Abstract
Background
Glioblastoma multiforme (GBM) is the most malignant and invasive human brain tumor, and it is characterized by a poor prognosis and short survival time. Current treatment strategies for GBM, using surgery, chemotherapy and/or radiotherapy, are ineffective. The PI3K/AKT/PTEN signaling pathway is frequently deregulated in this cancer, and it is connected with regulation of the cell cycle, apoptosis, and autophagy.
Objectives
The current study was undertaken to examine the effect of small interfering RNA (siRNA) targeting the AKT3 and PIK3CA genes on the susceptibility of T98G cells to temozolomide (TMZ) and carmustine (BCNU).
Methods
T98G cells were transfected with AKT3 or PI3KCA siRNA. Transfection efficiency was assessed using flow cytometry and fluorescence microscopy. The influence of AKT3 and PI3KCA siRNA in combination with TMZ and BCNU on T98G cell viability, proliferation, apoptosis, and autophagy was evaluated as well. Alterations in messenger RNA (mRNA) expression of apoptosis-related and autophagy-related genes were analyzed using quantitative reverse transcription polymerase chain reaction (QRT-PCR).
Results
Transfection of T98G cells with AKT3 or PI3KCA siRNA and exposure to TMZ and BCNU led to a significant reduction in cell viability, accumulation of subG1-phase cells, and reduction of cells in the S and G2/M phases, as well as induction of apoptosis or necrosis, and regulation of autophagy.
Conclusion
The siRNA-induced AKT3 and PI3KCA mRNA knockdown in combination with TMZ and BCNU inhibited proliferation and induced apoptosis and autophagy in T98G cells. Thus, knockdown of these genes in combination with TMZ and BCNU may offer a novel therapeutic strategy to more effectively control the growth of human GBM cells, but further studies are necessary to confirm a positive phenomenon for the treatment of GBM.
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References
Barciszewska AM, Gurda D, Głodowicz P, Nowak S, Naskręt-Barciszewska MZ. A new epigenetic mechanism of temozolomide action in glioma cells. PLoS One. 2015;10(8):e0136669. doi:10.1371/journal.pone.0136669 (eCollection 2015).
Nagpal S. The role of BCNU polymer wafers (Gliadel) in the treatment of malignant glioma. Neurosurg Clin N Am. 2012;23(2):289–95, ix. doi:10.1016/j.nec.2012.01.004.
Zou Y, Wang Q, Li B, Xie B, Wang W. Temozolomide induces autophagy via ATM–AMPK–ULK1 pathways in glioma. Mol Med Rep. 2014;1:411–6. doi:10.3892/mmr.2014.2151.
Jakubowicz-Gil J, Langner E, Bądziul D, Wertel I, Rzeski W. Apoptosis induction in human glioblastoma multiforme T98G cells upon temozolomide and quercetin treatment. Tumour Biol. 2013;34(4):2367–78. doi:10.1007/s13277-013-0785-0.
Knizhnik AV, Roos WP, Nikolova T, Quiros S, Tomaszowski KH, Christmann M, Kaina B. Survival and death strategies in glioma cells: autophagy, senescence and apoptosis triggered by a single type of temozolomide-induced DNA damage. PLoS One. 2013;8(1):e55665. doi:10.1371/journal.pone.0055665.
Chen MY, Clark AJ, Chan DC, Ware JL, Holt SE, Chidambaram A, Fillmore HL, Broaddus WC. Wilms’ tumor 1 silencing decreases the viability and chemoresistance of glioblastoma cells in vitro: a potential role for IGF-1R de-repression. J Neurooncol. 2011;103(1):87–102. doi:10.1007/s11060-010-0374-7.
Zhu Y, Zhao L, Liu L, Gao P, Tian W, Wang X, Jin H, Xu H, Chen Q. Beclin 1 cleavage by caspase-3 inactivates autophagy and promotes apoptosis. Protein Cell. 2010;1:468–77. doi:10.1007/s13238-010-0048-4.
Goldar S, Khaniani MS, Derakhshan SM, Baradaran B. Molecular mechanisms of apoptosis and roles in cancer development and treatment. Asian Pac J Cancer Prev. 2015;16(6):2129–44.
Ryter SW, Mizumura K, Choi AM. The impact of autophagy on cell death modalities. Int J Cell Biol. 2014:2014:502676. doi:10.1155/2014/502676.
Zhi X, Zhong Q. Autophagy in cancer. F1000 Prime Rep. 2015;7:18. doi:10.12703/P7-18.
Hönscheid P, Datta K, Muders MH. Autophagy: detection, regulation and its role in cancer and therapy response. Int J Radiat Biol. 2014;90(8):628–35. doi:10.3109/09553002.2014.907932.
Ryter SW, Cloonan SM, Choi AM. Autophagy: a critical regulator of cellular metabolism and homeostasis. Mol Cells. 2013;36(1):7–16. doi:10.1007/s10059-013-0140-8.
Polewska J. Autophagy—molecular mechanism, apoptosis and cancer. Postepy Hig Med Dosw (Online). 2012;66:921–36. doi:10.5604/17322693.1021109.
Thorburn A, Thamm DH, Gustafson DL. Autophagy and cancer therapy. Mol Pharmacol. 2014;85(6):830–8. doi:10.1124/mol.114.091850.
Zhang J. Teaching the basics of autophagy and mitophagy to redox biologists—mechanisms and experimental approaches. Redox Biol. 2015;4C:242–59. doi:10.1016/j.redox.2015.01.003.
Liu Y, Levine B. Autosis and autophagic cell death: the dark side of autophagy. Cell Death Differ. 2015;22(3):367–76. doi:10.1038/cdd.2014.143.
Tanaka S, Louis DN, Curry WT, Batchelor TT, Dietrich J. Diagnostic and therapeutic avenues for glioblastoma: no longer a dead end? Nat Rev Clin Oncol. 2013;10:14–26.
Wen PY, Lee EQ, Reardon DA, Ligon KL, Alfred Yung WK. Current clinical development of PI3K pathway inhibitors in glioblastoma. Neuro Oncol. 2012;14(7):819–29. doi:10.1093/neuonc/nos117.
Stupp R, Mason WP, van den Bent MJ, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 2005;352:987–96.
Burris HA. Overcoming acquired resistance to anticancer therapy: focus on the PI3K/AKT/mTOR pathway. Cancer Chemother Pharmacol. 2013;71:829–42.
Sami A, Karsy M. Targeting the PI3K/AKT/mTOR signaling pathway in glioblastoma: novel therapeutic agents and advances in understanding. Tumour Biol. 2013;34:1991–2002.
Hafsi S, Pezzino FM, Candido S, Ligresti G, Spandidos DA, Soua Z, et al. Gene alterations in the PI3K/PTEN/AKT pathway as a mechanism of drug-resistance (review). Int J Oncol. 2012;40:639–44.
Jin W, Wu L, Liang K, Liu B, Lu Y, Fan Z. Roles of the PI-3K and MEK pathways in Ras-mediated chemoresistance in breast cancer cells. Br J Cancer. 2003;89(1):185–91.
Krześlak A. Akt kinase: a key regulator of metabolism and progression of tumors. Postepy Hig Med Dosw. 2010;64:490–503.
Mure H, Matsuzaki K, Kitazato KT, Mizobuchi Y, Kuwayama K, Kageji T, et al. Akt2 and Akt3 play a pivotal role in malignant gliomas. Neuro Oncol. 2010;12:221–32.
Matheny RW, Adamo ML. Current perspectives on Akt Akt-ivation and Akt-ions. Exp Biol Med (Maywood). 2009;234:1264–70.
Young CD, Anderson SM. Sugar and fat—that’s where it’s at: metabolic changes in tumors. Breast Cancer Res. 2008;10:202.
Lefranc F, Facchini V, Kiss R. Proautophagic drugs: a novel means to combat apoptosis-resistant cancers, with a special emphasis on glioblastomas. Oncologist. 2007;12:1395–403.
Paul-Samojedny M, Suchanek R, Borkowska P, Pudełko A, Owczarek A, Kowalczyk M, Machnik G, Fila-Daniłow A, Kowalski J. Knockdown of AKT3 (PKBγ) and PI3KCA suppresses cell viability and proliferation and induces the apoptosis of glioblastoma multiforme T98G cells. Biomed Res Int. 2014;2014:768181. doi:10.1155/2014/768181.
Paul-Samojedny M, Pudełko A, Suchanek-Raif R, Kowalczyk M, Fila-Daniłow A, Borkowska P, Kowalski J. Knockdown of the AKT3 (PKBγ), PI3KCA, and VEGFR2 genes by RNA interference suppresses glioblastoma multiforme T98G cells invasiveness in vitro. Tumour Biol. 2014;36(5):3263–77. doi:10.1007/s13277-014-2955-0.
Stein GH. T98G: an anchorage-independent human tumor cell line that exhibits stationary phase G1 arrest in vitro. J Cell Physiol. 1979;99:43–54.
Nanegrungsunk D, Onchan W, Chattipakorn N, Chattipakorn SC. Current evidence of temozolomide and bevacizumab in treatment of gliomas. Neurol Res. 2015;37(2):167–83. doi:10.1179/1743132814Y.0000000423.
Hirose Y, Katayama M, Mirzoeva OK, Berger MS, Piper RO. Akt activation suppress Chk2-mediated, methylating agent-induced G2 arrest and protects from temozolomide-induced mitotic catastrophe and cellular senescence. Cancer Res. 2005;65:4861–9.
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;11:448–57.
Markiewicz-Żukowska R, Borawska MH, Fiedorowicz A, Naliwajko SK, Sawicka D, Car H. Propolis changes the anticancer activity of temozolomide in U87MG human glioblastoma cell line. BMC Complement Altern Med. 2013;13:50. doi:10.1186/1472-6882-13-50.
Khoshyomn S, Nathan D, Manske GC, Osler TM, Penar PL. Synergistic effect of genistein and BCNU on growth inhibition and cytotoxicity of glioblastoma cells. J Neurooncol. 2002;57(3):193–200.
Darzynkiewicz Z, Robinson JP, Crissman HA, editors. Flow cytometry. Methods in cell biology, 41, 42. Academic Press, Inc., San Diego; 1994.
Henery S, George T, Hall B, Basiji D, Ortyn W, Morrissey P. Quantitative image based apoptotic index measurement using multispectral imaging flow cytometry: a comparison with standard photometric methods. Apoptosis. 2008;13(8):1054–63.
Teicher BA, Menon K, Avarez E, Galbreath E, Shih C, Faul M. Antiangiogenic and antitumor effects of protein kinase Cb inhibitor in human T98G glioblastoma multiforme xenografts. Clin Cancer Res. 2001;7(3):634–40.
Opel D, Westhoff MA, Bender A, Braun V, Debatin KM, Fulda S. Phosphatidylinositol 3-kinase inhibition broadly sensitizes glioblastoma cells to death receptor- and drug-induced apoptosis. Cancer Res. 2008;68(15):6271–80. doi:10.1158/0008-5472.CAN-07-6769.
De Salvo M, Maresca G, D’Agnano I, Marchese R, Stigliano A, Gagliassi R, Brunetti E, Raza GH, De Paula U, Bucci B. Temozolomide induced c-Myc-mediated apoptosis via Akt signalling in MGMT expressing glioblastoma cells. Int J Radiat Biol. 2011;87(5):518–33. doi:10.3109/09553002.2011.556173.
Carmo A, Carvalheiro H, Crespo I, Nunes I, Lopes MC. Effect of temozolomide on the U-118 glioma cell line. Oncol Lett. 2011;2(6):1165–70.
Kanzawa T, Zhang L, Xiao L, Germano IM, Kondo Y, Kondo S. Arsenic trioxide induces autophagic cell death in malignant glioma cells by upregulation of mitochondrial cell death protein BNIP3. Oncogene. 2005;24:980–91.
Varna M, Ratajczak P, Bertheau P, Janin A. BCL2L1 (BCL2-like 1). Atlas Genet Cytogenet Oncol Haematol. 2010;14(9):866.
Scarlatti F, Granata R, Meijer AJ, Codogno P. Does autophagy have a license to kill mammalian cells? Cell Death Differ. 2009;16:12–20. doi:10.1038/cdd.2008.101.
Kondo Y, Kanzawa T, Sawaya R, Kondo S. The role of autophagy in cancer development and response to therapy. Nat Rev Cancer. 2005;5:726–34.
Hoyer-Hansen M, Bastholm L, Mathiasen IS, Elling F, Jaattela M. Vitamin D analog EB1089 triggers dramatic lysosomal changes and Beclin 1-mediated autophagic cell death. Cell Death Differ. 2005;12:1297–309.
Shimizu S, Kanaseki T, Mizushima N, Mizuta T, Arakawa-Kobayashi S, Thompson CB, Tsujimoto Y. Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes. Nat Cell Biol. 2004;6:1221–8.
Boya P, Gonzalez-Polo RA, Casares N, Perfettini JL, Dessen P, Larochette N, Métivier D, Meley D, Souquere S, Yoshimori T, Pierron G, Codogno P, Kroemer G. Inhibition of macroautophagy triggers apoptosis. Mol Cell Biol. 2005;25:1025–40.
Gonzalez-Polo RA, Boya P, Pauleau AL, Jalil A, Larochette N, Souquere S, Eskelinen EL, Pierron G, Saftig P, Kroemer G. The apoptosis/autophagy paradox: autophagic vacuolization before apoptotic death. J Cell Sci. 2005;118:3091–102.
Kroemer G, Jaattela M. Lysosomes and autophagy in cell death control. Nat Rev Cancer. 2005;5:886–97.
Yu L, Wan F, Dutta S, Welsh S, Liu Z, Freundt E, Baehrecke EH, Lenardo M. Autophagic programmed cell death by selective catalase degradation. Proc Natl Acad Sci USA. 2006;103:4952–7.
Roy B, Pattanaik AK, Das J, Bhutia SK, Behera B, Singh P, Maiti TK. Role of PI3K/Akt/mTOR and MEK/ERK pathway in concanavalin A induced autophagy in HeLa cells. Chem Biol Interact. 2014;210:96–102. doi:10.1016/j.cbi.2014.01.003.
Ren Y, Huang F, Liu Y, Yang Y, Jiang Q, Xu C. Autophagy inhibition through PI3K/Akt increases apoptosis by sodium selenite in NB4 cells. BMB Rep. 2009;42(9):599–604.
Chen S, Rehman SK, Zhang W, Wen A, Yao L, Zhang J. Autophagy is a therapeutic target in anticancer drug resistance. Biochim Biophys Acta. 2010;1806(2):220–9. doi:10.1016/j.bbcan.2010.07.003.
Paul-Samojedny M, Pudełko A, Kowalczyk M, Fila-Daniłow A, Suchanek-Raif R, Borkowska P, Kowalski J. Knockdown of AKT3 and PI3KCA by RNA interference changes the expression of the genes that are related to apoptosis and autophagy in T98G glioblastoma multiforme cells. Pharmacol Rep. 2015;67(6):1115–23. doi:10.1016/j.pharep.2015.04.012.
Su M, Mei Y, Sinha S. Role of the crosstalk between autophagy and apoptosis in cancer. J Oncol. 2013;2013:102735. doi:10.1155/2013/102735.
Alers S, Löffler AS, Wesselborg S, Gao P, Tian W, Wang X, Jin H, Xu H, Chen Q. Role of AMPK-mTOR-Ulk1/2 in the regulation of autophagy: cross talk, shortcuts, and feedbacks. Mol Cell Biol. 2012;32:2–11. doi:10.1128/MCB.06159-11.
Chang YY, Neufeld TP. An Atg1/Atg13 complex with multiple roles in TOR-mediated autophagy regulation. Mol. Biol. Cell. 2009;20:2004–14. doi:10.1091/mbc.E08-12-1250.
Yang Z, Klionsky DJ. Mammalian autophagy: core molecular machinery and signaling regulation. Curr Opin Cell Biol. 2010;22:124–31. doi:10.1016/j.ceb.2009.11.014.
He CK, Klionsky DJ. Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet. 2009;43:67–93. doi:10.1146/annurev-genet-102808-114910.
Jung CH, Seo M, Otto NM, Kim DH. ULK1 inhibits the kinase activity of mTORC1 and cell proliferation. Autophagy. 2011;7(10):1212–21. doi:10.4161/auto.7.10.16660.
Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G, et al. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy. 2008;4(2):151–75.
Sinha S, Levine B. The autophagy effector Beclin 1: a novel BH3-only protein. Oncogene. 2008;27(Suppl 1):S137–48. doi:10.1038/onc.2009.51.
Fimia GM, Stoykova A, Romagnoli A, Giunta L, Di Bartolomeo S, Nardacci R, Corazzari M, Fuoco C, Ucar A, Schwartz P, Gruss P, Piacentini M, Chowdhury K, Cecconi F. Ambra1 regulates autophagy and development of the nervous system. Nature. 2007;447:1121–5.
Fimia GM, Corazzari M, Antonioli M, Piacentini M. Ambra1 at the crossroad between autophagy and cell death. Oncogene. 2013;32:3311–8. doi:10.1038/onc.2012.455.
Kang R, Zeh HJ, Lotze MT, Tang D. The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ. 2011;18:571–80. doi:10.1038/cdd.2010.191.
Oberstein A, Jeffrey PD, Shi Y. Crystal structure of the Bcl-XL-Beclin 1 peptide complex: Beclin 1 is a novel BH3-only protein. J Biol Chem. 2007;282:13123–32.
Shimizu S, Kanaseki T, Mizushima N, Mizuta T, Arakawa-Kobayashi S, Thompson CB, Tsujimoto Y. Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes. Nat Cell Biol. 2004;6:1221–8.
Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, Packer M, Schneider MD, Levine B. Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell. 2005;122:927–39.
Pattingre S, Levine B. Bcl-2 inhibition of autophagy: a new route to cancer? Cancer Res. 2006;66:2885–8.
Krishan S, Richardson DR, Sahni S. Gene of the month: AMP kinase (PRKAA1). J Clin Pathol. 2014;67(9):758–63.
Mah LY, O’Prey J, Baudot AD, Hoekstra A, Ryan KM. DRAM-1 encodes multiple isoforms that regulate autophagy. Autophagy. 2012;8(1):18–28. doi:10.4161/auto.8.1.18077.
Guan JJ, Zhang XD, Sun W, Qi L, Wu JC, Qin ZH. DRAM1 regulates apoptosis through increasing protein levels and lysosomal localization of BAX. Cell Death Dis. 2015;6:e1624. doi:10.1038/cddis.2014.546.
Galavotti S, Bartesaghi S, Faccenda D, Shaked-Rabi M, Sanzone S, McEvoy A, Dinsdale D, Condorelli F, Brandner S, Campanella M, Grose R, Jones C, Salomoni P. The autophagy-associated factors DRAM1 and p62 regulate cell migration and invasion in glioblastoma stem cells. Oncogene. 2013;32(6):699–712. doi:10.1038/onc.2012.111.
Wang Q, Qian J, Wang J, Luo C, Chen J, Hu G, Lu Y. Knockdown of RLIP76 expression by RNA interference inhibits invasion, induces cell cycle arrest, and increases chemosensitivity to the anticancer drug temozolomide in glioma cells. J Neurooncol. 2013;112(1):73–82. doi:10.1007/s11060-013-1045-2.
Rubinfeld B, Albert I, Porfiri E, Fiol C, Munemitsu S, Polakis P. Binding of GSK3beta to the APC-beta-catenin complex and regulation of complex assembly. Science. 1996;272:1023–6.
Carmeliet P, Dor Y, Herbert JM, Fukumura D, Brusselmans K, Dewerchin M, Neeman M, Bono F, Abramovitch R, Maxwell P, Koch CJ, Ratcliffe P, Moons L, Jain RK, Collen D, Keshert E. Role of HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature. 1998;394:485–90.
Zhong H, De Marzo AM, Laughner E, Lim M, Hilton DA, Zagzag D, Buechler P, Isaacs WB, Semenza GL, Simons JW. Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their metastases. Cancer Res. 1999;59:5830–5.
Zhao Z, Ni D, Ghozalli I, Pirooz SD, Ma B, Liang C. UVRAG: at the crossroad of autophagy and genomic stability. Autophagy. 2012;8:1392–3. doi:10.4161/auto.21035.
Yin X, Cao L, Peng Y, Tan Y, Xie M, Kang R, Livesey KM, Tang D. A critical role for UVRAG in apoptosis. Autophagy. 2011;7:1242–4. doi:10.4161/auto.7.10.16507.
Hara T, Mizushima N. Role of ULK-FIP200 complex in mammalian autophagy: FIP200, a counterpart of yeast Atg17? Autophagy. 2009;5:85–7.
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This work was supported by a grant from the Medical University of Silesia. The University had no further role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication.
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Monika Paul-Samojedny, Adam Pudełko, Malgorzata Kowalczyk, Anna Fila-Daniłow, Renata Suchanek-Raif, Paulina Borkowska, and Jan Kowalski have no conflicts of interest that are directly relevant to the content of this study.
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Paul-Samojedny, M., Pudełko, A., Kowalczyk, M. et al. Combination Therapy with AKT3 and PI3KCA siRNA Enhances the Antitumor Effect of Temozolomide and Carmustine in T98G Glioblastoma Multiforme Cells. BioDrugs 30, 129–144 (2016). https://doi.org/10.1007/s40259-016-0160-y
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DOI: https://doi.org/10.1007/s40259-016-0160-y