Abstract
In this study we aimed to (1) screen phenothiazines for cytotoxic activity in glioma, neuroblastoma, and primary mouse brain tissue; and (2) determine the mechanism of the cytotoxic effect (apoptosis, necrosis) and the roles of calmodulin inhibition and σ receptor modulation. Rat glioma (C6) and human neuroblastoma (SHSY-5Y) cell lines were treated with different phenothiazines. All agents induced a dose-dependent decrease in viability and proliferation, with the highest activity elicited by thioridazine. Sensitivity to thioridazine of glioma and neuroblastoma cells was significantly higher (p<0.05) than that of primary mouse brain culture (IC50 11.2 and 15.1 µM vs 41.3 µM, respectively). The N-mustard fluphenazine induced significantly lower cytotoxicity in glioma cells, compared to fluphenazine. The σ receptor selective ligand (+)-SK&F10047 increased viability slightly while combined with fluphenazine; SK&F10047 did not alter fluphenazine activity. Flow cytometry of propidium iodide (PI)-stained glioma cells treated with thioridazine, fluphenazine, or perphenazine (6–50 µM) resulted in a concentration-dependent increase of fragmented DNA up to 94% vs 3% in controls by all agents. Thioridazine (12.5 µM)-treated glioma cells costained with PI and Hoechst 33342 revealed a red fluorescence of fragmented nuclei in treated cells and a blue fluorescence of intact control nuclei. After 4-h exposure to thioridazine (25 and 50 µM), a 25- to 30-fold increase in caspase-3 activity in neuroblastoma cells was noted. Overall, the marked apoptotic effect of phenothiazines in brain-derived cancer cells, and the low sensitivity of primary brain tissue suggest the potential use of selected agents as therapeutic modalities in brain cancer.
Similar content being viewed by others
References
Barancik M., Polekova L., Mrazova T., Breier A., Stankovicova T., and Slezak J. (1994) Reversal effects of several Ca(2+) entry blockers, neuroleptics and local anesthetics on p-glycoprotein-mediated vincristine resistance of L1210/VCR mouse leukemic cell-line. Drugs Exp. Clin. Res. 20, 13–18.
Behl C., Rupprecht R., Skutelia T., and Holsboer F. (1995) Haloperidol induced cell death- mechanism and protection with vitamin E “in vitro.” NeuroReport 7, 360–364.
Ben-Shachar D., Livne E., Spanier I., Leenders K. L., and Youdim M. B. (1994) Typical and atypical neuroleptics induce alteration in blood brain barrier and brain 59Fe c13 uptake. J. Neurochem. 62, 1112–1118.
Borenfreund E., and Puerner J. A. (1984) A simple quantitave procedure using monolayer cultures for cytotoxicity assays (HTD/NR-90). J. Tissue Culture Methods 9, 7–9.
Bossy-Wetzel E., Newmeyer D. D., and Green D. R. (1998) Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J. 17, 37–49.
Cameron L. B. (1992) Neuropsychotrophic drugs as adjuncts in the treatment of cancer pain. Oncology (Huntingt.) 6, 65–72.
Cohen M. E., Dembling B., and Schorling J. B. (2002) The association between schizophrenia and cancer: A population based mortality study. Schizophr. Res. 57, 139–146.
Deutsch S. I., Weizman A., Goldman M. E., and Morihisa J. M. (1988) The sigma receptor: a novel site implicated in psychosis and antipsychotic drug efficacy. Clin. Neuropharmacol. 11, 105–119.
Eriksson A., Yachnin J., Lewensohn R., Nilsson A., and Nilsson A. (2001) DNA dependent protein kinase is inhibited by trifluoperazine. Biochem Biophys. Res. Commun. 283, 726–731.
Galili Mosberg R., Gil-Ad I., Weizman A., Melamed E., and Offen D. (1999) Haloperidol-induced neurotoxicity—possible implication for tardive dyskinesia. J. Neural Transm. 107, 479–490.
Garcia-Calvo M., Peterson E. P., Leiting B., Ruel R., Nicholson D. W., and Thornberry N. A. (1998) Inhibition of human caspases by peptide-based and macromolecular inhibitors. J. Biol. Chem. 273, 32608–32613.
Gil-Ad I., Offen D., Shtaif B., Gallili-Mosberg R., and Weizman A. (1998) Haloperidol induces neurotoxicity in mouse embryo brain tissue. Evidence for oxidative damage mechanism, and implication for tardive dyskinesia,. in Progress in Alzheimer’s and Parkinson’s Diseases, Fisher, A., Yoshida, M., and Hanin, I., eds., Plenum Press, NY, pp. 163–169.
Gil-Ad I., Shtaif B., Shiloh R., and Weizman A. (2001) Evaluation of the neurotoxic activity typical and atypical neuroleptics: relevance to iatrogenic extrapyramidal symptoms. Cell. Mol. Neurobiol. 21, 705–716.
Hait W. N. and Lee A. L. (1985) Characteristics of the cytotoxic effects of the phenothiazine class of calmodulin antagonists. Biochem. Pharmacol. 34, 3973–3978.
Hait W. N., Glazer L., Kaiser C., Cross J., and Kennedy K. A. (1987) Pharmacological properties of fluphenazine-mustard, an irreversible calmodulin antagonist. Mol. Pharmacol. 32, 404–409.
Harel A., Bloch O., Vardi P., and Bloch K. (2002) Sensitivity of HaCat keratinocytes to diabetogenic toxins. Biochem. Pharmacol. 63, 171–178.
John C. S., Vilner B. J., Geyer B. C., Moody T., and Bowen W. D. (1999) Targeting sigma receptor-binding benzamides as in vivo diagnostic and therapeutic agents for human prostate tumors. Cancer Res. 59, 4578–4583.
Karmakar P., Natarajan A. T., Poddar R. K., and Dasgupta U. B. (2001) Induction of apoptosis by phenothiazines derivatives in V79 cells. Toxicol Lett. 15, 19–28.
Lang A., Soosaar A., Koks S., Volke V., Bourin M., Bradwejn J., and Vasar E. (1994) Pharmacological comparison of antipsychotic drugs and sigma antagonsits in rodents. Pharmacol. Toxicol. 75, 222–227.
Lee G. L., and Hait W. M. (1985) Inhibition of growth of C6 astrocytoma cells by inhibitors of calmodulin. Life Sci. 36, 347–354.
Makar T. K., Nedergraad M., Preuss A., Gelbard A., Perumal A. S., and Cooper A. J. L. (1994) Vitamin E, ascorbate, glutathione disulfide and enzymes of glutathione metabolism in cultures of chick astrocytes and neurons: evidence that astrocytes play an important role in antioxidative processes in the brain. J. Neurochem. 62, 45–53.
McGinnis K. M., Gnegy M. E., Falk N., Nath R., and Wang K. K. W. (2003) Cytochrome c translocation does not lead to caspase activation in maitotoxin-treated SH-SY5Y neuroblastoma cells. Neurochem. Int. 42, 517–523.
Moran J., Itoh T., Reddy U. R., Chen M., Alnemri E. S., and Pleasure I. (1999) Caspase-3 expression by cerebellar granule neurons is regulated by calcium and cycling AMP. Neurochemistry 73, 568–577.
Mortensen B. (1994) The occurrence of cancer in first admitted schizophrenic patients. Schizophr. Res. 12, 185–194.
Motohashi N., Kurihara T., Satoh K., Sakagami H., and Molnar J. (1997) Correlation between structure and diverse biological activities of “half mustard type” phenothiazines. Anticancer Res. 17, 4403–4406.
Motohashi N., Sakagami H., Kamata K., and Yamamoto Y. (1991) Cytotoxicity and differentiation-inducing activity of phenothiazine and benzo[a]phenothiazine derivatives. Anticancer Res. 11, 1933–1937.
Nicholson D. W. and Thornberry N. A. (1997) Caspases: killer proteases. Trends Biochem. Sci. 22, 299–306.
Nicolletti I., Migliorato G., Pagliacci M. C., Grimsani F., and Riccardi C. (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium—iodide staining and flow cytometry. J. Immunol. Methods 139, 271–279.
Nociari M. M., Shalev A., Benias P., and Russo C. (1998) A novel one-step highly sensitive fluorimetric assay to evaluate cell-mediated cytotoxicity. J. Immunol. Methods 213, 157–167.
Nordenberg J., Fenning E., Landau M., Weizman R., and Weizman A. (1999) Effect of psychotrophic drugs on cell proliferation and differentiation. Biochem. Pharmacol. 58, 1229–1236.
Patel A. J., Vertes Z. S., Lewis P. D., and Lai M. (1980) Effect of chlorpromazine on cell proliferation in the developing rat brain. A combined biochemical and morphological study. Brain Res. 202, 415–428.
Schleuning M., Brumme V., and Wilmanns W. (1993) Growth inhibition of human leukemic cell lines by the phenothiazine derivate fluphenazine. Anticancer Res. 13, 599–602.
Silver M. A., Yang Z. W., Ganguli R., and Nimgaonkar V. L. (1994) An inhibitory effect of psychoactive drugs on a human neuroblastoma cell line. Biol. Psychiatry 35, 824–826.
Sutherland R. L., Watts C. K., Hall R. E., and Ruenitz P. C. (1987) Mechanisms of growth inhibition by nonsteroidal antiestrogens in human breast cancer cells. J. Steroid Biochem. 27, 891–897.
Tam S. W. and Cook L. (1984) Sigma opiates and certain antipsychotic drugs mutually inhibit (+)-(3H)SKF10,047 and haloperidol binding in guinea pig brain membranes. Proc. Natl. Acad. Sci. USA 81, 5618–5621.
Vilner B. J., de Costa B. R., and Bowen W. D. (1995) Cytotoxic effects of sigma ligands: sigma receptor-mediated alteration in cellular morphology and viability. J. Neurosci. 15, 117–134.
Vilner B. J., John C. S., and Bowen W. D. (1995) Sigma-1 and sigma-2 receptors are expressed in a wide variety of human and rodent tumor cell-lines. Cancer Res. 15, 408–413.
Wuonola M. A., Palfreyman M. G., Motohashi N., Kawase M., Gabay S., and Gupta R. R. (1998) The primary in vitro anticancer activity of “half mustard type” phenothiazines in NCI’s revised anticancer screening paradigm. Anticancer Res. 18, 337–348.
Zhu H. G., Tayeh I., Israel L., and Castagna M. (1991) Different susceptibility of lung cell lines to inhibitors of tumor promotion and inducers of differentiation. J. Biol. Regul. Homeost. Agents 5, 52–58.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Gil-Ad, I., Shtaif, B., Levkovitz, Y. et al. Characterization of phenothiazine-induced apoptosis in neuroblastoma and glioma cell lines. J Mol Neurosci 22, 189–198 (2004). https://doi.org/10.1385/JMN:22:3:189
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1385/JMN:22:3:189