Modulation of Cisplatin Cytotoxicity and Cisplatin-Induced DNA Cross-Links in HepG2 Cells by Regulation of Glutathione-Related Mechanisms

Abstract

Glutathione (GSH), glutathione S-transferase (GST), and glutathione conjugate export pump (GS-X pump) have been shown to participate collectively in the detoxification of many anticancer drugs, including cisplatin. Identification and regulation of the rate-limiting step in the overall system for cisplatin detoxification is of crucial importance for sensitization of human tumor cells to cisplatin. In this study, the GSH content, GST activity, and GS-X pump activity were regulated separately to examine effects of the regulation on cisplatin cytotoxicity and cisplatin-induced DNA interstrand cross-links (ICL) in HepG2 cells. Seventy-percent depletion of GSH by buthionine sulfoximine (BSO) and 50% increase of GSH by monoethyl GSH ester (GSHe) potentiated and decreased cisplatin cytotoxicity, respectively. This was reflected by a significant decrease and increase of their respective IC(50) values by 62 and 107%. Cisplatin-induced ICL was also potentiated by depletion of GSH by BSO and decreased by enrichment of GSH by GSHe, as shown by a 125% increase and a 34% decrease of cross-linked DNA compared with control samples exposed to cisplatin alone (p = 0.008 and 0.03, respectively). On the other hand, inhibition of GST and GS-X pump by ethacrynic acid, quercetin, tannic acid, and indomethacin at concentrations that inhibited activities of GST and GS-X pump by more than 50% had no significant effects on cisplatin cytotoxicity and cisplatin-induced DNA ICL in these cells. The results showed that of the parameters measured, intracellular GSH seems to be the rate-limiting factor, and its regulation would provide a more promising strategy for sensitization of human liver tumor cells to cisplatin.

Hammerhead ribozyme against γ-glutamylcysteine synthetase sensitizes human colonic cancer cells to cisplatin by down-regulating both the glutathione synthesis and the expression of multidrug resistance proteins

“…8 It has been reported that an increase in the expression of GST , together with a high concentration of GSH, was involved in resistance to CDDP. 43,44 The activity of GST in HCT8DDP cells was higher than that in HCT8 cells, which are relatively sensitive to CDDP. 7 But the expression of GST mRNA did not change in HCT8DDP cells transfected with anti --GCSh ribozyme ( data not shown ).…”

Cell-interdependent cisplatin killing by Ku/DNA-dependent protein kinase signaling transduced through gap junctions

“…Factors described as playing a role in the cisplatin response, including high-mobility group proteins, DNA MMR factors, signaling molecules such as c-jun, c-abl, and p73 (4,10,26,27), and alterations in uptake and inactivation (1,7,8,28), were all thought to act within individual cells.…”

The Effect of Megestrol Acetate on Growth of HepG2 Cells In vitro and In vivo

“…In this study, the IC 50 of 260 M for megestrol acetate was much higher when compared with 42.8 M for cisplatin (29), which is a widely used anticancer drug for treatment of human malignancies, including HCC. This suggests that the direct effect of megestrol acetate on the growth of HepG2 cells can only be achieved at higher dosages, and high dosages are needed clinically for inhibition of growth of HCC in patients.…”

“…The IC 50 for megestrol acetate was calculated by a linear regression. The IC 50 of 260 M for megestrol acetate was much higher when compared with 42.8 M for cisplatin (29), which is a widely used anticancer drug for the treatment of human malignancies, including HCC. Incubation with 200 M megetrol acetate also showed timedependent inhibition on the growth of the human liver tumor cells (Fig.…”

“…Approximately 1 ϫ 10 4 cells were seeded in each well of 96-well tissue culture plates and incubated in a CO 2 incubator for 24 h. A 20 mM stock solution of megestrol acetate was prepared freshly in absolute ethanol, and aliquots of the stock solution were added to wells of the plates at desired concentrations. After incubation with megestrol acetate for various periods of time, survival cells in plate wells were determined by 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (Sigma) assay as reported previously (29). Twenty-five l of the 5 mg/ml stock solution of 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide were added to each well, and after 2 h of incubation at 37°C, 100 l of the extraction buffer [20% SDS w/v, in 50% N,N-dimethyl formamide v/v, 2.5% of 80% acetic acid and 2.5% of 1 N HCl (pH 4.7)] were added.…”