RS-33295-198: a novel, potent modulator of P-glycoprotein-mediated multidrug resistance.

A novel multidrug resistance modulator, RS-33295-198, circumvented drug resistance in human, mouse, and Chinese hamster cell lines overexpressing P-glycoprotein. It enhanced the antiproliferative activity of doxorubicin, vincristine, etoposide, and paclitaxel and increased doxorubicin retention in multidrug-resistant hamster CHRC5 cells. RS-33295-198 modulated doxorubicin resistance in a murine P388/ADR leukemia model when administered ip via continuous minipump delivery, ip by bolus injection, and orally; it also improved the efficacy of vincristine toward P388/VCR leukemia when given ip or po. RS-33295-198 showed weak activity in enhancing doxorubicin efficacy against a multidrug-resistant human sarcoma xenograft.

Effect of verapamil on doxorubicin cardiotoxicity: altered muscle gene expression in cultured neonatal rat cardiomyocytes.

Verapamil reverses multidrug resistance acquired by cancer cells during treatment with chemotherapeutic agents such as doxorubicin by inhibiting the function of P-glycoprotein. Verapamil has also been suggested to potentiate the cardiotoxicity of doxorubicin. We have recently demonstrated that selective inhibition of cardiac muscle gene expression is among the earliest events in doxorubicin cardiotoxicity. To explore the influence of verapamil on doxorubicin cardiotoxicity, we evaluated [14C]-doxorubicin accumulation, cardiac muscle gene expression by Northern blot analysis, and ultrastructural changes in cultured cardiomyocytes in the presence and absence of verapamil. Treatment with a combination of doxorubicin and verapamil for 24 h did not augment doxorubicin accumulation in cardiomyocytes, although substantial augmentation of doxorubicin accumulation by verapamil in cardiac fibroblasts was observed. Further, treatment with verapamil for 24 h did not augment the decrease in expression of muscle genes induced by doxorubicin (myosin light chain 2 slow, troponin I, M isoform creatine kinase). However, we found that verapamil reduced alpha-actin gene expression in a direct, doxorubicin-independent manner. Furthermore, the effect of doxorubicin plus verapamil on alpha-actin gene expression was additive over a wide range of doxorubicin and verapamil concentrations, resulting in a selective augmentation of doxorubicin-induced inhibition of gene expression for this single muscle protein gene. This was reflected in a substantial increase in cardiac myocyte damage when treatment with verapamil and doxorubicin was compared to treatment with doxorubicin alone by thin section electron microscopy. This suggests a possible mechanism by which verapamil may potentiate doxorubicin cardiotoxicity.

Multifactorial resistance in LS174T human colon carcinoma cells selected with doxorubicin.

A series of doxorubicin-resistant variants of the human LS174T colon carcinoma cell line was generated by stepwise selection. These variants also exhibited increased resistance to vinblastine, etoposide, cis-platinum, and melphalan, suggesting that resistance was multifactorial. The parental LS174T cell line and 3 resistant variants were examined for over-expression of P-glycoprotein, changes in total cellular glutathione content, and the level of topoisomerase-II expression. Changes in all of these parameters were observed in the doxorubicin-selectants, along with a marked shift in the intracellular distribution of doxorubicin. P-glycoprotein RNA and protein levels were increased 2- to 3-fold in the resistant variants, while total glutathione levels increased 1.4- to 2.1-fold. Treatment with DL-buthionine-[S,R]-sulfoximine, an inhibitor of glutathione biosynthesis, was able to reverse resistance to cis-platinum and melphalan in these variants, but had little effect on doxorubicin resistance. Immunoblot analysis of cell extracts indicated that the level of DNA topoisomerase II (EC 5.99.1.3) in the doxorubicin-resistant LS174T cells was decreased by approximately 50% compared with the parental cell line. Doxorubicin was mainly localized to the cytoplasm in resistant cells, while in the parent line it was mostly found in the nucleus. This constellation of changes suggests that selection with doxorubicin activated several mechanisms of resistance involving drug transport, metabolism, and ability to reach nuclear target sites.

Effect of exposure to calcium entry blockers on doxorubicin accumulation and cytotoxicity in multidrug-resistant cells.

Flow cytometry has been used to measure doxorubicin (DOX) retention in several pairs of drug-sensitive and multidrug-resistant (MDR) cell lines and in unselected human tumor cell lines. Co-exposure to several agents that have been reported to reverse multidrug resistance, particularly calcium entry blockers (CEBs), produced a dose-dependent increase in DOX accumulation in MDR cell lines. In MDR Chinese hamster ovary cells (CHRC5), DOX levels declined rapidly following removal of CEBs, reaching a plateau value above that found in cells treated with DOX alone; this small increase probably represents DOX that is not accessible to the p170 efflux pump overexpressed in these cells. Increased DOX retention could be observed even after brief exposure to CEBs and washout and correlates with a decrease in cell proliferation over a 3-day growth assay. These results suggest that only a brief inhibition of drug efflux is sufficient to produce a meaningful reversal of drug resistance.

Isolation and characterization of doxorubicin-resistant Lewis lung carcinoma variants.

A series of drug-resistant variants of the murine Lewis lung carcinoma (3LL-CK) cell line has been isolated using stepwise selection in increasing concentrations of doxorubicin. These variants exhibited classical multidrug resistance as evidenced by decreased doxorubicin accumulation, cross-resistance to vinblastine, reversibility of resistance by verapamil, and overexpression of P-glycoprotein. When the doxorubicin-resistant 3LL-CK cell populations were injected into the tail veins of B6D2F1 mice, their metastatic abilities were consistently reduced compared with that of the parental line.

Prevention of doxorubicin-induced hematotoxicity in mice by interleukin 1.

Interleukin 1 alpha and interleukin 1 beta induce peripheral neutrophilia with stimulation of granulopoiesis in bone marrow. The continuous administration of interleukin 1 (100 ng/day) to mice for 7 days by s.c.-implanted Alzet osmotic minipumps induced marked stimulation of granulopoiesis in marrow and spleen in normal mice, and protected against the marked depletion of myeloid and erythroid cells in bone marrow of mice treated with single injections of either 20 or 30 mg/kg doxorubicin (DXN). Interleukin 1 beta infusion also protected against DXN-induced atrophy of thymus and secondary lymphoid organs. Single i.p. injection of either interleukin 1 alpha or interleukin 1 beta at doses up to 1000 ng 24 h prior to treatment with DXN did not protect against the hematopoietic and lymphoid toxicities of DXN.

Enhanced antitumor efficacy of adriamycin in combination with interferon-gamma: effects on macrophages.

The combination of the immunomodulator interferon-gamma (IFN-gamma) with the chemotherapeutic drug adriamycin (ADM) was assessed in vitro and in vivo in murine tumor models. When tested in vivo against the murine Lewis lung carcinoma, significantly greater reduction of spontaneous pulmonary metastases was obtained by combination treatment with IFN-gamma, followed 1 day later by ADM. Intraperitoneal ADM treatment also resulted in an increased recruitment of peritoneal mononuclear cells. It is noteworthy that, although the antitumor efficacy was significantly increased by the IFN-gamma/ADM combination treatment, gross toxicity of ADM was not increased. Thus, a net increase in the therapeutic index of ADM was achieved. In vitro, the effects of ADM on the ability of murine peritoneal macrophages, with or without the addition of immunological macrophage activators, to kill tumor cells was studied. Resident macrophages were able to sequester ADM (when present at 10 micrograms/ml) from the medium, and could subsequently mediate killing of target tumor cells. However, incubation of macrophages with low (ineffective by themselves) doses of ADM (1 microgram/ml) prevented their simultaneous or subsequent activation to the tumoricidal state after incubation with the normal macrophage-activating mixture of IFN-gamma plus a muramyl dipeptide (MDP) analog. When the order of addition of reagents was reversed such that the macrophages were preincubated for 24 hr with IFN-gamma (100 U/ml) plus the MDP analog (0.1-10 micrograms/ml), no antagonism of tumoricidal activity was obtained upon subsequent incubation with ADM. There were no interactions between IFN-gamma and ADM on the direct proliferation of tumor cells. Taken together, these results suggest that the enhanced antitumor efficacy of IFN-gamma/ADM combinations in vivo was not due to direct antiproliferative effects on the tumor cells, but rather may be mediated by direct cytotoxicity of ADM on tumor cells enhanced by phagocytic mononuclear cells.