Int J Clin Exp Med 2016;9(6):10544-10550www.ijcem.com /ISSN:1940-5901/IJCEM0022871

Original ArticleExpression of CDC6 in ovarian cancer and its effect on proliferation of ovarian cancer cells

Ting-Yi Sun, Hong-Jian Xie, Zhen Li, Hui He, Ling-Fei Kong

Department of Pathology, Henan Provincial People’s Hospital, Zhengzhou, China

Received December 28, 2015; Accepted March 22, 2016; Epub June 15, 2016; Published June 30, 2016

Abstract: Cdc6 is an important component of pre-RC and plays an important role in DNA replication and the regula-tion process of mitosis. Our study found that the protein expression of Cdc6 was significantly higher (P < 0.05) in ovarian cancer tissues compared with normal tissues, which was associated with the differentiation degree and clinical stage of patients (P < 0.05). Cdc6 siRNA was used to inhibit the expression of Cdc6 in ovarian cancer cell line SKOV3. Results showed that after transfection of siRNA, protein expression of Cdc6 was significantly lower (P < 0.05) than in the blank group and negative control (NC) group. Cell Counting Kit-8 (CCK8), colony formation assay and flow cytometry assay were used to test the biology effect of ovarian cancer cells after the inhibition of Cdc6 expression. Results showed that after transfection of siRNA, proliferation of SKOV3 cells decreased while apoptosis rates increased, suggesting that Cdc6 acted as an oncogene in the occurrence and development process of ovar-ian cancer. Inhibiting the expression of Cdc6 reduced proliferation and increased apoptosis of ovarian cancer cells. Therefore, Cdc6 may serve as a potential target in the diagnosis and treatment of ovarian cancer.

Keywords: Ovarian cancer, Cdc6, proliferation, siRNA

Introduction

Ovarian cancer is one of the malignant female genital cancers, the incidence and mortality rate of which increased gradually in recent years [1]. Due to the lack of typical symptoms, effective early diagnosis and poor prognosis, the clinical stage is usually late when patients are diagnosed with ovarian cancer [2], in addi-tion, 5-year survival rate of which was only about 30% [3]. There is no suitable serum tumor marker with high sensitivity and specific-ity for screening for ovarian cancer by now [4], thus looking for typical biological markers for early diagnosis and evaluation of prognosis of ovarian cancer is very important.

Duplication of eukaryotic cells requires the binding of DNA origin with a plurality of related regulatory proteins according to characteristics order. These regulatory proteins are also known as pre-replication complexes, pre-RC [5]. Pre-RC includes Origin Recognition Complexes (ORCs), Cell division cycle protein (Cdc6), Cdcl0 dependent transcript l (Cdtl) and Minichro- mosome maintenance proteins (Mcms), etc.

After the sequentially assembling of pre-RC in the replication starting position of eukaryotic cells, cells acquire the ability to replicate [6, 7].

Cdc6 is an important component of pre-RC and plays an important role in the regulation of DNA replication process [8]. Cdc6 is also involved in regulatory process of cell mitosis, which pre-vents cells from entering the process of mitosis by promoting ATR signal [9, 10]. Besides, in mitosis anaphase, it can also inactivate CDK and promote mitotic exit by inhibiting CDK1-Clb2 pathway [11, 12]. Abnormal expression of Cdc6 can lead to malignant cell proliferation. Studies demonstrated that the expression level of Cdc6 was high in cervical cancer [13, 14], lung cancer [15, 16], oral squamous cell carci-noma [17] and prostate cancer [18], etc., which was related to the malignancy degree of tumor tissue. Currently, the expression level of Cdc6 in ovarian cancer is still unclear. In our study, the expression level of Cdc6 in ovarian cancer was analyzed, so was the effect on proliferation and apoptosis of ovarian cancer cells after inhibiting Cdc6 expression, to provide experi-mental evidence and theoretical basis for

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exploring new diagnostic biological markers and molecular targeted therapy sites of ovarian cancer.

Materials and methods

Clinical samples

46 cases of ovarian cancer and 13 cases of normal ovarian tissue samples were collected via surgical resection in The People’s Hospital of Henan Province from September 2013 to September 2014. All samples were confirmed by pathological diagnosis without chemothera-py, radiotherapy or immunotherapy. Of ovarian cancer cases, there were 11 well-differentiated cases, 16 moderately-differentiated cases and 19 poorly-differentiated cases; there were 6 cases in Phase I, 8 cases in Phase II, 17 cases in Phase III and 15 cases in Phase IV according to the Federation International of Gynecology and Obstetrics (FIGO) ovarian cancer clinical staging criteria [19]; there were 31 cases of

serous adenocarcinoma and 15 cases of muci-nous adenocarcinoma; there were 19 cases of < 50-year-old and 27 cases of ≥ 50-year-old. All patients signed informed consent. The study was approved by the Ethics Committee.

Cell lines and cell culture

Ovarian cancer cell line SKOV3 was purchased from cell bank Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI 1640 cul-ture medium containing 10% fetal bovine serum (FBS; Gibco BRL, Gaithersburg, MD, USA), 1000 U/ml penicillin and 100 mg/ml streptavidin Pigment, and placed in an incuba-tor thermostat at 37°C with 5% CO2.

Immunohistochemistry

3 μm sections were cut after ovarian cancer specimens fixed in 4% formaldehyde and paraf-fin-embedded. Cdc6 antibody (Abcam) was

Figure 1. Immunohistochemistry was used to detect Cdc6 in ovarian cancer tissues. Cdc6 positive cells expressed brown staining or granule in the nucleus or cytoplasm of ovarian cancer tissues. CDC6 positive cells existed in se-rous adenocarcinoma (A, × 100 and B, × 200 EnVision); CDC6 positive cells existed in mucinous adenocarcinoma (C, × 100 and D, × 200 EnVision).

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used as the primary antibody for staining according to the immunohistochemistry kit operating specifications (DAKO company, En-

dase-labeled IgG, Abcam) diluted at 1:1000. Chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ) was used

Table 1. Relationship between Cdc6 expression in ovarian cancer and clinicopathological characteristics

Clinicopathological characteristics n

Cdc6 expression x2 P value

+ -Age (years) < 50 19 11 8 0.356 0.544 ≥ 50 27 18 9Tissue Type Serous adenocarcinoma 31 20 11 0.088 0.766 Mucinous adenocarcinoma 15 9 6Differentiation Degree Well-differentiated 11 4 7 0.026*

Moderately-differentiated 16 9 7 7.331 Poorly-differentiated 19 16 3Clinical stage I-II 14 5 9 6.451 0.011*

III-IV 32 24 8Lymph node metastasis Without 18 10 8 0.712 0.399 With 28 19 9Notes: *Represents that there are statistical differences between the two groups (P < 0.05).

Figure 2. Protein expression of Cdc6 was inhibited in siRNA trans-fected SKOV3 cells. Cdc6 protein in SiRNA1 and siRNA2 transfect-ed SKOV3 cells was detected using western blot. Results showed protein levels of Cdc6 were significantly lower with siRNA transfec-tion compared with NC group and blank group. *Represents that there are statistical differences (P < 0.05).

Vision K5007). Instead of primary anti-body, PBS was used as negative con-trol. Cells expressed brown staining or granules in the nucleus or cytoplasm were considered Cdc6 positive cells.

siRNA synthesis and transfection as-says

According to the Cdc6 sequence (NM_ 001025779) retrieved from GeneBank, the target sequence siRNA1: 5’-C- ACTCTCCGAATGTAAATC-3’, siRNA2: 5’- GGAGAGCTATTGAAATTGT-3’ of Cdc6 were designed using online siRNA design software of Angela siRNA com-pany; in addition, a negative control (NC) sequence: 5’-GTTCTCCGAACGTGT- CACGT-3’ was designed since no homologous sequences was found in NCBI BLAST website. The designed oli-gonucleotides were sent to synthesis in Shanghai GenePharma Co. Ltd.

siRNA and lipid complexes were formu-lated according to LipofectamineTM 2000 (Invitrogen) kit instructions and were transfected into SKOV3 cells. This study consisted of four groups: trans-fection of PBS as blank group, trans-fection of nonsense sequence as nega-tive control (NC) group, transfection of Cdc6 siRNA1 as siRNA1 group, trans-fection of Cdc6siRNA2 as siRNA2 group.

Western blotting

Protein samples were collected from cells after transfection for 48 h, bicin-choninic acid (BCA) methods was used to measure the quantitation. After pre-pared in sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellu-lose membrane, primary antibody (Cdc6 antibody, Abcam) diluted at 1:500 was used to incubate at 4°C overnight. Membranes were then washed with Tris-Buffered Saline and Tween 20 (TBST) three times, 15 min each time, and incubated with the sec-ondary antibody (horseradish peroxi-

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to detect the signal. β-actin (Santa Cruz) was used as internal reference to calculate the rela-tive expression levels of protein samples.

CCK8 assay

Cell viability was detected according to CCK-8 kit specification (Dojindo Laboratories, Japan). Cells in logarithmic growth phase were seeded in 96-well plates at a concentration of 2 × 103 cells per well. Each group contained triplicate wells. 10 μL CCK-8 solution was added at 24 h, 48 h, 72 h and 96 h respectively to detect cell activity. OD values were measured at a wave-length of 450 nm, which represented for the corresponding numbers of viable cells.

Clone formation assay

RPMI 1640 medium containing 0.6% agarose gel and 10% FBS was added to 6-well plates for solidification at room temperature. Cells in log-arithmic growth phase after transfection were suspended in RPMI 1640 medium containing 0.3% low melting point agarose gel and 10% FBS, was then added to the solidified lower gel of 6-well plates, cultured at 37°C with 5% CO2 for 12 days. Cell clones were calculated after crystal violet staining. One monoclonal commu-nity should contain more than 50 cells. Cloning efficiency = numbers of formed clones/inocu-

lated cells × 100%. The assay was repeated 3 times and average was calculated.

Apoptosis detection

FITC Annexin V apoptosis detection kit (BestBio, Shanghai, China) was used to detect apoptosis. Cells in different groups were digested into sin-gle cell suspension in accordance with the instructions. After centrifugation and resuspen-sion, 1 × 106 cells/ml cell solution was pre-pared. Propidium iodide (PI) and FITC Annexin V were added. Flow cytometry was used to detected cell apoptosis. Detection completed in 30 min.

Statistical analyses

SPSS19.0 statistical software package was used for data analysis. Results of immuno- histochemistry were calculated using χ2 test. Results of western blotting, CCK8, colony for-mation assay and apoptosis experiment were analyzed using ANOVA. P < 0.05 was statisti-cally significant.

Results

Cdc6 expression in ovarian cancer

Cdc6 expression in ovarian cancer was detect-ed using immunohistochemistry (Figure 1). Cells expressed brown staining or granules in the nucleus or cytoplasm were considered Cdc6 positive cells. In 46 cases of ovarian car-cinoma, 29 cases expressed Cdc6 (63.0%), while all 13 cases of normal ovarian tissues showed no Cdc6 staining (P < 0.05).

Analysis of Cdc6 expression and pathological factors in patients with ovarian cancer tissues indicated that in 46 cases of ovarian cancer samples, Cdc6 expression was associated with tissue differentiation degree and clinical stage (P < 0.05), but not related to age, pathological type or lymph node metastasis (P > 0.05) (Table 1).

Effect of Cdc6 siRNA on Cdc6 expression in ovarian cancer cells

Protein expression of Cdc6 in Cdc6 siRNA transfected SKOV3 cells was detected using western blot with β-Actin used as control. Results showed that levels of Cdc6 protein expression decreased in siRNA1 and siRNA2

Figure 3. siRNA was used to silence Cdc6 expression and inhibit proliferation of SKOV3 cells. CCK-8 was used to detect proliferation of SKOV3 cells in siRNA1 group, siRNA2 group, NC group and blank group. Results showed that after siRNA transfection for 3 days, proliferation rates were significantly reduced compared with NC group and blank group (P < 0.05). *Represents that there are statistical differences (P < 0.05).

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group, while there was no significant change in NC group. Results of gray scale analysis showed that levels of protein expression in siRNA1 and siRNA2 group were significantly lower com-pared with blank group and NC group (P < 0.05) (Figure 2).

Effects of Cdc6 expression silencing on prolif-eration of ovarian cancer cells

CCK-8 and clone formation assay were used to detect the change of proliferation ability of ovarian cancer cell line SKOV3 with Cdc6 expression silencing. Results of CCK-8 showed that after three days of siRNA1 and siRNA2 transfection, OD values of that were significant-ly lower compared with the NC and blank con-trol (P < 0.05), as seen in Figure 3. Results of clone formation assay showed that numbers of colony formation in siRNA-transfected group significantly reduced (P < 0.05), as seen in Figure 4. These results indicated that Cdc6 expression silencing inhibited the proliferation of ovarian cancer cell line SKOV3.

Effects of Cdc6 expression silencing on apop-tosis of ovarian cancer cells

AnncxinV-FITC/PI double-labeled flow cytome-try was used to analysis changes of apoptosis rate in Cdc6 expression silencing ovarian can-cer cell line SKOV3. Results showed that com-pared with the NC group and blank group, apop-tosis rates in siRNA-transfected cells we- re significantly increased (P < 0.05), as seen in Figure 5. This indicated that Cdc6 expression silencing promoted apoptosis of ovarian cancer cell line SKOV3.

phage, and the research of which had become a hotspot in recent years.

Currently, research about Cdc6 expression in ovarian cancer has not been reported. Our study detected Cdc6 expression in ovarian can-cer tissues and normal ovarian tissues using immunohistochemistry. Results showed that expression level of Cdc6 in ovarian cancer tis-sues was significantly higher than in normal tis-sue, meanwhile the expression level of which was associated with differentiation degree and clinical stage. These results suggested that Cdc6 functioned as an oncogene, played an important role in the development process of ovarian cancer.

siRNA is a new technology in studying gene function and specific gene therapy, which effec-tively and specifically inhibits the expression of targeted gene in cells [24]. To further study the biological characteristic effect of Cdc6 on ovar-ian cancer cells, Cdc6-targeted siRNA was designed, transfected into SKOV3 ovarian can-cer cells by liposome, resulting in successful Cdc6 gene silencing.

Results of CCK8 and cloning formation assay showed that after Cdc6 expression silencing, proliferation ability was significantly lower than in the control group and blank group. Apoptosis rates detected using flow cytometry in ovarian cancer cells after silencing the expression of Cdc6 were significant increased. Cdc6 involving in pre-RC assembly and playing a regulatory role in mitosis were considered to be the pos-sible mechanism. Studies had shown that in addition to the regulation of cells entering S

Figure 4. siRNA was used to silence Cdc6 expression and inhibit colony forma-tion of SKOV3 cells. Colony formation was significantly lower in siRNA1 and siRNA2 transfected SKOV3 cells (P < 0.05). *Represents that there are statisti-cal differences (P < 0.05).

Discussion

Cdc6 located in human chromosome 17q21.3 [20], was considered to be an important component of pre-RC, played an important role in the regulation of DNA replication [21]. Previous studies found that by micro-injection of protein Cdc6 into cells in G1 phase, cells were prevented from enter-ing S phase [22, 23]. Thus, Cdc6 was considered one of the key regulators of cells entering S phase from G1

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phase, Cdc6 was still bind to chromatin when cells in S phage. Cdc6, as a critical protein of S-G2/M checkpoint, coordinated the process of S phase to M phase [25-27]. Previous stud-ies had proved that high expression of Cdc6 could induce DNA synthesis, while low expres-sion of which lead to chromosome loss and anomaly of progeny cells [28]. Other studies demonstrated that Cdc6, as substrates of Caspase-3, the degradation of which inhibited the replication of DNA and induced apoptosis [29].

Overall, Cdc6 gene acts as an oncogene in the development and progression of ovarian can-cer, therefore can be a useful target for diagno-sis of ovarian cancer, which is also likely to become a candidate target gene in ovarian can-cer therapy, since reducing the expression of Cdc6 can promote apoptosis and inhibit prolif-eration, invasion and metastasis. Currently, regulation of Cdc6 expression and mechanism how Cdc6 functions in development and pro-gression of tumor are not clear, therefore require further study.

Disclosure of conflict of interest

None.

Address correspondence to: Ting-Yi Sun, Depart- ment of Pathology, Henan Provincial People’s Hos- pital, Zhengzhou, China. Tel: +86(0371) 65897519; E-mail: docsun2041@163.com

References

[1] Siegel RL, Miller KD and Jemal A. Cancer sta-tistics, 2015. CA Cancer J Clin 2015; 65: 5-29.

[2] Barnett B, Kryczek I, Cheng P, Zou W and Curiel TJ. Regulatory T cells in ovarian cancer: biology and therapeutic potential. Am J Reprod Immunol 2005; 54: 369-377.

[3] Baldwin LA, Huang B, Miller RW, Tucker T, Goodrich ST, Podzielinski I, DeSimone CP, Ueland FR, van Nagell JR and Seamon LG. Ten-year relative survival for epithelial ovarian can-cer. Obstet Gynecol 2012; 120: 612-618.

[4] Etzioni R, Urban N, Ramsey S, McIntosh M, Schwartz S, Reid B, Radich J, Anderson G and Hartwell L. The case for early detection. Nat Rev Cancer 2003; 3: 243-252.

[5] Fragkos M, Ganier O, Coulombe P and Mechali M. DNA replication origin activation in space and time. Nat Rev Mol Cell Biol 2015; 16: 360-374.

[6] Riera A, Fernandez-Cid A and Speck C. The ORC/Cdc6/MCM2-7 complex, a new power player for regulated helicase loading. Cell Cycle 2013; 12: 2155-2156.

[7] Wu L, Liu Y and Kong D. Mechanism of chro-mosomal DNA replication initiation and repli-

Figure 5. siRNA was used to silence Cdc6 expression and promote apoptosis of SKOV3 cells. Flow cytometry was used to detected apoptosis rates in siRNA1 group, siRNA2 group, NC group and Blank group. Results showed apoptosis rates were significantly increased in siRNA-transfected cells. *Rep-resents that there are statistical differences (P < 0.05).

CDC6 and its effect in ovarian cancer

10550 Int J Clin Exp Med 2016;9(6):10544-10550

cation fork stabilization in eukaryotes. Sci China Life Sci 2014; 57: 482-487.

[8] Borlado LR and Mendez J. CDC6: from DNA replication to cell cycle checkpoints and onco-genesis. Carcinogenesis 2008; 29: 237-243.

[9] Chen S, Wan P, Ding W, Li F, He C, Chen P, Li H, Hu Z, Tan W and Li J. Norcantharidin inhibits DNA replication and induces mitotic catastro-phe by degrading initiation protein Cdc6. Int J Mol Med 2013; 32: 43-50.

[10] Yoshida K, Sugimoto N, Iwahori S, Yugawa T, Narisawa-Saito M, Kiyono T and Fujita M. CDC6 interaction with ATR regulates activation of a replication checkpoint in higher eukaryotic cells. J Cell Sci 2010; 123: 225-235.

[11] Daldello EM, Le T, Poulhe R, Jessus C, Haccard O and Dupre A. Control of Cdc6 accumulation by Cdk1 and MAPK is essential for completion of oocyte meiotic divisions in Xenopus. J Cell Sci 2015; 128: 2482-2496.

[12] Yim H and Erikson RL. Regulation of the final stage of mitosis by components of the pre-rep-licative complex and a polo kinase. Cell Cycle 2011; 10: 1374-1377.

[13] Xiong XD, Zeng LQ, Xiong QY, Lu SX, Zhang ZZ, Luo XP and Liu XG. Association between the CDC6 G1321A polymorphism and the risk of cervical cancer. Int J Gynecol Cancer 2010; 20: 856-861.

[14] Martin CM, Astbury K, McEvoy L, O’Toole S, Sheils O and O’Leary JJ. Gene expression pro-filing in cervical cancer: identification of novel markers for disease diagnosis and therapy. Methods Mol Biol 2009; 511: 333-359.

[15] Karakaidos P, Taraviras S, Vassiliou LV, Zacharatos P, Kastrinakis NG, Kougiou D, Kouloukoussa M, Nishitani H, Papavassiliou AG, Lygerou Z and Gorgoulis VG. Overexpression of the replication licensing regulators hCdt1 and hCdc6 characterizes a subset of non-small-cell lung carcinomas: synergistic effect with mutant p53 on tumor growth and chromo-somal instability--evidence of E2F-1 transcrip-tional control over hCdt1. Am J Pathol 2004; 165: 1351-1365.

[16] Zhang X, Xiao D, Wang Z, Zou Y, Huang L, Lin W, Deng Q, Pan H, Zhou J, Liang C and He J. MicroRNA-26a/b regulate DNA replication li-censing, tumorigenesis, and prognosis by tar-geting CDC6 in lung cancer. Mol Cancer Res 2014; 12: 1535-1546.

[17] Feng CJ, Li HJ, Li JN, Lu YJ and Liao GQ. Expression of Mcm7 and Cdc6 in oral squa-mous cell carcinoma and precancerous le-sions. Anticancer Res 2008; 28: 3763-3769.

[18] Liu Y, Gong Z, Sun L and Li X. FOXM1 and an-drogen receptor co-regulate CDC6 gene tran-scription and DNA replication in prostate can-cer cells. Biochim Biophys Acta 2014; 1839: 297-305.

[19] Kandukuri SR and Rao J. FIGO 2013 staging system for ovarian cancer: what is new in com-parison to the 1988 staging system? Curr Opin Obstet Gynecol 2015; 27: 48-52.

[20] Williams RS, Shohet RV and Stillman B. A hu-man protein related to yeast Cdc6p. Proc Natl Acad Sci U S A 1997; 94: 142-147.

[21] Niimi S, Arakawa-Takeuchi S, Uranbileg B, Park JH, Jinno S and Okayama H. Cdc6 protein ob-structs apoptosome assembly and consequent cell death by forming stable complexes with activated Apaf-1 molecules. J Biol Chem 2015; 290: 4130.

[22] Hateboer G, Wobst A, Petersen BO, Le Cam L, Vigo E, Sardet C and Helin K. Cell cycle-regulat-ed expression of mammalian CDC6 is depen-dent on E2F. Mol Cell Biol 1998; 18: 6679-6697.

[23] Yan Z, DeGregori J, Shohet R, Leone G, Stillman B, Nevins JR and Williams RS. Cdc6 is regulat-ed by E2F and is essential for DNA replication in mammalian cells. Proc Natl Acad Sci U S A 1998; 95: 3603-3608.

[24] Sarett SM, Nelson CE and Duvall CL. Technologies for controlled, local delivery of siRNA. J Control Release 2015; 218: 94-113.

[25] Kim GS, Kang J, Bang SW and Hwang DS. Cdc6 localizes to S- and G2-phase centrosomes in a cell cycle-dependent manner. Biochem Bio- phys Res Commun 2015; 456: 763-767.

[26] Coverley D, Pelizon C, Trewick S and Laskey RA. Chromatin-bound Cdc6 persists in S and G2 phases in human cells, while soluble Cdc6 is destroyed in a cyclin A-cdk2 dependent pro-cess. J Cell Sci 2000; 113: 1929-1938.

[27] Illenye S and Heintz NH. Functional analysis of bacterial artificial chromosomes in mammali-an cells: mouse Cdc6 is associated with the mitotic spindle apparatus. Genomics 2004; 83: 66-75.

[28] Kundu LR, Kumata Y, Kakusho N, Watanabe S, Furukohri A, Waga S, Seki M, Masai H, Enomoto T and Tada S. Deregulated Cdc6 inhibits DNA replication and suppresses Cdc7-mediated phosphorylation of Mcm2-7 complex. Nucleic Acids Res 2010; 38: 5409-5418.

[29] Yim H, Jin YH, Park BD, Choi HJ and Lee SK. Caspase-3-mediated cleavage of Cdc6 induc-es nuclear localization of p49-truncated Cdc6 and apoptosis. Mol Biol Cell 2003; 14: 4250-4259.