1 pathway and apoptosis in
HeLa xenografts did not show obvious differences. G.H.I. The biochemical indicators (ALT, AST, ALB, ALP, BUN, CR, LDH-L, CK) were detected from blood samples
and the morphologic of heart, liver and kidney were studied and showed that there were no serious adverse effects in xenograft models treated with LEE011.

8
Y. Xiong, et al. Biomedicine & Pharmacotherapy 112 (2019) 108602

Fig. 5. (continued)

9
Y. Xiong, et al.
Fig. 5. (continued)
cancers caused by HPV infection highly expressed p16 which is one of
tumor suppressor gene in human cancers and inhibited the activity of
CDK4 and activated Rb which led to cell cycle arrest [34,36]. Specific
somatic loss of p16 through mutations or deletions, and silencing of p16
through its promoter methylation, have been reported at high fre-
quency in many human cancers [35]. The biological activity of p16 in
HPV-associated with cancers is more likely an oncogene and is depen-
dent on its ability to inhibit CDK4/6 activity which is important for the
survival of cervical carcinoma cell lines containing HPV [37]. It has
been found that the viral proteins control the activity of E2F tran-
scription factors which lead to the inactivation of Rb [38]. Our result
which reported that LEE011 have no significant effect on HeLa cells
supports the report of Jirawatnotai et al. where it has been proved that
depletion of cyclin D1 did not affect the cell cycle and proliferation of
HeLa cells in vitro and had no impact on the rate of tumour growth in
vivo [39]. Interestingly, the use of Ribociclib has been well tolerated in
vivo because, apart from the weight loss in mice, there are no changes in
the liver, heart and kidney enzymes after the blood analysis. This
finding is not in correlation with the report of Hortobagyi et al. where
the grade 3 or 4 elevations level of alanine and aspartate amino-
transferase, as the liver enzymes were respectively reported in 9.3%
10
Biomedicine & Pharmacotherapy 112 (2019) 108602
and 5.7% in the patient treated with Ribociclib [21].
In sum, Ribociclib can be considered as a potential treatment
strategy for the improvement of new therapeutic measures for the
treatment of HPV-negative cervical cancer. Ribociclib has an anti-pro-
liferative effect on the certain type of cervical cancer cell lines by tar-
geting cell cycle and cell apoptosis. Ribociclib is also well tolerated in
the mice suggesting that the use of Ribociclib as a therapy for patients
with cervical cancer is encouraged in future. However, more study
needs to be carried out to know more about the adverse effect of
Ribociclib in cervical cancer patients and to find out the effect of
Ribociclib on different kinds of human cervical cancer cell line espe-
cially the HeLa cancer cell line.
Authors’ contributions
F.X.Z. and Y.F.Z designed experiments. Y.D.X. performed major
experiments and analyzed data. T.Q.L. assisted with animal experi-
ments. H.L and Q.Z. assisted with immunofluorescence assay. Y.Y.Z.
assisted with western blotting. Y.D.X. wrote the manuscript. G.A. was
involved in the writing and in the reading of the manuscript.
Y. Xiong, et al. Biomedicine & Pharmacotherapy 112 (2019) 108602

Fig. 5. (continued)

Conflict of interests Biological Behaviors for helping to process the study.

The authors declare no conflict of interest. Appendix A. Supplementary data

Supplementary material related to this article can be found, in the

Acknowledgements online version, at doi:https://doi.org/10.1016/j.biopha.2019.108602.

This research was funded by National Natural Science Foundation of References

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