Investigation of Non‐Caspase Mediated Mechanisms of Death in Response to Treatment with Novel Chalcone in Glioblastoma Cell Lines

Abstract

Despite recent advances in cancer treatments, there is still a paucity of therapeutic options for patients with glioblastoma. We previously reported the efficacy of the synthetic chalcone, RK6, against A‐172, T98G, and U87‐MG human glioblastoma cell lines (IC50 values: 10–20 μM). Despite the moderately potent anticancer activity of RK6, the mechanism of death and putative drug target has yet to be elucidated. Previous efforts to identify potential mechanisms of death included evaluation of executioner caspase activation and production of reactive oxygen species. However, these pathways were found to be not largely involved. As such, further investigation of non‐caspase mediated forms of cell death were performed.To rule out necrosis as a mechanism of death for RK6, a trypan blue exclusion assay was conducted to examine membrane destabilization. Cellular membranes remained intact following treatment with RK6 at concentrations greater than the IC50 for six hours, suggesting necrosis is not the mechanism of death.We then sought to determine if cysteine proteases such as calpains or cathepsins were involved in the observed cell death following RK6 exposure. For this, cells were co‐treated with RK6 along with selective and non‐selective calpain and cathepsin inhibitors followed by measurement of cell viability using the XTT cell viability assay. Sublethal concentrations of the calpain and cathepsin inhibitor, N‐Acetyl‐Leu‐Leu‐Norleucinal (ALLN), selective calpain inhibitor, calpeptin, and selective cathepsin B inhibitor, CA‐074‐Me, were used to interrogate these signaling pathways. Inhibition of calpains and cathepsins concomitantly using ALLN had no effect on the efficacy of RK6 in the A‐172, T98G, and U87‐MG glioblastoma cell lines. Similarly, inhibition of cathepsin B with CA‐074‐Me did not affect the efficacy of RK6. Interestingly, inhibition of calpains with calpeptin resulted in a synergistic cytotoxic effect in all three cell lines. The results of these studies suggest that inhibition of calpains further sensitizes the cells to RK6, however concomitant inhibition of calpain and cathepsins does not provide the same effect. Further investigations are ongoing to investigate the nature of the synergy between RK6 and calpeptin and the potential role of other forms of mediated cell death, such as necroptosis, caused by this synthetic chalcone.Support or Funding InformationThis project was supported in part by an American Foundation for Pharmaceutical Education Gateway to Research Award for the second author.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.