Cytosolic free Ca 2+ in daunorubicin and vincristine resistant ehrlich ascites tumor cells : Drug accumulation is independent of intracellular Ca 2+ changes

Abstract

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 Ca 2+ by the membrane permeable Ca 2+-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 2+ indicator fura-2 it is further shown that both drug-resistant daunorubicin (EHR2/DNR+) and vincristine (EHR/VCR+) sublines had lower (50–80 nM) concentration of cytosolic free calcium ([Ca 2+] i) 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 [Ca 2+] i of 60–80 nM. Furthermore, the total amount of Ca 2+ released to the cytosol with 10 μM ionomycin and 5 mM EGTA was 3–4-fold higher in EHR2 than in EHR2/ DNR+ or EHR2/VCR+. Mobilization of Ca 2+ with 1 μM ionomycin was almost identical in the presence and absence of Ca 2+ in the extracellular medium in EHR2 as well as in EHR2/DNR+ suggesting that the increase in [Ca 2+] i is mainly due to discharge of Ca 2+ from intracellular stores. Furthermore, the total cell calcium [Ca 2+] i concentration was slightly higher in EHR2/DNR+ and EHR2/VCR+ cells compared to EHR2. Incubation of the cells with the Ca 2+-channel blocker verapamil or the intracellular Ca 2+-antagonist TMB-8 causes depression of the Ca 2+-response in terms of rise in [Ca 2+] i caused by ionomycin. Sorcin, a major calcium-binding protein ( M r, 22 kDa), is shown to be overproduced in EHR2/DNR+ cells. The overproduction of this protein in resistant cells may be related to the difference in the intracellular calcium observed in this study. Thus, though handling of Ca 2+ 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 2+ per se have no influence on drug accumulation.