Bcl-2 siRNA Augments Taxol Mediated Apoptotic Death in Human Glioblastoma U138MG and U251MG Cells
- 363 Downloads
The anti-neoplastic drug taxol binds to β-tubulin to prevent tumor cell division, promoting cell death. However, high dose taxol treatment may induce cell death in normal cells too. The anti-apoptotic molecule Bcl-2 is upregulated in many cancer cells to protect them from apoptosis. In the current study, we knocked down Bcl-2 expression using cognate siRNA during low-dose taxol treatment to induce apoptosis in two human glioblastoma U138MG and U251MG cell lines. The cells were treated with either 100 nM taxol or 100 nM Bcl-2 siRNA or both for 72 h. Immunofluorescent stainings for calpain and active caspase-3 showed increases in expression and co-localization of these proteases in apoptotic cells. Fluorometric assays demonstrated increases in intracellular free [Ca2+], calpain, and caspase-3 indicating augmentation of apoptosis. Western blotting demonstrated dramatic increases in the levels of Bax, Bak, tBid, active caspases, DNA fragmentation factor-40 (DFF40), cleaved fragments of lamin, fodrin, and poly(ADP-ribose) polymerase (PARP) during apoptosis. The events related to apoptosis were prominent more in combination therapy than in either treatment alone. Our current study demonstrated that Bcl-2 siRNA significantly augmented taxol mediated apoptosis in different human glioblastoma cells through induction of calpain and caspase proteolytic activities. Thus, combination of taxol and Bcl-2 siRNA offers a novel therapeutic strategy for controlling the malignant growth of human glioblastoma cells.
KeywordsApoptosis Bcl-2 siRNA Calpain Glioblastoma Taxol
This investigation was supported in part by the R01 grants (CA-91460 and NS-57811) from the National Institutes of Health (Bethesda, MD) to S.K.R.
- 2.Cryns VL, Bergeron L, Zhu H, Li H, Yuan J (1996) Specific cleavage of α-fodrin during Fas- and tumor necrosis factor-induced apoptosis is mediated by an interleukin-1β-converting enzyme/Ced-3 protease distinct from the poly(ADP-ribose) polymerase protease. J Biol Chem 271:31277–31282PubMedCrossRefGoogle Scholar
- 7.Ganesh T, Yang C, Norris A, Glass T, Bane S, Ravindra R, Banerjee A, Metaferia B, Thomas SL, Giannakakou P, Alcaraz AA, Lakdawala AS, Snyder JP, Kingston DG (2007) Evaluation of the tubulin-bound paclitaxel conformation: synthesis, biology, and SAR studies of C-4 to C-3′ bridged paclitaxel analogues. J Med Chem 50:713–725PubMedCrossRefGoogle Scholar
- 19.Nath R, Raser KJ, Stafford D, Hajimohammadreza I, Posner A, Allen H Talanian RV, Yuen P, Gilbertsen RB, Wang KK (1996) Non-erythroid α-spectrin breakdown by calpain and interleukin-1β-converting-enzyme-like protease(s) in apoptotic cells: contributory roles of both protease families in neuronal apoptosis. Biochem J 319:683–690PubMedGoogle Scholar
- 28.Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD, Wang HG, Reed JC, Nicholson DW, Alnemri ES, Green DR, Martin SJ (1999) Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J Cell Biol 144:281–292PubMedCrossRefGoogle Scholar
- 29.Slee EA, Keogh SA, Martin SJ (2000) Cleavage of Bid during cytotoxic drug and UV radiation-induced apoptosis occurs downstream of the point of Bcl-2 action and is catalysed by caspase-3: a potential feedback loop for amplification of apoptosis-associated mitochondrial cytochrome c release. Cell Death Differ 7:556–565PubMedCrossRefGoogle Scholar