Skip to main content

Advertisement

SpringerLink
Log in

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

  • Original research article
  • Published:
Pharmacological Reports Aims and scope Submit manuscript

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. The PI3K/AKT/PTEN signaling pathway plays a crucial role in GBM development and it is connected with the regulation of apoptosis and autophagy. Akt is involved in various aspects of cancer cell biology such as cell survival, in addition to both apoptosis and autophagy.

The current study was undertaken to examine the effect of the siRNAs that target AKT3 and PI3KCA genes on the apoptosis and autophagy of T98G cells.

Methods

T98G cells were transfected with AKT3 and/or PI3KCA siRNAs. Alterations in the mRNA expression of apoptosis- and autophagy-related genes were analyzed using QRT-PCR. LC3IIA protein-positive cells were identified using flow cytometry with specific antibodies.

Results

Our findings demonstrate for the first time that the siRNAs that target AKT3 and PI3KCA change the expression of the genes that are related to apoptosis and autophagy and change the expression of the LC3IIA protein in T98G cells.

Conclusions

Thus, there is a high probability that the knockdown of these genes induces apoptosis and autophagy in T98G cells, but further studies are necessary in order to clarify and check whether autophagy induction is a positive phenomenon for the treatment of GBM.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Mrugala MM. Advances and challenges in the treatment of glioblastoma: a clinician’s perspective. Discov Med 2013;15:221–30.

    PubMed  Google Scholar 

  2. Burris HA. Overcoming acquired resistance to anticancer therapy: focus on the PI3K/AKT/mTOR pathway. Cancer Chemother Pharmacol 2013;71:829–42.

    Article  CAS  PubMed  Google Scholar 

  3. Sami A, Karsy M. Targeting the PI3K/AKT/mTOR signaling pathway in glioblastoma: novel therapeutic agents and advances in understanding. Tumour Biol 2013;34:1991–2.

    Article  CAS  PubMed  Google Scholar 

  4. 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.

    Article  CAS  PubMed  Google Scholar 

  5. 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. Int J Oncol 2012;40:639–44 [Review].

    CAS  PubMed  Google Scholar 

  6. 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.

    Article  CAS  PubMed  Google Scholar 

  7. Weber GL, Parat M-O, Binder ZA, Gallia GL, Riggins GJ. Abrogation of PI3KCA or PIK3R1 reduces proliferation, migration and invasion in glioblastoma multiforme cells. Oncotarget 2011;2:833–49.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Zhou X-K, Tang S-S, Yi G, Hou M, Chen J-H, Yang B, et al. RNAi knockdown of PI3KCA preferentially inhibits invasion of mutant PI3KCA cells. World J Gastroenterol 2011;17:3700–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Mitsutoshi N, Daisuke K, Takuya W, Yutaka H, Lei T, Ilya VP. Aberrant signaling pathways in glioma. Cancers 2011;3(3):3242–78.

    Article  CAS  Google Scholar 

  10. Knobbe CB, Reifenberger G. Genetic alterations and aberrant expression of genes related to the phosphatidyl-inositol-3′-kinase/protein kinase B (Akt) signal transduction pathway in glioblastomas. Brain Pathol 2003;13:507–18.

    Article  CAS  PubMed  Google Scholar 

  11. Mendoza MC, Blenis J. PHLPPing it off: phosphatases get in the Akt. Mol Cell 2007;25:798–800.

    Article  CAS  PubMed  Google Scholar 

  12. Duronio V. The life of a cell: apoptosis regulation by the PI3K/PKB pathway. Biochem J 2008;415:333–44.

    Article  CAS  PubMed  Google Scholar 

  13. Parcellier A, Tintignac LA, Zhuravleva E, Hemmings BA. PKB and the mitochondria: AKTing on apoptosis. Cell Signal 2008;20:21–30.

    Article  CAS  PubMed  Google Scholar 

  14. Fulda S. Modulation of mitochondrial apoptosis by PI3K inhibitors. Mitochondrion 2013;13:195–8.

    Article  CAS  PubMed  Google Scholar 

  15. Fan Q-W, Weiss WA. Targeting the RTK-PI3K-mTOR axis in malignant glioma: overcoming resistance. Curr Top Microbiol Immunol 2010;347:279–96.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Laplante M, Sabatini DM. Regulation of mTORC1 and its impact on gene expression at a glance. J Cell Sci 2013;126:1713–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 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.

    Article  CAS  PubMed  Google Scholar 

  18. Paul-Samojedny M, Suchanek R, Borkowska P, Pudełko A, Owczarek A, Kowalczyk M, et al. Knockdown of AKT3 (PKBγ) and PI3KCA suppresses cell viability and proliferation and induces the apoptosis of glioblastoma multiforme T98G cells. Biomed Res Int 2014;2014:1–12 [ID 768181].

    Article  Google Scholar 

  19. Tanida I, Ueno T, Kominami E. LC3 conjugation system in mammalian autophagy. Int J Biochem Cell Biol 2004;36:2503–18.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. He H, Dang Y, Dai F, Guo Z, Wu J, She X, et al. Post-translational modifications of three members of the human MAP1LC3 family and detection of a novel type of modification for MAP1LC3B. J Biol Chem 2003;278(31):29278–87.

    Article  CAS  PubMed  Google Scholar 

  21. Sukhdeo K, Paramban RI, Vidal JG, Elia J, Martin J, Rivera M, et al. Multiplex flow cytometry barcoding and antibody arrays identify surface antigen profiles of primary and metastatic colon cancer cell lines. PLOS ONE 2013;8(1):e53015.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Ziegler DS, Kung AL, Kieran MW. Anti-apoptosis mechanisms in malignant gliomas. J Clin Oncol 2008;26:493–500.

    Article  CAS  PubMed  Google Scholar 

  23. 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.

    Article  CAS  PubMed  Google Scholar 

  24. Gozuacik D, Kimchi A. Autophagy as a cell death and tumor suppressor mechanism. Oncogene 2004;23:2891–6.

    Article  CAS  PubMed  Google Scholar 

  25. 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;42:599–604.

    Article  CAS  PubMed  Google Scholar 

  26. Klionsky DJ, Meijer AJ, Codogno P. Autophagy and p70S6 kinase. Autophagy 2005;1:59–60.

    Article  CAS  PubMed  Google Scholar 

  27. Degenhardt K, Mathew R, Beaudoin B, Bray K, Anderson D, Chen G, et al. Autophagy promotes tumor cell survival and restricts necrosis, inflammation and tumorigenesis. Cancer Cell 2006;10:51–64.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Degtyarev M, De Mazière A, Klumperman J, Bray K, Anderson D, Chen G, et al. Autophagy, an achilles’ heel AKTing against cancer? Autophagy 2009;5:415–8.

    Article  CAS  PubMed  Google Scholar 

  29. González-Polo R-A, Boya P, Pauleau A-L, Jalil A, Larochette N, Souquère S, et al. The apoptosis/autophagy paradox: autophagic vacuolization before apoptotic death. J Cell Sci 2005;118:3091–102.

    Article  PubMed  CAS  Google Scholar 

  30. Janku F, McConkey DJ, Hong DS, Kurzrock R. Autophagy as a target for anticancer therapy. Nat Rev Clin Oncol 2011;8:528–39.

    Article  CAS  PubMed  Google Scholar 

  31. Amaravadi RK, Yu D, Lum JJ, Bui T, Christophorou MA, Evan GI, et al. Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma. J Clin Investig 2007;117:326–36.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Lemasters JJ, Nieminen AL, Qian T, Trost LC, Elmore SP, Nishimura Y, et al. The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. Biochim Biophys Acta 1998;1366:177–96.

    Article  CAS  PubMed  Google Scholar 

  33. Zhu Y, Zhao L, Liu L, Gao P, Tian W, Wang X, et al. Beclin 1 cleavage by caspase-3 inactivates autophagy and promotes apoptosis. Protein Cell 2010;1:468–77.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Alers S, Löffler AS, Wesselborg S, Gao P, Tian W, Wang X, et al. Role of AMPK-mTOR-Ulk1/2 in the regulation of autophagy: cross talk, shortcuts and feedbacks. Mol Cell Biol 2012;32:2–11.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Chan EY, Tooze SA. Evolution of Atg1 function and regulation. Autophagy 2009;5:758–65.

    Article  CAS  PubMed  Google Scholar 

  36. Hara T, Mizushima N. Role of ULK-FIP200 complex in mammalian autophagy: FIP200, a counterpart of yeast Atg17? Autophagy 2009;5:85–7.

    Article  CAS  PubMed  Google Scholar 

  37. Sinha S, Levine B. The autophagy effector Beclin 1: a novel BH3-only protein. Oncogene 2008;27(Suppl. 1):S137–48.

    Article  CAS  Google Scholar 

  38. Strappazzon F, Vietri-Rudan M, Campello S, Nazio F, Florenzano F, Fimia GM. Mitochondrial BCL-2 inhibits AMBRA1-induced autophagy. EMBO J 2011;30: 1195–208.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Fimia GM, Stoykova A, Romagnoli A, Giunta L, Di Bartolomeo S, Nardacci R, et al. Ambra1 regulates autophagy and development of the nervous system. Nature 2007;447:1121–5.

    Article  CAS  PubMed  Google Scholar 

  40. Fimia GM, Corazzari M, Antonioli M, Piacentini M. Ambra1 at the crossroad between autophagy and cell death. Oncogene 2013;32:3311–8.

    Article  CAS  PubMed  Google Scholar 

  41. 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.

    Article  CAS  PubMed  Google Scholar 

  42. Kang R, Zeh HJ, Lotze MT, Tang D. The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ 2011;18:571–80.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. 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.

    Article  CAS  PubMed  Google Scholar 

  44. Pattingre S, Levine B. Bcl-2 inhibition of autophagy: a new route to cancer? Cancer Res 2006;66:2885–8.

    Article  CAS  PubMed  Google Scholar 

  45. Miracco C, Cosci E, Oliveri G, Luzi P, Pacenti L, Monciatti I, et al. Protein and mRNA expression of autophagy gene Beclin 1 in human brain tumours. Int J Oncol 2007;30:429–36.

    CAS  PubMed  Google Scholar 

  46. Sun Q, Fan W, Zhong Q. Regulation of Beclin 1 in autophagy. Autophagy 2009;5:713–6.

    Article  CAS  PubMed  Google Scholar 

  47. 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Yin X, Cao L, Peng Y, Tan Y, Xie M, Kang R, et al. A critical role for UVRAG in apoptosis. Autophagy 2011;7:1242–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. 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.

    Article  CAS  PubMed  Google Scholar 

  50. Burton TR, Eisenstat DD, Gibson SB. BNIP3 (Bcl-2 19 kDa interacting protein) acts as transcriptional repressor of apoptosis-inducing factor expression preventing cell death in human malignant gliomas. J Neurosci 2009;29:4189–99.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Seglen PO, Gordon PB. 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Natl Acad Sci U S A 1982;79:1889–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Yamamoto A, Tagawa Y, Yoshimori T, Moriyama Y, Masaki R, Tashiro Y. Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct Funct 1998;23:33–42.

    Article  CAS  PubMed  Google Scholar 

  53. 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.

    Article  CAS  PubMed  Google Scholar 

  54. Fimia GM, Piacentini M. Regulation of autophagy in mammals and its interplay with apoptosis. Cell Mol Life Sci 2010;201(67):1581–8.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Medical Genetics, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, Katowice, Poland

    Monika Paul-Samojedny, Małgorzata Kowalczyk, Anna Fila-Daniłow, Renata Suchanek-Raif, Paulina Borkowska & Jan Kowalski

  2. Department of Clinical Chemistry and Laboratory Diagnostics, Medical University of Silesia, Katowice, Poland

    Adam Pudełko

Authors
  1. Monika Paul-Samojedny
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Adam Pudełko
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Małgorzata Kowalczyk
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anna Fila-Daniłow
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Renata Suchanek-Raif
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Paulina Borkowska
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jan Kowalski
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Monika Paul-Samojedny.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Paul-Samojedny, M., Pudełko, A., Kowalczyk, M. et al. 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 67, 1115–1123 (2015). https://doi.org/10.1016/j.pharep.2015.04.012

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1016/j.pharep.2015.04.012

Keywords

Search

Navigation