Effect of Ku80 on the radiosensitization of cisplatin in the cervical carcinoma cell line HeLa

Zhuang,Liang; Liu,Fei; Peng,Ping; Xiong,Huihua; Qiu,Hong; Fu,Xiugen; Xiao,Zhiping; Huang,Xiaoyuan

doi:10.3892/ol.2017.7304

Effect of Ku80 on the radiosensitization of cisplatin in the cervical carcinoma cell line HeLa

Authors:
- Liang Zhuang
- Fei Liu
- Ping Peng
- Huihua Xiong
- Hong Qiu
- Xiugen Fu
- Zhiping Xiao
- Xiaoyuan Huang
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Affiliations: Cancer Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China, Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China
Published online on: October 31, 2017 https://doi.org/10.3892/ol.2017.7304
Pages: 147-154
Copyright: © Zhuang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Cisplatin chemotherapy in combination with radiotherapy is the primary therapeutic strategy for the treatment of cervical cancer; however, the underlying molecular mechanism for cisplatin radiosensitization remains unknown. The aim of the present study was to investigate the effect of Ku80, a DNA double‑strand break (DSB) repair protein, on cisplatin radiosensitization in cervical cancer. The pre‑established Ku80 suppression cervical cancer cell line HeLa/Ku80‑siRNA and the normal HeLa cell line underwent 6 MV X‑ray irradiation (6 Gy) individually or in combination with 5 µg/ml cisplatin treatment. Alterations in apoptosis, the cell cycle and γH2AX expression were detected. Following irradiation individually and combined with cisplatin, compared with normal HeLa cells, HeLa/Ku80-siRNAexhibited an increased rate of apoptosis (P<0.05). It was identified that the earlier cisplatin was administered following irradiation, the higher the rate of apoptosis. Cell cycle analysis indicated that, following irradiation combined with cisplatin, the cells were arrested in G1 and S phase rather than in G2/M phase following irradiation alone. Microscopic imaging of immunofluorescence staining and western blotting identified that HeLa/Ku80‑siRNA cells exhibited more γH2AX foci remaining following treatment with irradiation and cisplatin, particularly in the group treated with 6 Gy irradiation for 1 h together with 23 h of exposure to cisplatin. Irradiation in combination with cisplatin promoted the apoptosis of HeLa cells in association with the inhibition of Ku80, and it was identified that the earlier cisplatin was administered following irradiation, the more apoptosis was induced. This maybe because irradiation combined with cisplatin is able to arrest cells in G1 and S phase to rapidly repair damaged DNA, and the lack of Ku80 induces the inability to repair DSB, resulting in increased apoptosis. The results of the present study suggest that Ku80 may be a potent molecular target in cisplatin radiosensitization.

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1	Petera J, Sirak I, Beranek M, Vosmik M, Drastikova M, Paulikova S and Soumarova R: Molecular predictive factors of outcome of radiotherapy in cervical cancer. Neoplasma. 58:469–475. 2011. View Article : Google Scholar : PubMed/NCBI
2	Kartalou M and Essigmann JM: Mechanisms of resistance to cisplatin. Mutat Res. 478:23–43. 2001. View Article : Google Scholar : PubMed/NCBI
3	Siddik ZH: Cisplatin: Mode of cytotoxic action and molecular basis of resistance. Oncogene. 22:7265–7279. 2003. View Article : Google Scholar : PubMed/NCBI
4	Wu X, Fan W, Xu S and Zhou Y: Sensitization to the cytotoxicity of cisplatin by transfection with nucleotide excision repair gene xeroderma pigmentosun group a antisense RNA in human lung adenocarcinoma cells. Clin Cancer Res. 9:5874–5879. 2003.PubMed/NCBI
5	Guggenheim ER, Xu D, Zhang CX, Chang PV and Lippard SJ: Photoaffinity isolation and identification of proteins in cancer cell extracts that bind to platinum-modified DNA. Chembiochem. 10:141–157. 2009. View Article : Google Scholar : PubMed/NCBI
6	Wang C and Lees-Miller SP: Detection and repair of ionizing radiation-induced DNA double strand breaks: New developments in nonhomologous end joining. Int J Radiat Oncol Biol Phys. 86:440–449. 2013. View Article : Google Scholar : PubMed/NCBI
7	Collis SJ, DeWeese TL, Jeggo PA and Parker AR: The life and death of DNA-PK. Oncogene. 24:949–961. 2005. View Article : Google Scholar : PubMed/NCBI
8	Burma S and Chen DJ: Role of DNA-PK in the cellular response to DNA double-strand breaks. DNA Repair (Amst). 3:909–918. 2004. View Article : Google Scholar : PubMed/NCBI
9	Lieber MR, Ma Y, Pannicke U and Schwarz K: Mechanism and regulation of human non-homologous DNA end-joining. Nat Rev Mol Cell Biol. 4:712–720. 2003. View Article : Google Scholar : PubMed/NCBI
10	Goodwin JF and Knudsen KE: Beyond DNA repair: DNA-PK function in cancer. Cancer Discov. 4:1126–1139. 2014. View Article : Google Scholar : PubMed/NCBI
11	Myint WK, Ng C and Raaphorst GP: Examining the non-homologous repair process following cisplatin and radiation treatments. Int J Radiat Biol. 78:417–424. 2002. View Article : Google Scholar : PubMed/NCBI
12	Boeckman HJ, Trego KS and Turchi JJ: Cisplatin sensitizes cancer cells to ionizing radiation via inhibition of nonhomologous end joining. Mol Cancer Res. 3:277–285. 2005. View Article : Google Scholar : PubMed/NCBI
13	Sears CR and Turchi JJ: Complex cisplatin-double strand break (DSB) lesions directly impair cellular non-homologous end-joining (NHEJ) independent of downstream damage response (DDR) pathways. J Biol Chem. 287:24263–24272. 2012. View Article : Google Scholar : PubMed/NCBI
14	Zhuang L, Yu SY, Huang XY, Gao QL, Xiong H and Leng Y: Effect of Ku80 expression inhibition by RNA interference on proliferation of cervical carcinoma cell line HeLa. Ai Zheng. 26:252–257. 2007.PubMed/NCBI
15	Jayakumar S, Bhilwade HN, Pandey BN, Sandur SK and Chaubey RC: The potential value of the neutral comet assay and the expression of genes associated with DNA damage in assessing the radiosensitivity of tumor cells. Mutat Res. 748:52–59. 2012. View Article : Google Scholar : PubMed/NCBI
16	Valerie K and Povirk LF: Regulation and mechanisms of mammalian double-strand break repair. Oncogene. 22:5792–5812. 2003. View Article : Google Scholar : PubMed/NCBI
17	Williams GJ, Hammel M, Radhakrishnan SK, Ramsden D, Lees-Miller SP and Tainer JA: Structural insights into NHEJ: Building up an integrated picture of the dynamic DSB repair super complex, one component and interaction at a time. DNA Repair (Amst). 17:110–120. 2014. View Article : Google Scholar : PubMed/NCBI
18	Gullo C, Au M, Feng G and Teoh G: The biology of Ku and its potential oncogenic role incancer. Biochim Biophys Acta. 1765:223–234. 2006.PubMed/NCBI
19	Zhuang L, Cao Y, Xiong H, Gao Q, Cao Z, Liu F, Qiu H, Yu S and Huang X: Suppression of DNA-PKcs and Ku80 individually and in combination: Different effects of radiobiology in HeLa cells. Int J Oncol. 39:443–451. 2011.PubMed/NCBI
20	Melo J and Toczyski D: A unified view of the DNA-damage checkpoint. Curr Opin Cell Biol. 14:237–245. 2002. View Article : Google Scholar : PubMed/NCBI
21	Wieringa HW, van der Zee AG, de Vries EG and van Vugt MA: Breaking the DNA damage response to improve cervical cancer treatment. Cancer Treat Rev. 42:30–40. 2016. View Article : Google Scholar : PubMed/NCBI
22	Fillingham J, Keogh MC and Krogan NJ: GammaH2AX and its role in DNA double-strand break repair. Biochem Cell Biol. 84:568–577. 2006. View Article : Google Scholar : PubMed/NCBI
23	Turchi JJ and Henkels K: Human Ku autoantigen binds cisplatin-damaged DNA but fails to stimulate human DNA-activated protein kinase. J Biol Chem. 271:13861–13867. 1996. View Article : Google Scholar : PubMed/NCBI
24	Jensen R and Glazer PM: Cell-interdependent cisplatin killing by Ku/DNA-dependent protein kinase signaling transduced through gap junctions. Proc Natl Acad Sci USA. 101:pp. 6134–6139. 2004; View Article : Google Scholar : PubMed/NCBI
25	Moll U, Lau R, Sypes MA, Gupta MM and Anderson CW: DNA-PK, the DNA-activated protein kinase, is differentially expressed in normal and malignant human tissues. Oncogene. 18:3114–3126. 1999. View Article : Google Scholar : PubMed/NCBI
26	Harima Y, Sawada S, Miyazaki Y, Kin K, Ishihara H, Imamura M, Sougawa M, Shikata N and Ohnishi T: Expression of Ku80 in cervical cancer correlates with response to radiotherapy and survival. Am J Clin Oncol. 26:e80–e85. 2003. View Article : Google Scholar : PubMed/NCBI
27	Pavón MA, Parreño M, León X, Sancho FJ, Céspedes MV, Casanova I, Lopez-Pousa A, Mangues MA, Quer M, Barnadas A and Mangues R: Ku70 predicts response and primary tumor recurrence after therapy in locally advanced head and neck cancer. Int J Cancer. 123:1068–1079. 2008. View Article : Google Scholar : PubMed/NCBI
28	Hayashi J, Sakata KI, Someya M, Matsumoto Y, Satoh M, Nakata K, Hori M, Takagi M, Kondoh A, Himi T and Hareyama M: Analysis and results of Ku and XRCC4 expression in hypopharyngeal cancer tissues treated with chemoradiotherapy. Oncol Lett. 4:151–155. 2012.PubMed/NCBI
29	Ma Q, Li P, Xu M, Yin J, Su Z, Li W and Zhang J: Ku80 is highly expressed in lung adenocarcinoma and promotes cisplatin resistance. J Exp Clin Cancer Res. 31:992012. View Article : Google Scholar : PubMed/NCBI