Proteasome inhibitors are emerging as a new class of anticancer agents. In this work, we examined the mechanisms underlying cytotoxicity, selectivity and adjuvant potential of the proteasome inhibitor MG132 in a panel of glioblastoma (GBM) cells (U138MG, C6, U87 and U373) and in normal astrocytes. MG132 markedly inhibited GBM cells growth irrespective of the p53 or PTEN mutational status of the cells whereas astrocytic viability was not affected, suggesting a selective toxicity of MG132 to cancerous glial cells. Mechanistically, MG132 arrested cells in G2/M phase of the cell cycle and increased p21WAF1 protein immunocontent. Following cell arrest, cells become apoptotic as shown by annexin-V binding, caspase-3 activation, chromatin condensation and formation of sub-G1 apoptotic cells. MG132 promoted mitochondrial depolarization and decreased the mitochondrial antiapoptotic protein bcl-xL; it also induced activation of JNK and p38, and inhibition of NFkappaB and PI3K/Akt survival pathways. Pre-treatment of GBMs with the mitochondrial permeability transition pore inhibitor, bongkrekic acid, or pharmacological inhibitors of JNK1/2 and p38, SP600125 and SB203580, attenuated MG132-induced cell death. Besides its apoptotic effect alone, MG132 also enhanced the antiglioma effect of the chemotherapeutics cisplatin, taxol and doxorubicin in C6 and U138MG cells, indicating an adjuvant/chemosensitizer potential. In summary, MG132 exerted profound and selective toxicity in GBMs, being a potential agent for further testing in animal models of the disease.
MG132 Glioblastoma Apoptosis Chemotherapy
This is a preview of subscription content, log in to check access.
We acknowledge the Brazilian funds CAPES, CNPq, and FINEP/IBNNet (01060842–00) for financial support. We thank Dr. Rafael Roesler (HCPA, Porto Alegre, Brazil) for gently provided U87 and U373 cell lines.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Adams J, Palombella VJ, Sausville EA, Johnson J, Destree A, Lazarus DD, Maas J, Pien CS, Prakash S, Elliott PJ (1999) Proteasome inhibitors: a novel class of potent and effective antitumor agents. Cancer Res 59:2615–2622PubMedGoogle Scholar
Baiz D, Pozzato G, Dapas B, Farra R, Scaggiante B, Grassi M, Uxa L, Giansante C, Zennaro C, Guarnieri G, Grassi G (2009) Bortezomib arrests the proliferation of hepatocellular carcinoma cells HepG2 and JHH6 by differentially affecting E2F1, p21 and p27 levels. Biochimie 91:373–382. doi:10.1016/j.biochi.2008.10.015PubMedCrossRefGoogle Scholar
Bredel M, Bredel C, Juric D, Duran GE, Yu RX, Harsh GR, Vogel H, Recht LD, Scheck AC, Sikic BI (2006) Tumor necrosis factor-alpha-induced protein 3 as a putative regulator of nuclear factor-kappaB-mediated resistance to O6-alkylating agents in human glioblastomas. J Clin Oncol 24:274–287. doi:10.1200/JCO.2005.02.9405PubMedCrossRefGoogle Scholar
Brennan C, Momota H, Hambardzumyan D, Ozawa T, Tandon A, Pedraza A, Holland E (2009) Glioblastoma subclasses can be defined by activity among signal transduction pathways and associated genomic alterations. PLoS One 4:e7752. doi:10.1371/journal.pone.0007752PubMedCrossRefGoogle Scholar
Brown RE, Law A (2006) Morphoproteomic demonstration of constitutive nuclear factor-kappaB activation in glioblastoma multiforme with genomic correlates and therapeutic implications. Ann Clin Lab Sci 36:421–426PubMedGoogle Scholar
Bubici C, Papa S, Pham CG, Zazzeroni F, Franzoso G (2006) The NF-kappaB-mediated control of ROS and JNK signaling. Histol Histopathol 21:69–80PubMedGoogle Scholar
da Frota ML, Braganhol E, Canedo AD, Klamt F, Apel MA, Mothes B, Lerner C, Battastini AM, Henriques AT, Moreira JC (2009) Brazilian marine sponge Polymastia janeirensis induces apoptotic cell death in human U138MG glioma cell line, but not in a normal cell culture. Invest New Drugs 27:13–20. doi:10.1007/s10637-008-9134-3PubMedCrossRefGoogle Scholar
Foti C, Florean C, Pezzutto A, Roncaglia P, Tomasella A, Gustincich S, Brancolini C (2009) Characterization of caspase-dependent and caspase-independent deaths in glioblastoma cells treated with inhibitors of the ubiquitin-proteasome system. Mol Cancer Ther 8:3140–3150. doi:10.1158/1535-7163.MCT-09-0431PubMedCrossRefGoogle Scholar
Frankel A, Man S, Elliott P, Adams J, Kerbel RS (2000) Lack of multicellular drug resistance observed in human ovarian and prostate carcinoma treated with the proteasome inhibitor PS-341. Clin Cancer Res 6:3719–3728PubMedGoogle Scholar
Ko BS, Chang TC, Chen CH, Liu CC, Kuo CC, Hsu C, Shen YC, Shen TL, Golubovskaya VM, Chang CC, Shyue SK, Liou JY (2010) Bortezomib suppresses focal adhesion kinase expression via interrupting nuclear factor-kappa B. Life Sci 86:199–206. doi:10.1016/j.lfs.2009.12.003PubMedCrossRefGoogle Scholar
Koschny R, Holland H, Sykora J, Haas TL, Sprick MR, Ganten TM, Krupp W, Bauer M, Ahnert P, Meixensberger J, Walczak H (2007) Bortezomib sensitizes primary human astrocytoma cells of WHO grades I to IV for tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. Clin Cancer Res 13:3403–3412. doi:10.1158/1078-0432.CCR-07-0251PubMedCrossRefGoogle Scholar
La Ferla-Brühl K, Westhoff MA, Karl S, Kasperczyk H, Zwacka RM, Debatin KM, Fulda S (2007) NF-kappaB-independent sensitization of glioblastoma cells for TRAIL-induced apoptosis by proteasome inhibition. Oncogene 26:571–582. doi:10.1038/sj.onc.1209841PubMedCrossRefGoogle Scholar
Lauricella M, Emanuele S, D’Anneo A, Calvaruso G, Vassallo B, Carlisi D, Portanova P, Vento R, Tesoriere G (2006) JNK and AP-1 mediate apoptosis induced by bortezomib in HepG2 cells via FasL/caspase-8 and mitochondria-dependent pathways. Apoptosis 11:607–625. doi:10.1007/s10495-006-4689-yPubMedCrossRefGoogle Scholar
Ling YH, Liebes L, Jiang JD, Holland JF, Elliott PJ, Adams J, Muggia FM, Perez-Soler R (2003) Mechanisms of proteasome inhibitor PS-341-induced G(2)-M-phase arrest and apoptosis in human non-small cell lung cancer cell lines. Clin Cancer Res 9:1145–1154PubMedGoogle Scholar
Lu G, Punj V, Chaudhary PM (2008) Proteasome inhibitor Bortezomib induces cell cycle arrest and apoptosis in cell lines derived from Ewing’s sarcoma family of tumors and synergizes with TRAIL. Cancer Biol Ther 7: 603–608. doi: http://dx.doi.org/10.4161/cbt.7.4.5564
Monticone M, Biollo E, Fabiano A, Fabbi M, Daga A, Romeo F, Maffei M, Melotti A, Giaretti W, Corte G, Castagnola P (2009) z-Leucinyl-leucinyl-norleucinal induces apoptosis of human glioblastoma tumor-initiating cells by proteasome inhibition and mitotic arrest response. Mol Cancer Res 7:1822–1834. doi:10.1158/1541-7786.MCR-09-0225PubMedCrossRefGoogle Scholar
Nagane M, Levitzki A, Gazit A, Cavenee WK, Huang HJ (1998) Drug resistance of human glioblastoma cells conferred by a tumor-specific mutant epidermal growth factor receptor through modulation of Bcl-XL and caspase-3-like proteases. Proc Natl Acad Sci USA 95:5724–5729PubMedCrossRefGoogle Scholar
Robe PA, Bentires-Alj M, Bonif M, Rogister B, Deprez M, Haddada H, Khac MT, Jolois O, Erkmen K, Merville MP, Black PM, Bours V (2004) In vitro and in vivo activity of the nuclear factor-kappaB inhibitor sulfasalazine in human glioblastomas. Clin Cancer Res 10:5595–5603. doi:10.1158/1078-0432.CCR-03-0392PubMedCrossRefGoogle Scholar
Thaker NG, Zhang F, McDonald PR, Shun TY, Lewen MD, Pollack IF, Lazo JS (2009) Identification of survival genes in human glioblastoma cells by small interfering RNA screening. Mol Pharmacol 76:1246–1255. doi:10.1124/mol.109.058024PubMedCrossRefGoogle Scholar
Vaziri SA, Hill J, Chikamori K, Grabowski DR, Takigawa N, Chawla-Sarkar M, Rybicki LR, Gudkov AV, Mekhail T, Bukowski RM, Ganapathi MK, Ganapathi R (2005) Sensitization of DNA damage-induced apoptosis by the proteasome inhibitor PS-341 is p53 dependent and involves target proteins 14-3-3-sigma and survivin. Mol Cancer Ther 4:1880–1890. doi:10.1158/1535-7163.MCT-05-0222PubMedCrossRefGoogle Scholar
Yang Y, Shao N, Luo G, Li L, Zheng L, Nilsson-Ehle P, Xu N (2010) Mutations of PTEN gene in gliomas correlate to tumor differentiation and short-term survival rate. Anticancer Res 30:981–985PubMedGoogle Scholar
Yin D, Zhou H, Kumagai T, Liu G, Ong JM, Black KL, Koeffler HP (2005) Proteasome inhibitor PS-341 causes cell growth arrest and apoptosis in human glioblastoma multiforme (GBM). Oncogene 24:344–354. doi:10.1038/sj.onc.1208225PubMedCrossRefGoogle Scholar
Zanotto-Filho A, Delgado-Cañedo A, Schröder R, Becker M, Klamt F, Moreira JC (2010) The pharmacological NFkappaB inhibitors BAY117082 and MG132 induce cell arrest and apoptosis in leukemia cells through ROS-mitochondria pathway activation. Cancer Lett 288:192–203. doi:10.1016/j.canlet.2009.06.038PubMedCrossRefGoogle Scholar
Zanotto-Filho A, Gelain DP, Schröder R, Souza LF, Pasquali MA, Klamt F, Moreira JCF (2009) The NFkappaB-mediated control of RS and JNK signaling in vitamin A-treated cells: duration of JNK-AP-1 pathway activation may determine cell death or proliferation. Biochem Pharmacol 77:1291–1301. doi:10.1016/j.bcp.2008.12.010PubMedCrossRefGoogle Scholar