Differential Effect of the Calmodulm Inhibitor Trifluoperazine on Cellular Accumulation, Retention, and Cytotoxicity of Anthracyclines in Doxorubicin (Adriamycin)-resistant P388 Mouse Leukemia Cells1

Calmodulin inhibitors enhance cytotoxic effects of doxorubicin (DOX) in DOX-resistant (P388/DOX) P388 mouse leukemia cells by increasing cellular accumulation and retention of drug. In P388/DOX cells treated for 3 hr, cytotoxic effects (based on colony formation in soft agar) of daunorubicin (DAU) in the presence of trifluoperazine (TFP) were DAU concentration-de pendent and enhanced 2to 100-fold. Additionally, in the pres ence of TFP, on a molar basis, equitoxic doses of DAU were 4fold lower than DOX for P388/DOX cells. However, in P388/ DOX cells treated for 3 hr with other anthracyclines, except for a slight enhancement in the cytotoxic effects of aclacinomycin A (ACM) with TFP, colony formation in soft agar of cells treated with A/-trifluoroacetyladriamycin-14-valerate (AD32) and A/-trifluoroacetyladriamycin were similar in the absence and presence of TFP. In DOX-sensitive (P388/S) P388 mouse leukemia cells treated for 3 hr, some enhancement in the cytotoxic effects due to TFP were observed with DAU and DOX but not with ACM, AD32, or N-trifluoroacetyladriamycin. Although accumulation of ACM and AD32 in P388/S and P388/DOX cells was similar and unaffected by TFP, the retention of ACM but not AD32 was enhanced 1.5-fold only in TFP-treated P388/DOX cells. In con trast, DAU accumulation in P388/S cells was 4-fold higher than in similarly treated P388/DOX cells, and the 2and 4-fold increase due to TFP in the accumulation and retention, respectively, of DAU in P388/DOX cells was not observed in P388/S cells. Results from this study indicate that in P388/DOX cells, the calmodulin inhibitor TFP is more effective with DAU than DOX, significantly less effective with ACM, and ineffective with AD32 and W-trifluoroacetyladriamycin. INTRODUCTION The anthracycline antibiotics DAU3 and DOX play a prominent role in the chemotherapeutic management of human cancers (5, 28). An inevitable reality limiting the success of chemotherapy is the presence of intrinsically drug-resistant cells and/or the emerg ence of resistant clones following repeated courses of chemo therapy (21,29). In order to understand the mechanisms involved with cellular resistance to anthracyclines, tumor model systems 1Supported by USPHS Grant 1R01 CA35531, awarded by the National Cancer Institute, Department of Health and Human Services, and the Cleveland Foundation. 2To whom requests for reprints should be addressed, at the Research Division, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44106. 3The abbreviations used are: DAU, daunorubicin; DOX, doxorubicin; AD32, W-tnfluoroacetyladriamycin-14-valerate; AD41, W-trifluoroacetyiadriamycin; ACM, aclacinomycin A; TFP, trifluoperazine dihydrochloride; FBS, fetal bovine serum; DMSO, dimethyl sulfoxide. Received January 31,1984; accepted August 8,1984. have been developed, and a common characteristic of these resistant sublines is the reduced accumulation and/or retention of cytotoxic concentrations of the drug (9,14,17,24,27). Recent studies by us (12, 13) and Tsuruo ef al. (30, 31) indicate that calmodulin inhibitors and calcium antagonists are capable of partially reversing resistance in DOX-resistant sublines of mouse leukemia P388, primarily by increasing cellular retention of drug. In the search for anthracyclines with broader spectrum of antitumor activity and less cardiotoxicity than DAU and DOX, a number of naturally occurring and semisynthetic anthracyclines with significantly different cellular pharmacokinetics, antitumor activity, and toxicity characteristics have been evaluated (8, 33). Although reduced drug accumulation appears to be a primary reason for resistance, anthracyclines with potent antitumor activ ity, and which are rapidly accumulated in cells due to their high lipophilicity, have been of limited value in circumventing resist ance in DOX-resistant sublines (18). In the present study, we have attempted to determine, in DOX-resistant cells, the rela tionship between cellular drug levels and cytotoxicity of anthra cyclines and the potential role for TFP in modulating these effects. Specifically, using the DOX-sensitive (P388/S) and DOXresistant (P388/DOX) P388 mouse leukemia model system, the effect of the calmodulin inhibitor TFP on the cellular accumula tion, retention, and cytotoxicity of the anthracyclines, namely, DAU, AD32, AD41, and ACM, which are distinctly different from DOX, was determined. MATERIALS AND METHODS The source of the P388/S and P388/DOX cells and conditions for their maintenance in vitro are similar to those reported previously (12, 14). TFP was a generous gift from Dr. Carl Kaiser, Smith, Kline & French Laboratories, Philadelphia, PA. DAU and ACM were provided by Dr. Ven Narayanan, Chief, Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda, MD. AD32 and AD41 were a generous gift from Dr. Mervyn Israel, Department of Pharmacology, University of Tennessee Center for Health Sciences, Memphis, TN. Anthracycline Cytotoxicity in Vitro. Cytotoxicity studies were carried out using a soft-agar colony-forming assay. Stock solutions of DOX, DAU, ACM, and TFP were prepared in sterile-glass distilled water and in the case of AD32 and AD41, stock solutions were prepared in DMSO. Working dilutions were made in RPM11640 supplemented with 25 rtiM /V^-hydroxyethylpiperazine-W-ethanesulfonic acid buffer (M. A. Btoproducts, Walkersville, MD) and 10% FBS (Sterile Systems, Inc., Logan, UT). The final concentration of DMSO for AD32and AD41 -treated cells was 1%. Cells in RPMI 1640 supplemented with 10% FBS were treated at 37°with the various anthracyclines in the absence and presence of 5 //M TFP for 3 hr. The anthracyclines and the range of concentrations used were as follows: DOX and DAU 0.005 to 0.1 /¿g/ml for P388/S cells 5056 CANCER RESEARCH VOL. 44 Research. on November 8, 2017. © 1984 American Association for Cancer cancerres.aacrjournals.org Downloaded from