Differential effect of the calmodulin inhibitor trifluoperazine in modulating cellular accumulation, retention and cytotoxicity of doxorubicin in progressively doxorubicin-resistant L1210 mouse leukemia cells: Lack of correlation between cellular doxorubicin levels and expression of resistance

Abstract

Calmodulin inhibitors are effective in enhancing cytotoxic effects of doxorubicin (DOX) in DOX-resistant cells, possibly by enhancing cellular levels of drug. In the present study, L1210 mouse leukemia cells adapted to grow in vitro, in the presence of 0.025 to 0.25 μg/ml DOX, and identified as L1210/DOX0.025, L1210/DOX0.05, L1210/DOX0.1, and L1210/DOX0.25 were approximately 5-, 10-, 20-, and 40-fold DOX resistant, respectively, compared to parent-sensitive cells (L1210/S). Using a soft agar colony assay and 3-hr drug exposure, the ic 50 concentration of DOX in the progressively DOX-resistant (5- to 40-fold) L1210 cells ranged from 0.25 to 2.0 μg/ml and from 0.08 to 0.25 μg/ml in the absence and presence of a non-cytotoxic concentration of 5 μM trifluoperazine (TFP) respectively. Further, based on the observed in vitro cytotoxic response, the ic 50 concentration of DOX in the presence of 5 μM TFP was 2.5-, 4-, 6.7- and 8-fold lower than DOX without 5 μM TFP in the L1210/ DOX0.025, L1210/DOX0.05, L1210/DOX0.1, and L1210/DOX0.25 resistant sublines respectively. In contrast, the ic 50 of DOX in L1210/S cells was approximately 0.05 μg/ml with or without 5 μM TFP. Cellular accumulation of DOX was 15–50% lower in the progressively resistant L1210 sublines compared to similarly treated L1210/S cells. However, in the presence of 5 μM TFP, cellular accumulation of DOX in the L1210/DOX0.05 and L1210/DOX0.1 but not L1210/DOX0.25 was comparable to the L1210/S cells. Cellular retention of DOX in the absence or presence of 5 μM TFP was comparable in similarly treated L1210/S, L1210/DOX0.05 and L1210/DOX0.1 cells, and a 2-fold reduction in the retention of DOX in the absence versus the presence of 5 μM TFP was apparent only in L1210/DOX0.25 cells. At the ic 50 of DOX in the presence of 5 μM TFP, although cellular accumulation of DOX was concentration dependent over the range of 1–20 μM TFP, enhancement in cytotoxicity of DOX was dose dependent at 1–5 μM TFP but not 5–20 μM TFP. In cells treated for 3 hr at the ic 50 concentration of DOX alone or DOX plus 5 μM TFP, cellular accumulation of DOX was 7- to 14-fold and 2.5- to 3.5-fold higher, respectively, in resistant than in sensitive cells. Additionally, following treatment for 3hr at the ic 50 dose of DOX in the absence or presence of 5 μM TFP, drug retention at 3 hr was 4- to 6-fold and 1.5-fold higher, respectively, in the resistant versus sensitive cells. Results from this study demonstrate that: (1) the effect of TFP on DOX cytotoxicity was dependent on the level of acquired resistance; (2) in contrast to sensitive cells, the resistant sublines required much higher cellular levels of DOX in the absence versus the presence of TFP to achieve equivalent cytotoxicity; and (3) differences in cellular levels of DOX during accumulation or retention in sensitive versus resistant sublines did not appear to correlate with the magnitude in expression of resistance.