Mechanism of resistance to oxidative stress in doxorubicin resistant cells.

Doxorubicin (DOX) is an anthracycline drug widely used in chemotherapy for cancer patients, but it often gives rise to multidrug resistance in cancer cells. The purpose of this work was to study the effect of hydrogen peroxide in DOX-sensitive mouse P388/S leukemia cells and in the DOX-resistant cell line. Hydrogen peroxide induced a significant increase in dose- and time-response cell death in cultured P388/S cells. The degree of cell death in P388/DOX cells induced by hydrogen peroxide was less than that in P388/S cells treated with hydrogen peroxide. Parent cells exposed to 3 mM of hydrogen peroxide showed a loss of mitochondrial membrane potential correlated with cell death. Hydrogen peroxide at a concentration greater than 0.3 mM increased the intracellular Ca2+ of P388/S cells dose-dependently; however, no change following addition of hydrogen peroxide (0.3-1 mM) was observed in the resistant cells. Hydrogen peroxide (0.1 and 1 mM) treatment also induced the production of intracellular ROS in P388/S cells, while no such increase was produced by this substance in P388/DOX cells. Resistant cells also showed a significant level of glutathione (GSH) compared with the parent cells. In addition, N-acetyl-L-cysteine and reduced GSH antioxidants abolished death of P388/S cells caused by hydrogen peroxide. Therefore, it is believed that the reduced effect of oxidative stress towards the resistant cells may be related to an increase in intracellular GSH level.

Effect of glutathione depletion by buthionine sulfoximine on doxorubicin toxicity in mice.

The role of the glutathione (GSH) system in vivo or in drug resistance has received much attention, since GSH is a major component of the cellular detoxification system. We Studied the effect of GSH depletion by buthionine sulfoximine (BSO), a potent inhibitor of gamma-glutamylcysteine synthetase, on doxorubicin (DOX) toxicity in mice. The administration of BSO (30 mM in drinking water for 5 days) significantly decreased the tissue GSH. The GSH depletion in various tissues by BSO was associated with a decrease in the detoxification of DOX in mice. A single dose of 20 mg/kg of DOX significantly reduced body weight and rectal temperature in mice 3 days after injection. The combination with BSO and cepharanthine (biscoclaurine alkaloid), a P-glycoprotein (P-gp) inhibitor, significantly potentiated decrease in body and hypothermia induced by DOX. The study demonstrates that BSO markedly increases the toxicological effect of DOX with the alterations in GSH of tissues and Suggests that the intracellular accumulation of DOX is not a factor.

Combined effects of buthionine sulfoximine and cepharanthine on cytotoxic activity of doxorubicin to multidrug-resistant cells.

We studied the potentiation of doxorubicin (DOX) activity in multidrug-resistant (MDR) cells by buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, and by cepharanthine (CE), which interacts with P-glycoprotein. The glutathione (GSH) of MDR cells was approximately 1.5-fold greater than that of the parental cell line. BSO reduced GSH content of MDR cells compared to that of the sensitive ones. The BSO treatment (50 microM) enhanced the effect of DOX by 1.8-fold, while CE caused a greater reversal of drug resistance. The combination of BSO with CE produced further potentiation of DOX activity in an antiproliferative effect. Pretreatment of cells with BSO did not alter the cellular accumulation of DOX in the absence or presence of CE. The addition of BSO (30 mM) to the drinking water of mice reduced the tissue levels of GSH in tumor cells, suggesting that the marked decrease in GSH might diminish the ability of that tumor to resist DOX. Combined administration of CE and DOX resulted in enhancement of DOX antitumor activity and prolongation of survival time. The survival of mice treated with BSO and CE as a supplement to DOX treatment was superior that of mice receiving DOX alone. These studies demonstrated that the combinations of BSO with CE may be useful for killing drug-resistant tumor cells.

Antitumor activity of acemetacin in mice bearing colon 26 carcinoma: a preliminary report.

The antitumor activity of acemetacin (ACM) was examined in mice bearing colon 26 carcinoma and its effect was compared with indomethacin (IND). The addition of ACM (0.001-0.003%) to drinking water prolonged the survival time of mice bearing colon 26. On the other hand, prostaglandin E2 (PGE2) reduced the effect of ACM. The results suggest that the antitumor activity of ACM may be associated with the reduction of PGE2 levels.

[Assay of flow cytometry for the effect of cepharanthine on resistance to doxorubicin].

The biochemical activity of cepharanthine and the possible mechanism by which it reverses the resistance to doxorubicin in P388 leukemia cells were examined in vitro. The microfluorometric analysis of the cellular level of doxorubicin in drug-resistant cells showed that cepharanthine markedly enhanced the sensitivity of doxorubicin against resistant cells in the cellular level. Cepharanthine also enhanced the inhibitory effect of doxorubicin on the incorporation of thymidine into DNA in resistant cells. The analysis of DNA histogram obtained by flow cytometry showed that doxorubicin exerted its growth-inhibitory effect by blocking the cell cycle at the G2 phase in P388 cells. At higher concentrations, doxorubicin prolonged the S phase and inhibited cell cycle progression to the G2/M phase in cells. The treatment with cepharanthine potentiated these blocking effects induced by doxorubicin in cells. It seems that the modifications of the biological effect of doxorubicin by cepharanthine are due to the change of their ability to induce DNA damage in cells.