Abstract 811: Schweinfurthin activity enhanced by verapamil

Abstract

Abstract Schweinfurthins are intriguing natural product anti-cancer agents with incompletely defined mechanisms of action. Schweinfurthins increase ER stress and result in apoptosis in vitro. To augment schweinfurthins’ growth inhibitory activity we hypothesized that an inhibitor of P-glycoprotein (Pgp) may increase the intracellular concentration of the schweinfurthins and lead to synergistic growth inhibition. The schweinfurthin sensitive glioblastoma multiforme cell line SF-295 and the relatively schweinfurthin insensitive lung carcinoma cell line A549 were treated with two schweinfurthin isoforms: methyl-G and 3-deoxyschweinfurthin B P-nitro bis stilbene (3dSB-PNBS). Methyl-G and 3dSB-PNBS (1 nM - 1 μM) decreased MTT activity at 48 hours but not at 24-hour incubation in both cell lines. The addition of verapamil (1μM - 1 mM) strongly induced synergy at 24 hours in SF-295 cells, yet verapamil did not greatly potentiate the schweinfurthin effect at 48 hours. This pattern was not evident in A549 cells. Isobologram analysis revealed combination indices of 0.38 and 0.47 at 24 hours in SF-295 cells for methyl-G and 3dSB-PNBS respectively. Fluorescent detection of 3dSB-PNBS demonstrated that verapamil increased the intracellular concentration of 3dSB-PNBS 3-fold in SF-295 and 2-fold in A549 cells. This effect occurred in a Pgp-independent manner as the specific Pgp inhibitor, CP 100356, failed to increase the intracellular concentration of 3dSB-PNBS. Flow cytometric analysis of intracellular calcium release demonstrated that neither methyl-G, verapamil, nor the combination increased intracellular calcium. However, Western blotting revealed that verapamil enhanced the ER stress caused by methyl-G in that there was increased expression of glucose-regulated protein 78 and increased phosphorylation of eukaryotic initiation factor 2α. Pgp expression was increased by verapamil in SF-295 cells, yet this increase was not present when methyl-G was combined with verapamil. Methyl-G also increased the intracellular concentration of a known Pgp substrate, Rhodamine 123 (R123), in SF-295 cells. Pgp requires cholesterol to function properly as a reduction in cellular cholesterol leads to the accumulation of Pgp substrates. Interestingly, since 3dSB depletes intracellular cholesterol and up-regulates the cholesterol efflux pump ABCA1, add-back of cholesterol rescued the methyl-G induced increase in R123 intracellular concentration. These studies demonstrate verapamil is able to potentiate the schweinfurthin growth inhibitory effect by increasing its intracellular concentration and by enhancing schweinfurthin induced ER stress; cholesterol is also identified as a key component of schweinfurthin action. Citation Format: Ryan M. Sheehy, Zoe C. Bachman, Raymond J. Hohl. Schweinfurthin activity enhanced by verapamil. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 811. doi:10.1158/1538-7445.AM2014-811