Radiosensitization by irinotecan is attributed to G2/M phase arrest, followed by enhanced apoptosis, probably through the ATM/Chk/Cdc25C/Cdc2 pathway in p53-mutant colorectal cancer cells

Wang,Yaqi; Yang,Lifeng; Zhang,Jing; Zhou,Menglong; Shen,Lijun; Deng,Weijuan; Liang,Liping; Hu,Ran; Yang,Wang; Yao,Ye; Zhang,Hui; Zhang,Zhen

doi:10.3892/ijo.2018.4514

Radiosensitization by irinotecan is attributed to G2/M phase arrest, followed by enhanced apoptosis, probably through the ATM/Chk/Cdc25C/Cdc2 pathway in p53-mutant colorectal cancer cells

Authors:
- Yaqi Wang
- Lifeng Yang
- Jing Zhang
- Menglong Zhou
- Lijun Shen
- Weijuan Deng
- Liping Liang
- Ran Hu
- Wang Yang
- Ye Yao
- Hui Zhang
- Zhen Zhang
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Affiliations: Department of Radiation Oncology, Fudan University Shanghai Cancer Center, Shanghai 200032, P.R. China
Published online on: August 3, 2018 https://doi.org/10.3892/ijo.2018.4514
Pages: 1667-1680

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Abstract

Irinotecan, an analog of camptothecin, which is an inhibitor of topoisomerase I, is currently used in the treatment of metastatic colorectal cancer. Camptothecin derivatives have been demonstrated to exert radiosensitizing effects on several types of cancer cells. However, to date, at least to the best of our knowledge, few studies have examined these effects in colorectal cancer cell lines. In the present study, we examined the radiosensitizing effects of irinotecan on the p53-mutant colorectal cancer cell lines, HT29 and SW620, and explored the potential underlying mechanisms. Drug cytotoxicity tests revealed that the 24 h half-maximal inhibitory concentrations (IC50s) of irinotecan as a single agent were 39.84 µg/ml (HT29 95% CI, 38.27-41.48) and 96.86 µg/ml (SW620 95% CI, 89.04-105.4); finally, concentrations <2 µg/ml were used in the subsequent experiments. Clonogenic assays revealed that irinotecan exerted radiosensitizing effects on the HT29 and SW620 cells, and the sensitivity enhancement ratios (SERs) at 2 Gy increased with increasing concentrations (SER at 2 Gy, 1.41 for the HT29 cells, 1.87 for the SW620 cells; with irinotecan at 2 µg/ml). Subsequently, the cells were divided into 4 groups: The control group, irinotecan group, radiation group and combination group. Compared with the control, irinotecan and radiation groups, the combination group had the slowest cell growth rate and the most obvious foci of Ser139p‑γH2AX. Combined treatment resulted in a firstly decreased and then increased M phase arrest and led to the most significant G2/M phase arrest, followed by the most significant increase in apoptosis. The results of western blot analysis indicated that the expression levels of proteins related to the DNA damage response system (Ser1981p‑ATM, Ser345p‑Chk1, Thr68p‑Chk2 and Ser139p‑γH2AX) and the cell cycle (Tyr15p‑Cdc2 and cyclin B1) exhibited the greatest increase in the combined group. In addition, the expression of Ser216p‑Cdc25C was also increased in the combined group, indicating that irinotecan likely radiosensitized the p53-mutant HT29 and SW620 cells through the ATM/Chk/Cdc25C/Cdc2 pathway.

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1	Siegel RL, Miller KD and Jemal A: Cancer Statistics, 2017. CA Cancer J Clin. 67:7–30. 2017. View Article : Google Scholar : PubMed/NCBI
2	Sauer R, Becker H, Hohenberger W, Rödel C, Wittekind C, Fietkau R, Martus P, Tschmelitsch J, Hager E, Hess CF, et al German Rectal Cancer Study Group: Preoperative versus postoperative chemoradiotherapy for rectal cancer. N Engl J Med. 351:1731–1740. 2004. View Article : Google Scholar : PubMed/NCBI
3	Bosset JF, Calais G, Mineur L, Maingon P, Radosevic-Jelic L, Daban A, Bardet E, Beny A, Briffaux A and Collette L: Enhanced tumorocidal effect of chemotherapy with preoperative radiotherapy for rectal cancer: Preliminary results - EORTC 22921. J Clin Oncol. 23:5620–5627. 2005. View Article : Google Scholar : PubMed/NCBI
4	Bosset JF, Collette L, Calais G, Mineur L, Maingon P, Radosevic-Jelic L, Daban A, Bardet E, Beny A and Ollier JC; EORTC Radiotherapy Group Trial 22921: Chemotherapy with preoperative radiotherapy in rectal cancer. N Engl J Med. 355:1114–1123. 2006. View Article : Google Scholar : PubMed/NCBI
5	Ma B, Gao P, Wang H, Xu Q, Song Y, Huang X, Sun J, Zhao J, Luo J, Sun Y, et al: What has preoperative radio(chemo) therapy brought to localized rectal cancer patients in terms of perioperative and long-term outcomes over the past decades? A systematic review and meta-analysis based on 41,121 patients. Int J Cancer. 141:1052–1065. 2017. View Article : Google Scholar : PubMed/NCBI
6	Greenhalgh TA, Dearman C and Sharma RA: Combination of novel agents with radiotherapy to treat rectal cancer. Clin Oncol (R Coll Radiol). 28:116–139. 2016. View Article : Google Scholar
7	Aschele C, Cionini L, Lonardi S, Pinto C, Cordio S, Rosati G, Artale S, Tagliagambe A, Ambrosini G, Rosetti P, et al: Primary tumor response to preoperative chemoradiation with or without oxaliplatin in locally advanced rectal cancer: Pathologic results of the STAR-01 randomized phase III trial. J Clin Oncol. 29:2773–2780. 2011. View Article : Google Scholar : PubMed/NCBI
8	Gérard JP, Azria D, Gourgou-Bourgade S, Martel-Laffay I, Hennequin C, Etienne PL, Vendrely V, François E, de La Roche G, Bouché O, et al: Comparison of two neoadjuvant chemoradiotherapy regimens for locally advanced rectal cancer: Results of the phase III trial ACCORD 12/0405-Prodige 2. J Clin Oncol. 28:1638–1644. 2010. View Article : Google Scholar : PubMed/NCBI
9	Rödel C, Liersch T, Becker H, Fietkau R, Hohenberger W, Hothorn T, Graeven U, Arnold D, Lang-Welzenbach M, Raab HR, et al German Rectal Cancer Study Group: Preoperative chemo-radiotherapy and postoperative chemotherapy with fluorouracil and oxaliplatin versus fluorouracil alone in locally advanced rectal cancer: Initial results of the German CAO/ARO/AIO-04 randomised phase 3 trial. Lancet Oncol. 13:679–687. 2012. View Article : Google Scholar
10	Allegra CJ, Yothers G, O'Connell MJ, Beart RW, Wozniak TF, Pitot HC, Shields AF, Landry JC, Ryan DP, Arora A, et al: Neoadjuvant 5-FU or capecitabine plus radiation with or without oxaliplatin in rectal cancer patients: A phase III randomized clinical trial. J Natl Cancer Inst. 107:1072015. View Article : Google Scholar
11	Fujita K, Kubota Y, Ishida H and Sasaki Y: Irinotecan, a key chemotherapeutic drug for metastatic colorectal cancer. World J Gastroenterol. 21:12234–12248. 2015. View Article : Google Scholar : PubMed/NCBI
12	Chabot GG: Clinical pharmacokinetics of irinotecan. Clin Pharmacokinet. 33:245–259. 1997. View Article : Google Scholar : PubMed/NCBI
13	Chen AY, Choy H and Rothenberg ML: DNA topoisomerase I-targeting drugs as radiation sensitizers. Oncology (Williston Park). 13(Suppl 5): 39–46. 1999.
14	Mattern MR, Hofmann GA, McCabe FL and Johnson RK: Synergistic cell killing by ionizing radiation and topoisomerase I inhibitor topotecan (SK&F 104864). Cancer Res. 51:5813–5816. 1991.PubMed/NCBI
15	Kim JH, Kim SH, Kolozsvary A and Khil MS: Potentiation of radiation response in human carcinoma cells in vitro and murine fibrosarcoma in vivo by topotecan, an inhibitor of DNA topoisomerase I. Int J Radiat Oncol Biol Phys. 22:515–518. 1992. View Article : Google Scholar : PubMed/NCBI
16	Boothman DA, Wang M, Schea RA, Burrows HL, Strickfaden S and Owens JK: Posttreatment exposure to camptothecin enhances the lethal effects of x-rays on radioresistant human malignant melanoma cells. Int J Radiat Oncol Biol Phys. 24:939–948. 1992. View Article : Google Scholar : PubMed/NCBI
17	Hennequin C, Giocanti N, Balosso J and Favaudon V: Interaction of ionizing radiation with the topoisomerase I poison camptothecin in growing V-79 and HeLa cells. Cancer Res. 54:1720–1728. 1994.PubMed/NCBI
18	Chen AY, Scruggs PB, Geng L, Rothenberg ML and Hallahan DE: p53 and p21 are major cellular determinants for DNA topoisomerase I-mediated radiation sensitization in mammalian cells. Ann N Y Acad Sci. 922:298–300. 2000. View Article : Google Scholar
19	Yin L, Wu J, Wu J, Ye J, Jiang X, Chen M, Wang D, Wang X, Zong D, Gu J, et al: Radiosensitization effect of nedaplatin on nasopharyngeal carcinoma cells in different status of Epstein-Barr virus infection. BioMed Res Int. 2014:7136742014. View Article : Google Scholar : PubMed/NCBI
20	Jones L, Hoban P and Metcalfe P: The use of the linear quadratic model in radiotherapy: A review. Australas Phys Eng Sci Med. 24:132–146. 2001. View Article : Google Scholar
21	NCCN Clinical Practice Guidelines in Oncology Rectal Cancer Version 2.2018. J Natl Compr Canc Netw. 16:874–901. 2018. View Article : Google Scholar
22	Haug K, Kravik KL and De Angelis PM: Cellular response to irinotecan in colon cancer cell lines showing differential response to 5-fluorouracil. Anticancer Res. 28A:583–592. 2008.
23	Kaku Y, Tsuchiya A, Kanno T and Nishizaki T: Irinotecan induces cell cycle arrest, but not apoptosis or necrosis, in Caco-2 and CW2 colorectal cancer cell lines. Pharmacology. 95:154–159. 2015. View Article : Google Scholar : PubMed/NCBI
24	Pawlik TM and Keyomarsi K: Role of cell cycle in mediating sensitivity to radiotherapy. Int J Radiat Oncol Biol Phys. 59:928–942. 2004. View Article : Google Scholar : PubMed/NCBI
25	Stark GR and Taylor WR: Control of the G2/M transition. Mol Biotechnol. 32:227–248. 2006. View Article : Google Scholar : PubMed/NCBI
26	Taylor WR and Stark GR: Regulation of the G2/M transition by p53. Oncogene. 20:1803–1815. 2001. View Article : Google Scholar : PubMed/NCBI
27	Reinhardt HC and Yaffe MB: Kinases that control the cell cycle in response to DNA damage: Chk1, Chk2, and MK2. Curr Opin Cell Biol. 21:245–255. 2009. View Article : Google Scholar : PubMed/NCBI
28	Preuss U, Landsberg G and Scheidtmann KH: Novel mitosis-specific phosphorylation of histone H3 at Thr11 mediated by Dlk/ZIP kinase. Nucleic Acids Res. 31:878–885. 2003. View Article : Google Scholar : PubMed/NCBI
29	Bhonde MR, Hanski ML, Notter M, Gillissen BF, Daniel PT, Zeitz M and Hanski C: Equivalent effect of DNA damage-induced apoptotic cell death or long-term cell cycle arrest on colon carcinoma cell proliferation and tumour growth. Oncogene. 25:165–175. 2006. View Article : Google Scholar
30	Das P, Skibber JM, Rodriguez-Bigas MA, Feig BW, Chang GJ, Wolff RA, Eng C, Krishnan S, Janjan NA and Crane CH: Predictors of tumor response and downstaging in patients who receive preoperative chemoradiation for rectal cancer. Cancer. 109:1750–1755. 2007. View Article : Google Scholar : PubMed/NCBI
31	Chen AY, Chou R, Shih SJ, Lau D and Gandara D: Enhancement of radiotherapy with DNA topoisomerase I-targeted drugs. Crit Rev Oncol Hematol. 50:111–119. 2004. View Article : Google Scholar : PubMed/NCBI
32	Li TK and Liu LF: Tumor cell death induced by topoisomerase-targeting drugs. Annu Rev Pharmacol Toxicol. 41:53–77. 2001. View Article : Google Scholar : PubMed/NCBI
33	Chen AY, Okunieff P, Pommier Y and Mitchell JB: Mammalian DNA topoisomerase I mediates the enhancement of radiation cytotoxicity by camptothecin derivatives. Cancer Res. 57:1529–1536. 1997.PubMed/NCBI
34	Omura M, Torigoe S and Kubota N: SN-38, a metabolite of the camptothecin derivative CPT-11, potentiates the cytotoxic effect of radiation in human colon adenocarcinoma cells grown as spheroids. Radiother Oncol. 43:197–201. 1997. View Article : Google Scholar : PubMed/NCBI
35	Jo HJ, Song JD, Kim KM, Cho YH, Kim KH and Park YC: Diallyl disulfide induces reversible G2/M phase arrest on a p53-independent mechanism in human colon cancer HCT-116 cells. Oncol Rep. 19:275–280. 2008.
36	Sur S and Agrawal DK: Phosphatases and kinases regulating CDC25 activity in the cell cycle: Clinical implications of CDC25 overexpression and potential treatment strategies. Mol Cell Biochem. 416:33–46. 2016. View Article : Google Scholar : PubMed/NCBI
37	Bartek J and Lukas J: Chk1 and Chk2 kinases in checkpoint control and cancer. Cancer Cell. 3:421–429. 2003. View Article : Google Scholar : PubMed/NCBI
38	Hendzel MJ, Wei Y, Mancini MA, Van Hooser A, Ranalli T, Brinkley BR, Bazett-Jones DP and Allis CD: Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma. 106:348–360. 1997. View Article : Google Scholar
39	Magrini R, Bhonde MR, Hanski ML, Notter M, Scherübl H, Boland CR, Zeitz M and Hanski C: Cellular effects of CPT-11 on colon carcinoma cells: Dependence on p53 and hMLH1 status. Int J Cancer. 101:23–31. 2002. View Article : Google Scholar : PubMed/NCBI
40	Bhonde MR, Hanski ML, Budczies J, Cao M, Gillissen B, Moorthy D, Simonetta F, Scherübl H, Truss M, Hagemeier C, et al: DNA damage-induced expression of p53 suppresses mitotic checkpoint kinase hMps1: The lack of this suppression in p53MUT cells contributes to apoptosis. J Biol Chem. 281:8675–8685. 2006. View Article : Google Scholar : PubMed/NCBI
41	Mohiuddin M, Winter K, Mitchell E, Hanna N, Yuen A, Nichols C, Shane R, Hayostek C and Willett C; Radiation Therapy Oncology Group Trial 0012: Randomized phase II study of neoadjuvant combined-modality chemoradiation for distal rectal cancer: Radiation Therapy Oncology Group Trial 0012. J Clin Oncol. 24:650–655. 2006. View Article : Google Scholar : PubMed/NCBI
42	Wong SJ, Moughan J, Meropol NJ, Anne PR, Kachnic LA, Rashid A, Watson JC, Mitchell EP, Pollock J, Lee RJ, et al: Efficacy endpoints of radiation therapy group protocol 0247: A randomized, phase 2 study of neoadjuvant radiation therapy plus concurrent capecitabine and irinotecan or capecitabine and oxaliplatin for patients with locally advanced rectal cancer. Int J Radiat Oncol Biol Phys. 91:116–123. 2015. View Article : Google Scholar
43	Mehta VK, Cho C, Ford JM, Jambalos C, Poen J, Koong A, Lin A, Bastidas JA, Young H, Dunphy EP, et al: Phase II trial of preoperative 3D conformal radiotherapy, protracted venous infusion 5-fluorouracil, and weekly CPT-11, followed by surgery for ultrasound-staged T3 rectal cancer. Int J Radiat Oncol Biol Phys. 55:132–137. 2003. View Article : Google Scholar
44	Navarro M, Dotor E, Rivera F, Sánchez-Rovira P, Vega-Villegas ME, Cervantes A, García JL, Gallén M and Aranda E: A Phase II study of preoperative radiotherapy and concomitant weekly irinotecan in combination with protracted venous infusion 5-fluorouracil, for resectable locally advanced rectal cancer. Int J Radiat Oncol Biol Phys. 66:201–205. 2006. View Article : Google Scholar : PubMed/NCBI
45	Liu X, Cheng D, Kuang Q, Liu G and Xu W: Association of UGT1A1^*28 polymorphisms with irinotecan-induced toxicities in colorectal cancer: A meta-analysis in Caucasians. Pharmacogenomics J. 14:120–129. 2014. View Article : Google Scholar
46	Toffoli G, Cecchin E, Corona G, Russo A, Buonadonna A, D'Andrea M, Pasetto LM, Pessa S, Errante D, De Pangher V, et al: The role of UGT1A1^*28 polymorphism in the pharmacodynamics and pharmacokinetics of irinotecan in patients with metastatic colorectal cancer. J Clin Oncol. 24:3061–3068. 2006. View Article : Google Scholar : PubMed/NCBI
47	Innocenti F, Undevia SD, Iyer L, Chen PX, Das S, Kocherginsky M, Karrison T, Janisch L, Ramírez J, Rudin CM, et al: Genetic variants in the UDP-glucuronosyltransferase 1A1 gene predict the risk of severe neutropenia of irinotecan. J Clin Oncol. 22:1382–1388. 2004. View Article : Google Scholar : PubMed/NCBI
48	Zhu J, Li X, Shen Y, Guan Y, Gu W, Lian P, Sheng W, Cai S and Zhang Z: Genotype-driven phase I study of weekly irinotecan in combination with capecitabine-based neoadjuvant chemo-radiation for locally advanced rectal cancer. Radiother Oncol Radiother Oncol. S0167-8140:32749–32744. 2017.