Cytosolic free Ca2+ in daunorubicin and vincristine resistant ehrlich ascites tumor cells (original) (raw)

1991, Biochemical Pharmacology

The possible role of intracellular calcium on daunorubicin (DNR) accumulation in wild-type (EHR2) and multi-drug resistant (MDR) Ehrlich ascites tumor cell subline was investigated. DNR accumulation was not enhanced either by increasing the concentration of cellular calcium with the calcium ionophore ionomycin nor by chelating the cytosolic free Cat+ by the membrane permeable Ca2+-buffering agents BAPTA or MAPTAM. No effect was observed in the presence of extremely low extracellular calcium concentration that prevent transmembrane calcium influx or when the cells were calcium depleted using EGTA and ionomycin. Using the fluorescent Ca*+ indicator fura-it is further shown that both drug-resistant daunorubicin (EHRZ/DNR+) and vincristine (EHR/VCR+) sublines had lower (50-80 nM) concentration of cytosolic free calcium ([Ca*+]J compared to their corresponding wild-type parenteral tumors (140-180 nM). In calcium free medium, however, no significant difference was found, all cell lines having a [Ca2+li of 60-80 nM. Furthermore, the total amount of Ca"+ released to the cytosol with 10 FM ionomycin and 5 mM EGTA was 3-4-fold higher in EHR2 than in EHR2f DNR+ or EHRZ!/VCR+. Mobilization of Ca*+ with 1 PM ionomycin was almost identical in the presence and absence of Ca*+ in the extracellular medium in EHR2 as well as in EHRZ/DNR+ suggesting that the increase in [Ca2+li is mainly due to discharge of Ca 2+ from intracellular stores. Furthermore, the total cell calcium [Ca2+11 concentration was slightly higher in EHR2/DNR+ and EHR2/VCR+ cells compared to EHR2. Incubation of the cells with the Ca*+-channel blocker verapamil or the intracellular Ca*+-antagonist TMB-8 causes depression of the Ca2+-response in terms of rise in [Ca*+]j caused by ionomycin. Sorcin, a major calcium-binding protein (& 22 kDa), is shown to be overproduced in EHR2/DNR+ cells. The ove~roduction of this protein in resistant cells may be related to the difference in the intracellular calcium observed in this study. Thus, though handling of CaZ+ is different in wild-type and MDR cell lines, our data suggest that calcium is not involved directly in drug transport processes and the level of Ca*+ per se have no influence on drug accumulation. The emergence of resistance is a major problem in cancer chemotherapy. Acquired resistance to anthracyclines is invariably associated with cross resistance to the structurally unrelated vinca alkaloids and vice versa (MDR). The MDR phenotype is commonly associated with reduced drug accumulation, considered to be due, in part, to an energy dependent efflux mechanism in resistant cells [l]. Since the original observation by Tsuruo et al. [Z, 31 that the calcium channel blocker verapamil was able to both modulate resistance and inhibit active drug efflux in resistant cells, there have been numerous confirmatory reports on the abilities of both calcium channel blockers and calmodulin inhibitors to reverse MDR. Both verapamii and trifluoperazine have been shown to be specific inhibitors of voltage-dependent calcium channel and calmodulin-dependent processes in mammalian cells, respectively [4,5]. However,