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.

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