Overcoming acquired doxorubicin resistance of ovarian carcinoma cells by verapamil-mediated promotion of DNA damage-driven cell death

The therapeutic efficacy of anticancer therapeutics is limited by acquired drug resistance of tumor cells. Here, we aim to characterize and overcome resistance mechanisms of ovarian cancer cells to the anthracycline derivative doxorubicin (Doxo). To this end, comparative analyses of Doxo-induced stress responses of parental A2780 and Doxo-resistant A2780ADR variant were performed. A2780ADR cells revealed cross-resistance to multiple compounds, including anticancer drugs (cisplatin (CisPt), etoposide (Eto)) and DNA repair/ DNA damage response (DDR) inhibitors (olaparib, niraparib, entinostat, prexasertib, rabusertib). A2780ADR cells formed significantly less DNA double-strand breaks (DSB) after Doxo exposure as compared to A2780, resulting in a mitigated DDR, reduced proliferation inhibition and attenuated apoptosis. Potential resistance mechanisms identified to contribute to Doxo resistance of A2780ADR cells include increased Doxo efflux due to increased MDR1 expression and reduced topoisomerase II protein expression. Substantial resensitization of A2780ADR cells to Doxo was achieved by both the RAC1 GTPase inhibitor EHT1864, the histone deacetylase inhibitor entinostat (Est) and, most effectively, the calcium channel blocker verapamil (Ver). Notably, Ver-mediated sensitization also pertains to Eto and CisPt. The synergistic effect of Ver in combination with Doxo, which is reflected by low combination index (CI), likely involves inhibition of MDR1-mediated drug transport, leading to increased intracellular steady state levels of Doxo and elevated DNA damage formation, eventually promoting pro-apoptotic DDR. However, combination treatment with Doxo and Ver also increased the cytotoxic response of non-malignant murine cardiomyocytes (HL-1), murine embryonic stem cells (mESC) and human induced pluripotent stem cells (hiPSC). Based on our data we suggest inhibition of MDR1-mediated Doxo efflux by Ver as a useful approach to overcome acquired drug resistance of A2780ADR cells because it promotes DDR-related pro-death mechanisms, yet at the price of a potentially increased risk of normal tissue toxicity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/650268v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@43c0e5org.highwire.dtl.DTLVardef@2d1c5forg.highwire.dtl.DTLVardef@1a1d97borg.highwire.dtl.DTLVardef@cd4522_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract:C_FLOATNO Verapamil-mediated resensitization of Doxo-resistant tumor cells.As concluded from our data we suggest that verapamil increases the anticancer efficacy of doxorubicin (Doxo) in a synergistic manner in anticancer drug resistant ovarian A2780ADR cells. This is likely due to inhibition of MDR1-mediated drug export by Ver, leading to higher intracellular steady-state concentrations of Doxo. In consequence, Doxo-mediated Topo II poisoning is promoted, eventually causing increased DNA damage (DSB) formation and activation of DDR-related signaling mechanims, which in turn impair cell proliferation and stimulate cell death. Apart from verapamil, inhibition of Rac1 GTPase-regulated signaling by EHT1864 and inhibition of HDACs class I by the HDACi entinostat are also useful to overcome Doxo resistance of A2780ADR cells, yet with the exact molecular mechanisms involved being still unclear. C_FIG