Abstract 3472: Molecular mechanisms of growth inhibition of human brain tumor stem cells and glioblastoma LN18 and U138MG cells by combination of curcumin and paclitaxel

Abstract

Glioblastoma is the deadliest human brain tumor that responds poorly to conventional chemotherapeutic agents and continues to form tumor mass because of existence of highly chemoresistant human brain tumor stem cells (HBTSC). There is an urgent need for new therapeutic strategy that can target HBTSC as well as other glioblastoma cells. We explored the synergistic efficacy of a low dose of curcumin (CCM) and a low dose of paclitaxel (PTX) to induce apoptosis and inhibit cell survival, proliferation, invasion, and angiogenesis in HBTSC and human glioblastoma LN18 (p53 mutant and PTEN proficient) and U138MG (p53 mutant and PTEN mutant) cells. Western blotting indicated the highest expression of the cancer stem cell markers aldehyde dehydrogenase 1 (ALDH1) and CD133 in HBTSC when compared with LN18 and U138MG cells. Further, confocal laser scanning immunofluorescence microscopy confirmed more expression of ALDH1, presently considered as a novel cancer stem cell marker, in HBTSC than that in LN18 and U138MG cells. Our MTT assay revealed that combination of 20 µM CCM and 10 nM PTX worked synergistically and more effectively decreased the cell viability than the single drug treatments in HBTSC, LN18, and U138MG cells. Our in situ Wright staining and light microscopy showed that combination of CCM and PTX was highly effective in inducing morphological features of apoptosis in these cells. We also confirmed the induction of the highest amounts of apoptosis by this combination therapy in the cells after Annexin V/PI staining. The molecular mechanisms of induction of apoptosis required activation of caspase-8, cleavage of Bid to tBid, increase in Bax:Bcl-2 ratio, and mitochondrial release of cytochrome c, Smac, and apoptosis-inducing factor (AIF). Further, increase in phosphorylation of Bcl-2 (p-Bcl-2) following combination therapy could promote Bax homodimerization for enhancing mitochondrial release of pro-apoptotic factors into the cytosol. Increased expression of calpain and caspase-3 and their proteolytic activities confirmed the completion of apoptotic process in all cell lines. Our in vitro Matrigel invasion assay showed efficacy of the combination therapy in inhibiting invasion of cells. Efficacy of this combination therapy was also evident in reduced expression of cell survival and proliferation factors (p-Akt, NF-κB, survivin, and hTERT), invasion factors (MMP-2 and MMP-9), and angiogenic factors (VEGF, b-FGF, and CD31) most prominently in HBTSC and very clearly in glioblastoma LN18 and U138MG cells. Moreover, our in vitro angiogenic network formation assay indicated that this combination therapy significantly reduced the angiogenic capability of HBTSC, LN-18, and U138MG cells. Taken together, our results clearly show that combination of CCM and PTX is a promising therapy for controlling malignant growth of HBTSC and other glioblastoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3472. doi:1538-7445.AM2012-3472