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.

National Institute of Health (NIH)
National Cancer Institute (NCI)
First Independent Research Support & Transition (FIRST) Awards (R29)
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Experimental Therapeutics Subcommittee 1 (ET)
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Johnson, George S
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University of Texas Medical Br Galveston
Internal Medicine/Medicine
Schools of Medicine
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Lin, J T; Sharma, R; Grady, J J et al. (2001) A flow cell assay for evaluation of whole cell drug efflux kinetics: analysis of paclitaxel efflux in CCRF-CEM leukemia cells overexpressing P-glycoprotein. Drug Metab Dispos 29:103-10
Awasthi, S; Pandya, U; Singhal, S S et al. (2000) Curcumin-glutathione interactions and the role of human glutathione S-transferase P1-1. Chem Biol Interact 128:19-38
Singhal, S S; Awasthi, S; Pandya, U et al. (1999) The effect of curcumin on glutathione-linked enzymes in K562 human leukemia cells. Toxicol Lett 109:87-95
Singh, S V; Benson, P J; Hu, X et al. (1998) Gender-related differences in susceptibility of A/J mouse to benzo[a]pyrene-induced pulmonary and forestomach tumorigenesis. Cancer Lett 128:197-204
Morrison, R J; Singhal, S S; Bidani, A et al. (1998) Glutathione S-transferases of rabbit lung macrophages. Toxicol Appl Pharmacol 148:229-36
He, N G; Awasthi, S; Singhal, S S et al. (1998) The role of glutathione S-transferases as a defense against reactive electrophiles in the blood vessel wall. Toxicol Appl Pharmacol 152:83-9
Piper, J T; Singhal, S S; Salameh, M S et al. (1998) Mechanisms of anticarcinogenic properties of curcumin: the effect of curcumin on glutathione linked detoxification enzymes in rat liver. Int J Biochem Cell Biol 30:445-56
Srivastava, S K; Hu, X; Xia, H et al. (1998) ATP-dependent transport of glutathione conjugate of 7beta, 8alpha-dihydroxy-9alpha,10alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene in murine hepatic canalicular plasma membrane vesicles. Biochem J 332 ( Pt 3):799-805
Awasthi, S; Singhal, S S; Srivastava, S K et al. (1998) ATP-Dependent human erythrocyte glutathione-conjugate transporter. I. Purification, photoaffinity labeling, and kinetic characteristics of ATPase activity. Biochemistry 37:5231-8
Awasthi, S; Singhal, S S; Pikula, S et al. (1998) ATP-Dependent human erythrocyte glutathione-conjugate transporter. II. Functional reconstitution of transport activity. Biochemistry 37:5239-48

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