Our preliminary studies have demonstrated that a glutathione-xenobiotic (GS-X) conjugate transporter is capable of mediating the ATP-dependent transport of doxorubicin (DOX) in the inside-out vesicles (IOVs) prepared from membranes of humans erythrocytes and of the H-128 human small cell lung cancer (SCLC) cell line. In these studies, we have also observed the inhibition of the ATP-dependent efflux of DOX by the conjugate of glutathione (GSH) and ethacrynic acid. Ethacrynic acid. Ethacrynic acid (EA), a commonly used non-cytotoxic loop diuretic drug, has been shown to enhance the cytotoxicity of the alkylating agent class of chemotherapeutic drugs through the inhibition of glutathione S- transferase (GSTs), important implication of our finding that EA-SC inhibits the transport of dox the GS-X transporter is that EA should also enhance the cytotoxicity of DOX towards noncancerous tissues through the EA-SG mediated inhibition of this efflux. Our preliminary studies have shown that EA is rapidly converted to EA-SG under physiologic conditions and that GST -eta0 is a significant determination of the rate of EA-SG both in vitro and in SCLC cell lines in situ. Previous studies have found that EA is less effective at enhancing alkylating agent cytotoxicity in malignant cells over-expressing GST-eta. The efficacy of EA for enhancing alkylating agent cytotoxicity would thus be limited since most malignancies express greater GST-eta content than normal tissues and many drug resistant malignancies display even greater GST-eta expression. In contrast, because the effect of EA on DOX cytotoxicity should depend on inhibition of DOX efflux by EA-SG, EA should preferentially enhance the cytotoxicity of DOX in malignant cells which express high GST-eta compared with normal tissues. We thus hypothesize that in addition to its effects mediated through inhibition of GSTs, EA should preferentially enhance DOX cytotoxicity of DOX through inhibition of DOX efflux by EA-SG and that it should preferentially enhance DOX cytotoxicity in cells over-expressing GST eta. To test our hypothesis, we have designed a model system for studying the cellular pharmacokinetics of DOX efflux, EA-SG formation and for evaluating its effect on DOX efflux in five human SCLC cell lines with varying amounts of GST-eta expression. Results of these studies will elucidate the role of the GS-X transporter as a determinant of DOX sensitivity and resistance and will provide valuable insight for design of clinical regimens utilizing EA or other drugs which undergo conjugation with GSH to enhance the anticancer efficacy of DOX through inhibition of the GS-X transporter.
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