Investigation of Methuosis in Glioblastoma Cell Lines in Response to Treatment with Novel Chalcones

Abstract

The knowledge of cell death mechanisms following cancer therapies is continuing to expand. What was formerly understood as either apoptotic or necrotic cell death has evolved with recent research into non-classical cell death pathways. Our interest in non-classical cell death pathways resulted from our recent investigations of synthetic chalcone derivatives in glioblastoma cell lines. We previously reported on the cytotoxicity of two of our synthetic chalcones, RK6 and RK15, in glioblastoma cell lines (IC50 values: 10-20 µM). In our investigations into the mechanisms of action for these chalcones, we found that they induced cell death through a non-caspase mediated mechanism. Others have suggested that treatment of glioblastoma cells with chalcone-like compounds may induce methuosis, a nonapoptotic form of cell death involving the formation of vacuoles from macropinosomes which leads to cell lysis. With this in mind, we sought to determine if cell death caused by RK6 and RK15 was mediated through methuosis. Methuosis involves activation of the Ras signaling pathway followed by activation of Rac1, leading to vacuole formation and ultimately cell death. Rac1 inhibitors, such as EHT1864, have been shown to inhibit methuosis by preventing vacuole formation. We hypothesized that EHT1864 would reduce the cytotoxic effects of RK6 and RK15 in glioblastoma cell lines. To test our hypothesis, we used the XTT assay to assess cellular viability after treating glioblastoma cell lines, A-172, T98G, and U87, with various concentrations of our synthetic chalcone compounds, RK6 and RK15, with and without EHT1864. In each of our experiments, EHT1864 failed to prevent cell death caused by RK6 and RK15, thereby suggesting our initial hypothesis was incorrect. Interestingly, in several instances, EHT1864 actually enhanced the cytotoxic effects of RK6 and RK15. This data suggests that methuosis is likely not involved as a cell death mechanism for RK6 and RK15. However, the enhancement of RK6 and RK15-induced cytotoxicity by the Rac1 inhibitor, EHT1864, warrants further investigation.