Resveratrol abrogates the Temozolomide-induced G2 arrest leading to mitotic catastrophe and reinforces the Temozolomide-induced senescence in glioma cells

BMC Cancer

Eduardo C Filippi-Chiela²  Marcos Paulo Thomé²  Mardja Manssur Bueno e Silva²  Alessandra Luíza Pelegrini²  Pitia Flores Ledur²  Bernardo Garicochea¹  Lauren L Zamin³  Guido Lenz⁴ 

[1] Oncology Center - Hospital Sirio Libanes, Sao Paulo, SP, Brazil

[2] Department of Biophysics, Universidade Federal do Rio Grande do Sul (UFRGS), Rua Bento Gonçalves, 9500, Prédio 43431 – Lab. 107, Porto Alegre, RS CEP 91501-970, Brazil

[3] Universidade Federal da Fronteira Sul (UFFS), Cerro Largo, RS, Brazil

[4] Center of Biotechnology, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil

关键词: Senescence;    Mitotic Catastrophe;    Autophagy;    Temozolomide;    Resveratrol;    Glioblastoma;   

Others  :  1079843
DOI  :  10.1186/1471-2407-13-147

 received in 2012-12-16, accepted in 2013-03-19,  发布年份 2013

【 摘 要 】

Background

Temozolomide (TMZ) is the most widely used drug to treat glioblastoma (GBM), which is the most common and aggressive primary tumor of the Central Nervous System and one of the hardest challenges in oncotherapy. TMZ is an alkylating agent that induces autophagy, apoptosis and senescence in GBM cells. However, therapy with TMZ increases survival after diagnosis only from 12 to 14.4 months, making the development of combined therapies to treat GBM fundamental. One candidate for GBM therapy is Resveratrol (Rsv), which has additive toxicity with TMZ in several glioma cells in vitro and in vivo. However, the mechanism of Rsv and TMZ additive toxicity, which is the aim of the present work, is not clear, especially concerning cell cycle dynamics and long term effects.

Methods

Glioma cell lines were treated with Rsv and TMZ, alone or in combinations, and the induction and the role of autophagy, apoptosis, cell cycle dynamics, protein expression and phosphorylation status were measured. We further evaluated the long term senescence induction and clonogenic capacity.

Results

As expected, temozolomide caused a G2 cell cycle arrest and extensive DNA damage response. Rsv did not reduced this response, even increasing pATM, pChk2 and gammaH2Ax levels, but abrogated the temozolomide-induced G2 arrest, increasing levels of cyclin B and pRb(S807/811) and reducing levels of pWee1(S642) and pCdk1(Y15). This suggests a cellular state of forced passage through G2 checkpoint despite large DNA damage, a scenario that may produce mitotic catastrophe. Indeed, the proportion of cells with high nuclear irregularity increased from 6 to 26% in 48 h after cotreatment. At a long term, a reduction in clonogenic capacity was observed, accompanied by a large induction of senescence.

Conclusion

The presence of Rsv forces cells treated with TMZ through mitosis leading to mitotic catastrophe and senescence, reducing the clonogenic capacity of glioma cells and increasing the chronic effects of temozolomide.

【 授权许可】

   
2013 Filippi-Chiela et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

【 参考文献 】

• [1]Furnari FB, Fenton T, Bachoo RM, Mukasa A, Stommel JM, Stegh A, Hahn WC, Ligon KL, Louis DN, Brennan C: Malignant astrocytic glioma: genetics, biology, and paths to treatment. Genes Dev 2007, 21(21):2683-2710.
• [2]Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M, Kros JM, Hainfellner JA, Mason W, Mariani L: MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 2005, 352(10):997-1003.
• [3]Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, Ludwin SK, Allgeier A, Fisher B, Belanger K: Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 2009, 10(5):459-466.
• [4]Tran B, Rosenthal MA: Survival comparison between glioblastoma multiforme and other incurable cancers. J Clin Neurosci 2010, 17(4):417-421.
• [5]Hirose Y, Berger MS, Pieper RO: p53 effects both the duration of G2/M arrest and the fate of temozolomide-treated human glioblastoma cells. Cancer Res 2001, 61(5):1957-1963.
• [6]Hermisson M, Klumpp A, Wick W, Wischhusen J, Nagel G, Roos W, Kaina B, Weller M: O6-methylguanine DNA methyltransferase and p53 status predict temozolomide sensitivity in human malignant glioma cells. J Neurochem 2006, 96(3):766-776.
• [7]Kanzawa T, Germano IM, Komata T, Ito H, Kondo Y, Kondo S: Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells. Cell Death Differ 2004, 11(4):448-457.
• [8]Newlands ES, Stevens MF, Wedge SR, Wheelhouse RT, Brock C: Temozolomide: a review of its discovery, chemical properties, pre-clinical development and clinical trials. Cancer Treat Rev 1997, 23(1):35-61.
• [9]Perry JA, Kornbluth S: Cdc25 and Wee1: analogous opposites? Cell Div 2007, 2:12. BioMed Central Full Text
• [10]Castedo M, Perfettini JL, Roumier T, Valent A, Raslova H, Yakushijin K, Horne D, Feunteun J, Lenoir G, Medema R: Mitotic catastrophe constitutes a special case of apoptosis whose suppression entails aneuploidy. Oncogene 2004, 23(25):4362-4370.
• [11]Huang H, Fletcher L, Beeharry N, Daniel R, Kao G, Yen TJ, Muschel RJ: Abnormal cytokinesis after X-irradiation in tumor cells that override the G2 DNA damage checkpoint. Cancer Res 2008, 68(10):3724-3732.
• [12]Lefranc F, Facchini V, Kiss R: Proautophagic drugs: a novel means to combat apoptosis-resistant cancers, with a special emphasis on glioblastomas. Oncologist 2007, 12(12):1395-1403.
• [13]Roninson IB, Broude EV, Chang BD: If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells. Drug Resist Updat 2001, 4(5):303-313.
• [14]Pervaiz S, Holme AL: Resveratrol: its biologic targets and functional activity. Antioxid Redox Signal 2009, 11(11):2851-2897.
• [15]Zamin LL, Dillenburg-Pilla P, Argenta-Comiran R, Horn AP, Simao F, Nassif M, Gerhardt D, Frozza RL, Salbego C: Protective effect of resveratrol against oxygen-glucose deprivation in organotypic hippocampal slice cultures: Involvement of PI3-K pathway. Neurobiol Dis 2006, 24(1):170-182.
• [16]Mahyar-Roemer M, Katsen A, Mestres P, Roemer K: Resveratrol induces colon tumor cell apoptosis independently of p53 and precede by epithelial differentiation, mitochondrial proliferation and membrane potential collapse. Int J Cancer 2001, 94(5):615-622.
• [17]Lu R, Serrero G: Resveratrol, a natural product derived from grape, exhibits antiestrogenic activity and inhibits the growth of human breast cancer cells. J Cell Physiol 1999, 179(3):297-304.
• [18]Fuggetta MP, D’Atri S, Lanzilli G, Tricarico M, Cannavo E, Zambruno G, Falchetti R, Ravagnan G: In vitro antitumour activity of resveratrol in human melanoma cells sensitive or resistant to temozolomide. Melanoma Res 2004, 14(3):189-196.
• [19]Tsan MF, White JE, Maheshwari JG, Chikkappa G: Anti-leukemia effect of resveratrol. Leuk Lymphoma 2002, 43(5):983-987.
• [20]Kuwajerwala N, Cifuentes E, Gautam S, Menon M, Barrack ER, Reddy GP: Resveratrol induces prostate cancer cell entry into s phase and inhibits DNA synthesis. Cancer Res 2002, 62(9):2488-2492.
• [21]Filippi-Chiela EC, Villodre ES, Zamin LL, Lenz G: Autophagy interplay with apoptosis and cell cycle regulation in the growth inhibiting effect of resveratrol in glioma cells. PLoS One 2011, 6(6):e20849.
• [22]Li J, Qin Z, Liang Z: The prosurvival role of autophagy in Resveratrol-induced cytotoxicity in human U251 glioma cells. BMC Cancer 2009, 9:215. BioMed Central Full Text
• [23]Jiang H, Zhang L, Kuo J, Kuo K, Gautam SC, Groc L, Rodriguez AI, Koubi D, Hunter TJ, Corcoran GB: Resveratrol-induced apoptotic death in human U251 glioma cells. Mol Cancer Ther 2005, 4(4):554-561.
• [24]Zamin LL, Filippi-Chiela EC, Dillenburg-Pilla P, Horn F, Salbego C, Lenz G: Resveratrol and quercetin cooperate to induce senescence-like growth arrest in C6 rat glioma cells. Cancer Sci 2009, 100(9):1655-1662.
• [25]Tyagi A, Singh RP, Agarwal C, Siriwardana S, Sclafani RA, Agarwal R: Resveratrol causes Cdc2-tyr15 phosphorylation via ATM/ATR-Chk1/2-Cdc25C pathway as a central mechanism for S phase arrest in human ovarian carcinoma Ovcar-3 cells. Carcinogenesis 2005, 26(11):1978-1987.
• [26]Zhang P, Li H, Wu ML, Chen XY, Kong QY, Wang XW, Sun Y, Wen S, Liu J: c-Myc downregulation: a critical molecular event in resveratrol-induced cell cycle arrest and apoptosis of human medulloblastoma cells. J Neurooncol 2006, 80(2):123-131.
• [27]Benitez DA, Pozo-Guisado E, Alvarez-Barrientos A, Fernandez-Salguero PM, Castellon EA: Mechanisms involved in resveratrol-induced apoptosis and cell cycle arrest in prostate cancer-derived cell lines. J Androl 2007, 28(2):282-293.
• [28]Ahmad N, Adhami VM, Afaq F, Feyes DK, Mukhtar H: Resveratrol causes WAF-1/p21-mediated G(1)-phase arrest of cell cycle and induction of apoptosis in human epidermoid carcinoma A431 cells. Clin Cancer Res 2001, 7(5):1466-1473.
• [29]Ragione FD, Cucciolla V, Borriello A, Pietra VD, Racioppi L, Soldati G, Manna C, Galletti P, Zappia V: Resveratrol arrests the cell division cycle at S/G2 phase transition. Biochem Biophys Res Commun 1998, 250(1):53-58.
• [30]Lin CJ, Lee CC, Shih YL, Lin TY, Wang SH, Lin YF, Shih CM: Resveratrol enhances the therapeutic effect of temozolomide against malignant glioma in vitro and in vivo by inhibiting autophagy. Free Radic Biol Med 2012, 52(2):377-391.
• [31]Yuan Y, Xue X, Guo RB, Sun XL, Hu G: Resveratrol enhances the antitumor effects of temozolomide in glioblastoma via ROS-dependent AMPK-TSC-mTOR signaling pathway. CNS Neurosci Ther 2012, 18(7):536-546.
• [32]Kil WJ, Cerna D, Burgan WE, Beam K, Carter D, Steeg PS, Tofilon PJ, Camphausen K: In vitro and in vivo radiosensitization induced by the DNA methylating agent temozolomide. Clin Cancer Res 2008, 14(3):931-938.
• [33]Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T: LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 2000, 19(21):5720-5728.
• [34]Mizushima N, Yoshimori T, Levine B: Methods in mammalian autophagy research. Cell 2010, 140(3):313-326.
• [35]Horvathova E, Dusinska M, Shaposhnikov S, Collins AR: DNA damage and repair measured in different genomic regions using the comet assay with fluorescent in situ hybridization. Mutagenesis 2004, 19(4):269-276.
• [36]Singh NP, McCoy MT, Tice RR, Schneider EL: A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988, 175(1):184-191.
• [37]Pelegrini AL, Moura DJ, Brenner BL, Ledur PF, Maques GP, Henriques JA, Saffi J, Lenz G: Nek1 silencing slows down DNA repair and blocks DNA damage-induced cell cycle arrest. Mutagenesis 2010, 25(5):447-454.
• [38]Nadin SB, Vargas-Roig LM, Ciocca DR: A silver staining method for single-cell gel assay. J Histochem Cytochem 2001, 49(9):1183-1186.
• [39]Ross GM, McMillan TJ, Wilcox P, Collins AR: The single cell microgel electrophoresis assay (comet assay): technical aspects and applications. Report on the 5th LH Gray Trust Workshop, Institute of Cancer Research, 1994. Mutat Res 1995, 337(1):57-60.
• [40]Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O: A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA 1995, 92(20):9363-9367.
• [41]Filippi-Chiela EC, Oliveira MM, Jurkovski B, Callegari-Jacques SM, da Silva VD, Lenz G: Nuclear Morphometric Analysis (NMA): Screening of Senescence. Apoptosis and Nuclear Irregularities. PLoS One 2012, 7(8):e42522.
• [42]Mineura K, Watanabe K, Yanagisawa T, Kowada M: Quantification of O6-methylguanine-DNA methyltransferase mRNA in human brain tumors. Biochim Biophys Acta 1996, 1289(1):105-109.
• [43]Gatz SA, Wiesmuller L: Take a break--resveratrol in action on DNA. Carcinogenesis 2008, 29(2):321-332.
• [44]O’Connell MJ, Raleigh JM, Verkade HM, Nurse P: Chk1 is a wee1 kinase in the G2 DNA damage checkpoint inhibiting cdc2 by Y15 phosphorylation. EMBO J 1997, 16(3):545-554.
• [45]Lindqvist A, van Zon W, Karlsson Rosenthal C, Wolthuis RM: Cyclin B1-Cdk1 activation continues after centrosome separation to control mitotic progression. PLoS Biol 2007, 5(5):e123.
• [46]Lundberg AS, Weinberg RA: Functional inactivation of the retinoblastoma protein requires sequential modification by at least two distinct cyclin-cdk complexes. Mol Cell Biol 1998, 18(2):753-761.
• [47]Tamrakar S, Rubin E, Ludlow JW: Role of pRB dephosphorylation in cell cycle regulation. Front Biosci 2000, 5:D121-137.
• [48]Potapova TA, Daum JR, Pittman BD, Hudson JR, Jones TN, Satinover DL, Stukenberg PT, Gorbsky GJ: The reversibility of mitotic exit in vertebrate cells. Nature 2006, 440(7086):954-958.
• [49]Eom YW, Kim MA, Park SS, Goo MJ, Kwon HJ, Sohn S, Kim WH, Yoon G, Choi KS: Two distinct modes of cell death induced by doxorubicin: apoptosis and cell death through mitotic catastrophe accompanied by senescence-like phenotype. Oncogene 2005, 24(30):4765-4777.
• [50]Ueno T, Ko SH, Grubbs E, Yoshimoto Y, Augustine C, Abdel-Wahab Z, Cheng TY, Abdel-Wahab OI, Pruitt SK, Friedman HS: Modulation of chemotherapy resistance in regional therapy: a novel therapeutic approach to advanced extremity melanoma using intra-arterial temozolomide in combination with systemic O6-benzylguanine. Mol Cancer Ther 2006, 5(3):732-738.
• [51]Hammond LA, Eckardt JR, Kuhn JG, Gerson SL, Johnson T, Smith L, Drengler RL, Campbell E, Weiss GR, Von Hoff DD: A randomized phase I and pharmacological trial of sequences of 1,3-bis(2-chloroethyl)-1-nitrosourea and temozolomide in patients with advanced solid neoplasms. Clin Cancer Res 2004, 10(5):1645-1656.
• [52]Chen M, Osman I, Orlow SJ: Antifolate activity of pyrimethamine enhances temozolomide-induced cytotoxicity in melanoma cells. Mol Cancer Res 2009, 7(5):703-712.
• [53]Chen M, Rose AE, Doudican N, Osman I, Orlow SJ: Celastrol synergistically enhances temozolomide cytotoxicity in melanoma cells. Mol Cancer Res 2009, 7(12):1946-1953.
• [54]O’Brien V, Brown R: Signalling cell cycle arrest and cell death through the MMR System. Carcinogenesis 2006, 27(4):682-692.
• [55]Mhaidat NM, Zhang XD, Allen J, Avery-Kiejda KA, Scott RJ, Hersey P: Temozolomide induces senescence but not apoptosis in human melanoma cells. Br J Cancer 2007, 97(9):1225-1233.
• [56]Castedo M, Perfettini JL, Roumier T, Yakushijin K, Horne D, Medema R, Kroemer G: The cell cycle checkpoint kinase Chk2 is a negative regulator of mitotic catastrophe. Oncogene 2004, 23(25):4353-4361.
• [57]Kandel ES, Skeen J, Majewski N, Di Cristofano A, Pandolfi PP, Feliciano CS, Gartel A, Hay N: Activation of Akt/protein kinase B overcomes a G(2)/m cell cycle checkpoint induced by DNA damage. Mol Cell Biol 2002, 22(22):7831-7841.
• [58]Vakifahmetoglu H, Olsson M, Zhivotovsky B: Death through a tragedy: mitotic catastrophe. Cell Death Differ 2008, 15(7):1153-1162.
• [59]Castedo M, Perfettini JL, Roumier T, Andreau K, Medema R, Kroemer G: Cell death by mitotic catastrophe: a molecular definition. Oncogene 2004, 23(16):2825-2837.
• [60]Lee J, Kumagai A, Dunphy WG: Positive regulation of Wee1 by Chk1 and 14-3-3 proteins. Mol Biol Cell 2001, 12(3):551-563.
• [61]Rothblum-Oviatt CJ, Ryan CE, Piwnica-Worms H: 14-3-3 binding regulates catalytic activity of human Wee1 kinase. Cell Growth Differ 2001, 12(12):581-589.
• [62]Lukas J, Bartkova J, Bartek J: Convergence of mitogenic signalling cascades from diverse classes of receptors at the cyclin D-cyclin-dependent kinase-pRb-controlled G1 checkpoint. Mol Cell Biol 1996, 16(12):6917-6925.
• [63]Ludlow JW, Glendening CL, Livingston DM, DeCarprio JA: Specific enzymatic dephosphorylation of the retinoblastoma protein. Mol Cell Biol 1993, 13(1):367-372.
• [64]Hendzel MJ, Wei Y, Mancini MA, Van Hooser A, Ranalli T, Brinkley BR, Bazett-Jones DP, 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 1997, 106(6):348-360.
• [65]Ianzini F, Domann FE, Kosmacek EA, Phillips SL, Mackey MA: Human glioblastoma U87MG cells transduced with a dominant negative p53 (TP53) adenovirus construct undergo radiation-induced mitotic catastrophe. Radiat Res 2007, 168(2):183-192.
• [66]Mir SE, De Witt Hamer PC, Krawczyk PM, Balaj L, Claes A, Niers JM, Van Tilborg AA, Zwinderman AH, Geerts D, Kaspers GJ: In silico analysis of kinase expression identifies WEE1 as a gatekeeper against mitotic catastrophe in glioblastoma. Cancer Cell 2010, 18(3):244-257.
• [67]Jean-Claude BJ, Mustafa A, Damian Z, De Marte J, Vasilescu DE, Yen R, Chan TH, Leyland-Jones B: Cytokinetics of a novel 1,2,3-triazene-containing heterocycle, 8-nitro-3-methyl-benzo-1,2,3,5-tetrazepin-4(3H)-one (NIME), in the human epithelial ovarian cancer cell line OVCAR-3. Biochem Pharmacol 1999, 57(7):753-762.
• [68]Yoshikawa R, Kusunoki M, Yanagi H, Noda M, Furuyama JI, Yamamura T, Hashimoto-Tamaoki T: Dual antitumor effects of 5-fluorouracil on the cell cycle in colorectal carcinoma cells: a novel target mechanism concept for pharmacokinetic modulating chemotherapy. Cancer Res 2001, 61(3):1029-1037.
• [69]Hirose Y, Katayama M, Mirzoeva OK, Berger MS, Pieper RO: Akt activation suppresses Chk2-mediated, methylating agent-induced G2 arrest and protects from temozolomide-induced mitotic catastrophe and cellular senescence. Cancer Res 2005, 65(11):4861-4869.
• [70]Ohgaki H, Kleihues P: Genetic pathways to primary and secondary glioblastoma. Am J Pathol 2007, 170(5):1445-1453.