The effect of modulation ofγ‐glutamyl transpeptidase and nitric oxide synthase activity on GSH homeostasis in HepG2 cells

Abstract

High glutathione (GSH) level and elevated gamma-glutamyl transpeptidase (gammaGT) activity are hallmarks of tumor cells. Toxicity of drugs and radiation to the cells is largely dependent on the level of thiols. In the present studies, we attempted to inhibit gammaGT activity in human hepatoblastoma (HepG2) cells to examine whether the administration of gammaGT inhibitors, acivicin (AC) and 1,2,3,4-tetrahydroisoquinoline (TIQ) influences cell proliferation and enhances cytostatic action of doxorubicin (DOX) and cisplatin (CP) on HepG2 cells. The effects of these inhibitors were determined by 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), BrdU and lactate dehydrogenase (LDH) tests and by estimation of GSH level. Additionally, we investigated the changes in caspase-3 activity, which is a marker of apoptosis. The obtained results showed that the gammaGT inhibitors introduced to the medium alone elicited cytotoxic effect, which was accompanied by an increase in GSH level in the cells. TIQ concomitantly increased caspase-3 activity. Doxorubicin and CP proved to be cytotoxic, and both inhibitors augmented this effect. As well DOX as CP radically decreased GSH levels, whereas gammaGT inhibitors had diverse effects. Therefore, the obtained results confirm that gammaGT inhibitors can enhance pharmacological action of DOX and CP, which may permit clinicians to decrease their doses thereby alleviating side effects. Aminoguanidine (nitric oxide synthase inhibitor) given alone was little cytotoxic to HepG2 cells, while its introduction to the medium together with DOX and CP significantly increased their cytotoxicity. Aminoguanidine on its own did not show any effect on GSH level in HepG2 cells, but markedly and significantly elevated its concentration when added in combination with CP but not with DOX. This indicates that when CP was used as a cytostatic, GSH level rose after treatment with its combination with both AC and aminoguanidine.