DNA strand breaks produced by etoposide (VP-16,213) in sensitive and resistant human breast tumor cells: implications for the mechanism of action.

Pleotropic resistant human breast cancer cells (MCF-7), selected for resistance to Adriamycin, were used to study the production of DNA strand breaks by etoposide (VP-16) and its relationship to drug cytotoxicity. It was shown that the resistant MCF-7 cell line was cross-resistant to VP-16, and the degree of resistance was found to be 125-200-fold. Alkaline elution studies indicated that the parental cell line was very sensitive to VP-16 which caused extensive DNA strand breakage. In contrast, little DNA strand breakage was detected in the resistant MCF-7 cells, even at very high drug concentrations, indicating a good agreement between strand breaks and cytotoxicity. Further studies indicated that the nuclei isolated from the parental cell line were more resistant to VP-16-induced DNA strand breaks than the intact cells, while the opposite was found in the resistant cell line. In addition, the alkaline elution studies in isolated nuclei showed only a 2-fold reduction of VP-16-induced DNA breaks in nuclei from the resistant cells. In agreement with this result, it was found that nuclear extract from the resistant cells produced 2-3-fold less VP-16-induced DNA breaks than that from the sensitive cells in 32P-end-labeled SV40 DNA. VP-16 uptake and efflux studies indicated that there was a 2-3-fold decrease in net cellular accumulation of VP-16 in the resistant cells. Although the reduced uptake of VP-16 and decreased drug sensitivity of topoisomerase II appear to contribute to the mechanism of action and the development of resistance to VP-16, they do not completely explain the degree of resistance to VP-16 in this multidrug-resistant MCF-7 cell line indicating that other biochemical factors, such as activation of VP-16, are also involved in drug resistance and suggesting that the resistance is multifactorial.

Differential oxygen radical susceptibility of adriamycin-sensitive and -resistant MCF-7 human breast tumor cells.

Recent evidence supports the concept that Adriamycin cytotoxicity may be mediated by drug semiquinone free radical and oxyradical generation. We tested this hypothesis further by exposing drug-sensitive (WT) and 500-fold Adriamycin-resistant MCF-7 human breast tumor cells (ADRR) to exogenous superoxide- and hydrogen peroxide-generating systems and subsequently monitored cell proliferation as a measure of cytotoxicity. The ADRR tumor cells tolerated a 4-fold greater exposure than sensitive cells to superoxide generated by the xanthine/xanthine oxidase system. Likewise, exposure to hydrogen peroxide produced by the action of glucose oxidase on glucose revealed a 4-fold diminished susceptibility of the drug-resistant cells to this reduced form of oxygen. Similar results were obtained by the direct application of hydrogen peroxide to cells. For both cell lines, cytotoxicity was dependent upon the magnitude and the duration of reactive oxygen exposure. When WT and ADRR cells were cultured under hyperoxia (95% O2:5% CO2), in order to stimulate the intracellular production of oxyradicals, proliferation was inhibited to a greater extent in the drug-sensitive cell line. Additionally, hyperoxia potentiated the cytotoxicity of Adriamycin to both sensitive and drug-resistant cells, but the effect depended upon the concentration of the drug. Under hyperoxic conditions, Adriamycin caused oxygen radical-dependent cytotoxicity to the WT tumor cells at clinically relevant drug concentrations as low as 2 to 3 nM. With ADRR tumor cells, hyperoxia increased the cytotoxicity of Adriamycin at concentrations above 5 microM. Paradoxically, both the WT and the ADRR tumor cells were equally susceptible to the cytotoxic effects of gamma irradiation. It is known that the Adriamycin-resistant MCF-7 cells greatly overexpress glutathione peroxidase and glutathione transferase activities; however, other biochemical defenses against reactive drug intermediates and oxygen radicals have been reported to be similar in the two cell lines. We have reexamined those observations in this report. The resistance of ADRR breast tumor cells to Adriamycin appears to be associated with a developed tolerance to superoxide, most likely because of a twofold increase in superoxide dismutase activity, and a decreased susceptibility to hydrogen peroxide, most likely because of 12-fold augmented selenium-dependent glutathione peroxidase activity. Acting in concert, these two enzymes would decrease the formation of hydroxyl radical from reduced molecular oxygen intermediates.(ABSTRACT TRUNCATED AT 400 WORDS)

Potentiation of doxorubicin cytotoxicity by buthionine sulfoximine in multidrug-resistant human breast tumor cells.

Resistance to antineoplastic drugs is a major problem in the clinical management of cancer. Previous studies have demonstrated that the cytotoxicity of certain anticancer drugs is increased by lowering the glutathione (GSH) levels with buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase. In this study we report that the resistance to doxorubicin, an anthracycline antibiotic and the most active agent in the treatment of breast cancer, can be partially reversed by exposing MCF-7 doxorubicin-resistant breast tumor cells (MCF-7/ADRR) to minimally cytotoxic doses of BSO. We found that the BSO treatment (50 microM, 48 h) of MCF-7/ADRR cells resulted in 80 to 90% depletion in total GSH concentrations. The toxicity of doxorubicin, as determined by growth inhibition and clonogenic assays, was significantly potentiated in the BSO-treated GSH-depleted cells relative to control breast tumor cells, and a dose-modifying factor of 5 to 7 was observed. Since the cytotoxicity of doxorubicin has been associated with its ability to undergo enzymatic activation and to form hydroxyl (OH) radicals in this cell line, we also quantitated the OH formation in the BSO-treated and untreated MCF-7/ADRR cells using electron spin resonance spintrapping techniques. These results show that doxorubicin stimulated at least 2-fold more OH formation in the tumor cells after GSH levels were decreased by 90%. These results indicate that GSH plays an important role in modulating doxorubicin-induced OH formation via the scavenging of hydrogen peroxide by glutathione peroxidase and thus partially protects MCF-7/ADRR cells from the cytotoxic effect of doxorubicin.

DNA interstrand cross-link and free radical formation in a human multidrug-resistant cell line from mitomycin C and its analogues.

A subline of the human breast tumor cell line (MCF-7), selected for resistance to Adriamycin and having the multidrug resistance phenotype, also developed significant cross-resistance to mitomycin C and its two analogues, BMY 25282 and BMY 25067. Because mitomycin C and the analogues contain both quinone and aziridine moieties, the mechanism of tumor cell kill is thought to involve alkylation and cross-linking of DNA molecules, hence they are not expected to show cross-resistance to cells selected for resistance to a DNA intercalator. Studies to understand this novel observation show that the resistant MCF-7 cells form significantly less hydroxyl radical and DNA cross-linking in the presence of mitomycin C and BMY 25282 than the sensitive cells. Although BMY 25067 formed less free radicals in the resistant cells, similar to the other two drugs, the formation of DNA cross-links was identical in both cell lines, indicating a somewhat different mechanism of tumor cell kill by this analogue. DNA cross-link formation increased slightly with time in the sensitive cells while there was a small decrease in the resistant cells. This difference in the formation of toxic intermediates appeared to result from enhanced detoxification of reactive species (hydrogen peroxide and alkylating intermediates) as a result of significantly higher glutathione peroxidase (14-fold) and glutathione S-transferase (44-fold) activities in the resistant cell line. These events, i.e., free radical formation and DNA alkylation, showed a good correlation with the cytotoxicity in drug-sensitive cells, indicating that both mechanisms contribute to cell killing of human breast tumor cells.

Resistance of paraquat and adriamycin in human breast tumor cells: role of free radical formation.

Generation and enhanced detoxification of toxic free radicals by glutathione peroxidase and glutathione transferase in human breast tumor cells have been suggested to play an important role in toxicity and in resistance to adriamycin. We have examined the biochemical basis of paraquat-induced free radical formation and the mechanism of resistance to this agent in human breast tumor cell lines. We have also compared the similarities and differences between adriamycin and paraquat in their mode of free radical formation and tumor cell kill. Anaerobic incubation of paraquat resulted in the formation of the paraquat cation radical in both the sensitive and resistant cells which increased with time and was enhanced by NADPH addition. Our studies show that while both adriamycin and paraquat form hydroxyl radicals (.OH) in these cell lines, adriamycin was 2-3 fold better at reducing oxygen. The formation of .OH was inhibited by exogenously added superoxide dismutase and catalase, indicating the involvement of both superoxide anion radical and hydrogen peroxide. In the adriamycin-resistant cell line, less .OH was formed by each of these drugs. While the .OH appeared to be formed outside by both adriamycin and paraquat in the drug-sensitive cells, experiments using chromium oxalate as a spin-broadening agent suggest that the drug-induced .OH formation in the resistant cells is an intracellular event. The adriamycin-resistant cell line was also cross-resistant to paraquat, suggesting a common mechanism of toxicity for both drugs. However, adriamycin was significantly more toxic (4000-times) to the sensitive cells suggesting that either other mechanisms or site-specific free radical formation are also important in biochemical mechanisms of adriamycin toxicity.

DNA damage, cytotoxicity and free radical formation by mitomycin C in human cells.

Mitomycin C (MMC), a quinone-containing antitumor drug, has been shown to alkylate DNA and to form DNA cross-links. The ability of MMC to alkylate O6-guanine and to form interstrand cross-links (ISC) has been studied using Mer+ and Mer- human embryonic cells. Mer+ (IMR-90) cells have been reported to contain an O6-alkylguanine transferase enzyme and are, in general, more resistant to alkylating agents than the Mer- (VA-13) cell line, which is deficient in the repair of O6-lesions in DNA. Studies reported here show that MMC is more cytotoxic to VA-13 cells compared to IMR-90 cells. The alkaline elution technique was used to quantify MMC-induced ISC, and double strand breaks (DSB) in these cells. The drug-dependent formation of DSB was significantly lower in IMR-90 cells than in VA-13 cells. In contrast, no significant difference in cross-linking could be detected at the end of 2-h drug treatment. Although a small increase in cross-link frequency was observed in the VA-13 cell line relative to the IMR-90 cell line 6 h post drug treatment, it is not clear whether monoalkylated adducts at the O6-position are formed, and contribute to cross-link formation for differential cytotoxicity in VA-13 cells. Electron spin resonance and spin-trapping technique were used to detect the formation of hydroxyl radical from MMC-treated cells. Our studies show that MMC significantly stimulated the formation of hydroxyl radical in VA-13 cells, but not in the IMR-90 cells. The formation of the hydroxyl radical was inhibited by superoxide dismutase (SOD) and catalase. In addition, the presence of these enzymes partially protected VA-13 cells from MMC toxicity but not IMR-90 cells. Further studies indicated that the decreased free radical formation and resistance to MMC may be due to the increased activities of catalase and glutathione transferase in the IMR-90 cell line. These results suggest that MMC-dependent DNA damage (alkylation and DNA DSB) and the stimulation of oxy-radical formation may play critical roles in the determination of MMC-induced cell killing.