Cytotoxic mechanism of chlorpromazine
Chlorpromazine (CPZ), a phenothiazine derivative, which has been extensively used in the treatment of schizophrenia for the last 50 years, also has recently been reported to have anticancer potential. Though it is one of the widely publicised compounds the information on the mechanism of its cytotoxicity is limited. Hence, an in vitro investigation was undertaken to study its cytotoxic mechanism. In a time and dose related study in SA3T3 cells the early statistically significant cytotoxicity was measured after a six-hour exposure with 50 uM and above (maximum concentration applied was 100 uM). The cytotoxicity increased with time and after 24 hours even 10 uM produced significant cytotoxicity and the I/C50 concentration was 40 uM. CPZ was found to induce dose-dependent statistically significant DNA single strand breaks in SA3T3 cells two hours before cell viability loss was measured, but no double strand breaks was noticed. The cytotoxic response in the presence of proadifen revealed that CPZ, but not any of its a number of metabolites, as suggested by many authors, is the molecule responsible for the cytotoxicity. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, known to induce N-demethylation and N-polydemethylation of tertiary and quaternary amines potentiated the cytotoxicity of CPZ. Potentiation of CPZ cytotoxicity was seen also in the presence of clorgyline, a monoamine oxidase (A) inhibitor. Increased cytotoxicity in the presence of these agents indicates that the dimethylamino moiety of the dimethylaminopropyl side chain of CPZ plays an important role in the process. Although the importance of dimethylamino moiety in the cytotoxicity of CPZ has not been previously reported, established cytotoxic drugs like dacarbazine, tamoxifen and ethylenimines possess this group(s) in their structure. Flow cytometric analysis and other cytotoxicity measurements have indicated that the cytotoxicity induced by CPZ is biphasic that at 50 uM and above it induces cell death and at 40 uM and below it inhibits cell proliferation. The cytostatic effect of 40 uM CPZ is the same as that induced by 25-150 uM cisplatin, 6-50 juM vinblastin or 12.5- 200 uM doxorubicin. None of these compounds potentiated the cytotoxicity of CPZ but 30 jaM menadione, which causes double strand breaks in SA3T3 cells, increased the cytotoxicity of 70-100 uM CPZ. However, all the cytotoxic effects remained reversible up to the first four hours, for it was possible to restore total cell viability by removing CPZ from the extracellular medium after the said period. A fluorimetric analysis was developed, using 2',7'-dichlorodihydroxy fluorescein diacetate to measure free radical generation, which turned out to be useful in assessing the redox status of cells. Thus CPZ was found to raise the redox status of SA3T3 cells while keeping the cellular glutathione (GSH) concentration unaltered. Involvement of reactive oxygen species in CPZ mediated cytotoxicity was investigated. The OH radical scavengers: sodium benzoate, ot-tocopherol, ascorbic acid or retinol; the iron chelator: deferoxamine; the iron and copper chelator: 1,10-phenanthroline; O2 inhibitors: superoxide dismutase or diphenyline iodonium chloride; H2O2 inhibitors: catalase, or sodium azide; or the singlet oxygen quencher: 2,2,6,6-tetramethyl 4- piperidone gave no protection against CPZ cytotoxicity. There was no nitric oxide generated in CPZ treated SA3T3 cells and the peroxidase inhibitor sodium azide did not give any protection. Thus, the involvement of peroxinitrite or hypochloride anion as cytotoxicity mediators also was excluded. tertiary-Butyl hydroperoxide (t-BH)-mediated cytotoxic response was fully restored by CPZ though the cells died within the next two hours. The cytotoxic effect of CPZ in precision cut kidney slices of Wistar rats was found to be greater than that of liver slices. CPZ (100 uM) abolished lipid peroxidation and decreased lactate dehydogenase leakage induced by 1.5 mM t-BH in both kidney and liver tissue slices. However, in contrast to the findings in SA3T3 cells, CPZ inhibited GSH, though not to the extent of causing cytotoxicity, and the MTT assay did not show any protective effect in the said tissue slices. The relevance of all these findings in establishing the cytotoxic mechanism of CPZ is discussed. Since induction of cytotoxicity is a beneficial effect in anticancer chemotherapy the significance of the mechanisms and cytotoxicity of CPZ in cancer treatment is also discussed.