N-acetylcysteine potentiates doxorubicin-induced ATM and p53 activation in ovarian cancer cells

Brum,Gabriella; Carbone,Thomas; Still,Eric; Correia,Vendita; Szulak,Kevin; Calianese,David; Best,Charles; Cammarata,Garret; Higgins,Katelyn; Ji,Fang; Di,Wen; Wan,Yinsheng

doi:10.3892/ijo.2012.1680

N-acetylcysteine potentiates doxorubicin-induced ATM and p53 activation in ovarian cancer cells

Authors:
- Gabriella Brum
- Thomas Carbone
- Eric Still
- Vendita Correia
- Kevin Szulak
- David Calianese
- Charles Best
- Garret Cammarata
- Katelyn Higgins
- Fang Ji
- Wen Di
- Yinsheng Wan
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Affiliations: Department of Biology, Providence College, Providence, RI 02918, USA, Department of Obstetrics and Gynecology, Renji Hospital of Shanghai Jiaotong University Medical School, Shanghai 200001, P.R. China
Published online on: October 26, 2012 https://doi.org/10.3892/ijo.2012.1680
Pages: 211-218

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Abstract

Doxorubicin has been used clinically to treat various types of cancer, and yet the molecular mode of actions of doxorubicin remains to be fully unraveled. In this study, we investigated the effect of doxorubicin on cultured ovarian cancer cells (CaOV3). MTT assay data showed that doxorubicin inhibits cell proliferation in a time- and dose-dependent manner. Phagokinetic cell motility assay data indicated that doxorubicin inhibits both basal level and EGF-induced cell migration in CaOV3 cells. Confocal microscopic data revealed that doxorubicin induces reorganization of cytoskeletal proteins including actin, tubulin and vimentin. Doxorubicin induces phosphorylation of p53 at Ser15 and 20, acetylation of p53 and ATM activation. Doxorubicin also induces phosphorylation of histone H2AX at Ser139. Interestingly, doxorubicin also inhibits mTOR activity, measured by phosphorylation of S6 ribosomal protein. Pretreatment of CaOV3 cells with antioxidant N-acetylcysteine (NAC), but not pyrrolidine dithiocarbamate (PDTC) potentiates doxorubicin-induced phosphorylation of p53 and ATM. Collectively, we conclude that doxorubicin induces ATM/p53 activation leading to reorganization of cytoskeletal networks, inhibition of mTOR activity, and inhibition of cell proliferation and migration. Our data also suggest that removal of oxidants by antioxidants such as NAC may enhance the efficacy of doxorubicin in vivo.

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1.	Sardao VA, Oliveira PJ, Holy J, Oliveira CR and Wallace KB: Morphological alterations induced by doxorubicin on H9c2 myoblasts: nuclear, mitochondrial, and cytoskeletal targets. Cell Biol Toxicol. 25:227–243. 2009. View Article : Google Scholar : PubMed/NCBI
2.	Hublarova P, Greplova K, Holcakova J, Vojtesek B and Hrstka R: Switching p53-dependent growth arrest to apoptosis via the inhibition of DNA damage-activated kinases. Cell Mol Biol Lett. 15:473–484. 2010. View Article : Google Scholar : PubMed/NCBI
3.	Eom YW, Kim MA, Park SS, Goo MJ, Kwon HJ, Sohn S, Kim WH, Yoon G and Choi KS: Two distinct modes of cell death induced by doxorubicin: apoptosis and cell death through mitotic catastrophe accompanied by senescence-like phenotype. Oncogene. 24:4765–4777. 2005. View Article : Google Scholar : PubMed/NCBI
4.	Kurz EU, Douglas P and Lees-Miller SP: Doxorubicin activates ATM-dependent phosphorylation of multiple downstream targets in part through the generation of reactive oxygen species. J Biol Chem. 279:53272–53281. 2004. View Article : Google Scholar : PubMed/NCBI
5.	Voisard R, Seitzer U, Baur R, Dartsch PC, Osterhues H, Hoher M and Hombach V: A prescreening system for potential antiproliferative agents: implications for local treatment strategies of postangioplasty restenosis. Int J Cardiol. 51:15–28. 1995. View Article : Google Scholar
6.	Otsuka Y, Tanaka T, Uchida D, Noguchi Y, Saeki N, Saito Y and Tatsuno I: Roles of cyclin-dependent kinase 4 and p53 in neuronal cell death induced by doxorubicin on cerebellar granule neurons in mouse. Neurosci Lett. 365:180–185. 2004. View Article : Google Scholar : PubMed/NCBI
7.	Panta GR, Kaur S, Cavin LG, Cortes ML, Mercurio F, Lothstein L, Sweatman TW, Israel M and Arsura M: ATM and the catalytic subunit of DNA-dependent protein kinase activate NF-kappaB through a common MEK/extracellular signal-regulated kinase/p90(rsk) signaling pathway in response to distinct forms of DNA damage. Mol Cell Biol. 24:1823–1835. 2004. View Article : Google Scholar
8.	Wu R, Hu TC, Rehemtulla A, Fearon ER and Cho KR: Preclinical testing of PI3K/AKT/mTOR signaling inhibitors in a mouse model of ovarian endometrioid adenocarcinoma. Clin Cancer Res. 17:7359–7372. 2011. View Article : Google Scholar : PubMed/NCBI
9.	Litwiniec A, Grzanka A, Helmin-Basa A, Gackowska L and Grzanka D: Features of senescence and cell death induced by doxorubicin in A549 cells: organization and level of selected cytoskeletal proteins. J Cancer Res Clin Oncol. 136:717–736. 2010. View Article : Google Scholar : PubMed/NCBI
10.	Grzanka A, Grzanka D and Orlikowska M: Cytoskeletal reorganization during process of apoptosis induced by cytostatic drugs in K-562 and HL-60 leukemia cell lines. Biochem Pharmacol. 66:1611–1617. 2003. View Article : Google Scholar : PubMed/NCBI
11.	Bijman MN, van Berkel MP, van Nieuw Amerongen GP and Boven E: Interference with actin dynamics is superior to disturbance of microtubule function in the inhibition of human ovarian cancer cell motility. Biochem Pharmacol. 76:707–716. 2008. View Article : Google Scholar : PubMed/NCBI
12.	Qiu L, Zhou C, Sun Y, Di W, Scheffler E, Healey S, Wanebo H, Kouttab N, Chu W and Wan Y: Paclitaxel and ceramide synergistically induce cell death with transient activation of EGFR and ERK pathway in pancreatic cancer cells. Oncol Rep. 16:907–913. 2006.PubMed/NCBI
13.	Albrecht-Buehler G: The phagokinetic tracks of 3T3 cells. Cell. 11:395–404. 1977. View Article : Google Scholar : PubMed/NCBI
14.	Qiu L, Wang Q, Di W, Jiang Q, Schefeller E, Derby S, Wanebo H, Yan B and Wan Y: Transient activation of EGFR/AKT cell survival pathway and expression of survivin contribute to reduced sensitivity of human melanoma cells to betulinic acid. Int J Oncol. 27:823–830. 2005.PubMed/NCBI
15.	Qiu L, Di W, Jiang Q, Scheffler E, Derby S, Yang J, Kouttab N, Wanebo H, Yan B and Wan Y: Targeted inhibition of transient activation of the EGFR-mediated cell survival pathway enhances paclitaxel-induced ovarian cancer cell death. Int J Oncol. 27:1441–1448. 2005.PubMed/NCBI
16.	Bagriacik EU, Uslu K, Yurtcu E, Stefek M and Karasu C: Stobadine inhibits doxorubicin-induced apoptosis through a caspase-9 dependent pathway in P815 mastocytoma cells. Cell Biol Int. 31:979–984. 2007. View Article : Google Scholar : PubMed/NCBI
17.	Rogalska A, Gajek A, Szwed M, Jozwiak Z and Marczak A: The role of reactive oxygen species in WP 631-induced death of human ovarian cancer cells: a comparison with the effect of doxorubicin. Toxicol In Vitro. 25:1712–1720. 2011. View Article : Google Scholar : PubMed/NCBI
18.	McLoon LK, Falkenberg JH, Dykstra D and Iaizzo PA: Doxorubicin chemomyectomy as a treatment for cervical dystonia: histological assessment after direct injection into the sternocleidomastoid muscle. Muscle Nerve. 21:1457–1464. 1998. View Article : Google Scholar
19.	Ji C, Cao C, Lu S, Kivlin R, Amaral A, Kouttab N, Yang H, Chu W, Bi Z, Di W and Wan Y: Curcumin attenuates EGF-induced AQP3 up-regulation and cell migration in human ovarian cancer cells. Cancer Chemother Pharmacol. 62:857–865. 2008. View Article : Google Scholar : PubMed/NCBI
20.	Necco A, Vailati G and Colombo R: Actin behaviour in doxorubicin treatment. Cell Biol Int Rep. 7:129–134. 1983. View Article : Google Scholar : PubMed/NCBI
21.	Dalledonne I, Milzani A and Colombo R: DXR depresses the alpha-actinin-induced formation of actin bundles. Cancer Biochem Biophys. 13:245–254. 1993.PubMed/NCBI
22.	Necco A, Usardi C, Scari G, Islam K, Melzner I and Vergani G: The effects of doxorubicin on embryonic spinal motoneurons cultured in vitro: cytoskeletal response. Cytotechnology. 11:133–141. 1993. View Article : Google Scholar : PubMed/NCBI
23.	Colombo R, Necco A, Vailati G, Saracco B, Milzani A and Scari G: Doxorubicin affects actin assembly in vitro. Cell Biol Int Rep. 8:127–135. 1984. View Article : Google Scholar : PubMed/NCBI
24.	Mariano R, Gonzalez B and Lewis W: Cardiac actin interactions with doxorubicin in vitro. Exp Mol Pathol. 44:7–13. 1986. View Article : Google Scholar : PubMed/NCBI
25.	Jaamaa S, Af Hallstrom TM, Sankila A, Rantanen V, Koistinen H, Stenman UH, Zhang Z, Yang Z, De Marzo AM, Taari K, Ruutu M, Andersson LC and Laiho M: DNA damage recognition via activated ATM and p53 pathway in nonproliferating human prostate tissue. Cancer Res. 70:8630–8641. 2010. View Article : Google Scholar : PubMed/NCBI
26.	Alexandre J, Hu Y, Lu W, Pelicano H and Huang P: Novel action of paclitaxel against cancer cells: bystander effect mediated by reactive oxygen species. Cancer Res. 67:3512–3517. 2007. View Article : Google Scholar : PubMed/NCBI
27.	Kim BM, Choi YJ, Lee YH, Joe YA and Hong SH: N,N-Dimethyl phytosphingosine sensitizes HL-60/MX2, a multidrug-resistant variant of HL-60 cells, to doxorubicin-induced cytotoxicity through ROS-mediated release of cytochrome c and AIF. Apoptosis. 15:982–993. 2010. View Article : Google Scholar
28.	Giannakakou P, Nakano M, Nicolaou KC, O’Brate A, Yu J, Blagosklonny MV, Greber UF and Fojo T: Enhanced microtubule-dependent trafficking and p53 nuclear accumulation by suppression of microtubule dynamics. Proc Natl Acad Sci USA. 99:10855–10860. 2002. View Article : Google Scholar : PubMed/NCBI
29.	Imreh G, Norberg HV, Imreh S and Zhivotovsky B: Chromosomal breaks during mitotic catastrophe trigger gammaH2AX-ATM-p53-mediated apoptosis. J Cell Sci. 124:2951–2963. 2011. View Article : Google Scholar : PubMed/NCBI
30.	Gokcimen A, Cim A, Tola HT, Bayram D, Kocak A, Ozguner F and Ayata A: Protective effect of N-acetylcysteine, caffeic acid and vitamin E on doxorubicin hepatotoxicity. Hum Exp Toxicol. 26:519–525. 2007. View Article : Google Scholar : PubMed/NCBI
31.	Ashley N and Poulton J: Anticancer DNA intercalators cause p53-dependent mitochondrial DNA nucleoid re-modelling. Oncogene. 28:3880–3891. 2009. View Article : Google Scholar : PubMed/NCBI
32.	Bichat F, Mouawad R, Solis-Recendez G, Khayat D and Bastian G: Cytoskeleton alteration in MCF7R cells, a multidrug resistant human breast cancer cell line. Anticancer Res. 17:3393–3401. 1997.PubMed/NCBI
33.	Grzanka A: Estimation of changes in vimentin filaments induced by etoposide and doxorubicin in human leukemia cell line K-562 by using immunofluorescence microscopy. Neoplasma. 48:285–289. 2001.PubMed/NCBI
34.	Grzanka A, Grzanka D and Orlikowska M: Fluorescence and ultrastructural localization of actin distribution patterns in the nucleus of HL-60 and K-562 cell lines treated with cytostatic drugs. Oncol Rep. 11:765–770. 2004.PubMed/NCBI
35.	Grzanka D, Marszalek A, Izdebska M, Gackowska L, Andrzej Szczepanski M and Grzanka A: Actin cytoskeleton reorganization correlates with cofilin nuclear expression and ultrastructural changes in cho aa8 cell line after apoptosis and mitotic catastrophe induction by doxorubicin. Ultrastruct Pathol. 35:130–138. 2011. View Article : Google Scholar
36.	Maxwell SA, Cherry EM and Bayless KJ: Akt, 14-3-3zeta, and vimentin mediate a drug-resistant invasive phenotype in diffuse large B-cell lymphoma. Leuk Lymphoma. 52:849–864. 2011. View Article : Google Scholar : PubMed/NCBI
37.	Akan I, Akan S, Akca H, Savas B and Ozben T: N-acetylcysteine enhances multidrug resistance-associated protein 1 mediated doxorubicin resistance. Eur J Clin Invest. 34:683–689. 2004. View Article : Google Scholar : PubMed/NCBI
38.	Herman EH, Ferrans VJ, Myers CE and Van Vleet JF: Comparison of the effectiveness of (+/−)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane (ICRF-187) and N-acetylcysteine in preventing chronic doxorubicin cardiotoxicity in beagles. Cancer Res. 45:276–281. 1985.
39.	Kockar MC, Naziroglu M, Celik O, Tola HT, Bayram D and Koyu A: N-acetylcysteine modulates doxorubicin-induced oxidative stress and antioxidant vitamin concentrations in liver of rats. Cell Biochem Funct. 28:673–677. 2010. View Article : Google Scholar : PubMed/NCBI
40.	Park ES, Kim SD, Lee MH, Lee HS, Lee IS, Sung JK and Yoon YS: Protective effects of N-acetylcysteine and selenium against doxorubicin toxicity in rats. J Vet Sci. 4:129–136. 2003.PubMed/NCBI
41.	Pichot CS, Hartig SM, Xia L, Arvanitis C, Monisvais D, Lee FY, Frost JA and Corey SJ: Dasatinib synergizes with doxorubicin to block growth, migration, and invasion of breast cancer cells. Br J Cancer. 101:38–47. 2009. View Article : Google Scholar : PubMed/NCBI
42.	Bulucu F, Oktenli C, Kenar L, Koc B, Ocal R, Karadurmus N, Inal V, Yamanel L, Sanisoglu YS and Aydin A: Detrimental effects of N-acetylcysteine plus desferoxamine combination in an experimental nephrotic syndrome model. Int J Toxicol. 26:525–532. 2007. View Article : Google Scholar : PubMed/NCBI