A Phospholipase C-Dependent Intracellular $Ca^{2+}$ Release Pathway Mediates the Capsaicin-Induced Apoptosis in HepG2 Human Hepatoma Cells 73

  • Published : 2005.01.01


The effect of capsaicin on apoptotic cell death was investigated in HepG2 human hepatoma cells. Capsaicin induced apoptosis in time- and dose-dependent manners. Capsaicin induced a rapid and sustained increase in intracellular $Ca^{2+}$ concentration, and BAPTA, an intracellular $Ca^{2+}$ chelator, significantly inhibited capsaicin-induced apoptosis. The capsaicin-induced increase in the intracellular $Ca^{2+}$ and apoptosis were not significantly affected by the extracellular $Ca^{2+}$ chelation with EGTA, whereas blockers of intracellular $Ca^{2+}$ release (dantrolene) and phospholipase C inhibitors, U-73122 and manoalide, profoundly reduced the capsaicin effects. Interestingly, treatment with the vanilloid receptor antagonist, capsazepine, did not inhibit either the increased capsaicin-induced $Ca^{2+}$ or apoptosis. Collectively, these results suggest that the capsaicin-induced apoptosis in the HepG2 cells may result from the activation of a PLC-dependent intracellular $Ca^{2+}$ release pathway, and it is further suggested that capsaicin may be valuable for the therapeutic intervention of human hepatomas.


  1. Agrawal, R. C., Wiessler, M., Hecker, E., and Bhide, S. V., Tumour-promoting effect of chilli extract in BALB/c mice. Int. J. Cancer, 38, 689-695 (1986) https://doi.org/10.1002/ijc.2910380512
  2. Aranda, F. J., Villalain, J., and Gomez-Fernandez, J. C., Capsaicin affects the structure and phase organization of phospholipid membranes. Biochim. Biophys. Acta, 1234, 225-234 (1995) https://doi.org/10.1016/0005-2736(94)00293-X
  3. Azizan, A. and Blevins, R. D., Mutagenicity and antimutagenicity testing of six chemicals associated with the pungent properties of specific spices as revealed by the Ames Salmonella/microsomal assay. Arch. Environ. Cont. Toxicol., 28, 248-258 (1995)
  4. Bennett, C. F., Mong, S., Wu, H. L., Clark, M. A., Wheeler, L., and Crooke, S. T., Inhibition of phosphoinositide-specific phospholipase C by manoalide. Mol. Pharmacol., 32, 587- 593 (1987)
  5. Bombeli, T., Karsan, A., Tait, J. F., and Harlan, J. M., Apoptotic vascular endothelial cells become procoagulant. Blood, 89, 2429-2442 (1997)
  6. Bonnefoy-Berard, N., Genestier, L., Flacher, M., and Revillard, J. P., The phosphoprotein phosphatase calcineurin controls calcium-dependent apoptosis in B cell lines. Eur. J. Immunol., 24, 325-329 (1994) https://doi.org/10.1002/eji.1830240208
  7. Cochereau, C., Sanchez, D., Bourhaoui, A., and Creppy, E. E., Capsaicin, a structural analog of tyrosine, inhibits the aminoacylation of tRNA(Tyr). Toxicol. Appl. Pharmacol., 141, 133-137 (1996)
  8. Cohen, J. J. and Duke, R. C., Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death. J. Immunol., 132, 38-42 (1984)
  9. Distelhorst, C. W. and Dubyak, G., Role of calcium in glucocorticosteroid- induced apoptosis of thymocytes and lymphoma cells: resurrection of old theories by new findings. Blood, 91, 731-734 (1998)
  10. Dray, A. and Urban, L., New pharmacological strategies for pain relief. Annu. Rev. Pharmacol. Toxicol., 36, 253-280 (1996) https://doi.org/10.1146/annurev.pa.36.040196.001345
  11. Ehrlich, B. E., Kaftan, E., Bezprozvannaya, S., and Bezprozvanny, I., The pharmacology of intracellular $Ca^{2+}$-release channels. Trends Pharmacol. Sci., 15, 145-149 (1994) https://doi.org/10.1016/0165-6147(94)90074-4
  12. Feigin, A. M., Aronov, E. V., Bryant, B. P., Teeter, J. H., and Brand, J. G., Capsaicin and its analogs induce ion channels in planar lipid bilayers. Neuroreport, 6, 2134-2136 (1995) https://doi.org/10.1097/00001756-199511000-00009
  13. Fesus, L., Thomazy, V., and Falus, A., Induction and activation of tissue transglutaminase during programmed cell death. FEBS Lett., 224, 104-108 (1987) https://doi.org/10.1016/0014-5793(87)80430-1
  14. Grynkiewicz, G., Poene, M., and Tsien, R. Y., A new generation of $Ca^{2+}$ indicators with greatly improved fluorescence properties. J. Biol. Chem., 260, 3440-3450 (1985)
  15. Harman, A. W. and Maxwell, M. J., An evaluation of the role of calcium in cell injury. Annu. Rev. Pharmacol. Toxicol., 35, 129-144 (1995) https://doi.org/10.1146/annurev.pa.35.040195.001021
  16. Holzer, P., Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons. Pharmacol. Rev., 43, 143-201 (1991)
  17. Hughes, A. R. and Putney, J. W. Jr., Inositol phosphate formation and its relationship to calcium signaling. Environ. Health Perspect., 84, 141-147 (1990) https://doi.org/10.2307/3430715
  18. Jin, W., Lo, T. M., Loh, H. H., and Thayer, S. A., U73122 inhibits phospholipase C-dependent calcium mobilization in neuronal cells. Brain Res., 642, 237-243 (1994) https://doi.org/10.1016/0006-8993(94)90927-X
  19. Jordan, J., Galindo, M. F., and Miller, R. J., Role of calpain and interleukin-1 beta converting enzyme-like proteases in the beta-amyloid-induced death of rat hippocampal neurons in culture. J. Neurochem., 68, 1612-1621 (1997) https://doi.org/10.1046/j.1471-4159.1997.68041612.x
  20. Kastan, M. B., Canman, C. E., and Leonard, C. J., P53, cell cycle control and apoptosis: implications for cancer. Cancer Metastasis Rev., 14, 3-15 (1995) https://doi.org/10.1007/BF00690207
  21. Kidd, V. J., Proteolytic activities that mediate apoptosis. Annu. Rev. Physiol., 60, 533-573 (1998) https://doi.org/10.1146/annurev.physiol.60.1.533
  22. Kornblau, S. M., The role of apoptosis in the pathogenesis, prognosis, and therapy of hematologic malignancies. Leukemia, 12 Suppl 1, S41-46 (1998)
  23. Kuenzi, F. M. and Dale, N., Effect of capsaicin and analogues on potassium and calcium currents and vanilloid receptors in Xenopus embryo spinal neurones. Br. J. Pharmacol., 119, 81-90 (1996) https://doi.org/10.1111/j.1476-5381.1996.tb15680.x
  24. Lawson, T. and Gannett, P., The mutagenicity of capsaicin and dihydrocapsaicin in V79 cells. Cancer Lett., 49, 109-113 (1989) https://doi.org/10.1016/0304-3835(89)90045-1
  25. Macho, A., Blazquez, M. V., Navas, P., and Munoz, E., Induction of apoptosis by vanilloid compounds does not require de novo gene transcription and activator protein 1 activity. Cell Growth Differ., 9, 277-286 (1998)
  26. Macho, A., Calzado, M. A., Munoz-Blanco, J., Gomez-Diaz, C., Gajate, C., Mollinedo, F., Navas, P. and Munoz, E., Selective induction of apoptosis by capsaicin in transformed cells: the role of reactive oxygen species and calcium. Cell Death Differ., 6, 155-165 (1999) https://doi.org/10.1038/sj.cdd.4400465
  27. McConkey, D. J. and Orrenius, S., The role of calcium in the regulation of apoptosis. J. Leukoc. Biol., 59, 775-783 (1996)
  28. Melino, G., Annicchiarico-Petruzzeli, M., Piredda, L., Candi, E., Gentile, V., Davies, P. J., and Piacentini, M., Tissue transglutaminase and apoptosis: Sense and antisense transfection studies with human neuroblastoma cells. Mol. Cell Biol., 14, 6584-6596 (1994)
  29. Milner, A. E., Levens, J. M., and Gregory, C. D., Flow cytometric methods of analyzing apoptotic cells. Methods Mol. Biol., 80, 347-354 (1998) https://doi.org/10.1007/978-1-59259-257-9_35
  30. Morre, D. J., Chueh, P. J., and Morre, D. M., Capsaicin inhibits preferentially the NADH oxidase and growth of transformed cells in culture. Proc. Natl. Acad. Sci. U.S.A., 92, 1831-1835 (1995) https://doi.org/10.1073/pnas.92.6.1831
  31. Morre, D. J., Sun, E., Geilen, C., Wu, L. Y., de Cabo, R., Krasagakis, K., Orfanos, C. E., and Morre, D. M., Capsaicin inhibits plasma membrane NADH oxidase and growth of human and mouse melanoma lines. Eur. J. Cancer, 32, 1995-2003 (1996) https://doi.org/10.1016/0959-8049(96)00234-1
  32. Nayfield, S. G., Karp, J. E., Ford, L. G., Dorr, F. A., and Kramer, B. S., Potential role of tamoxifen in prevention of breast cancer. J. Natl. Cancer. Inst., 83, 1450-1459 (1991) https://doi.org/10.1093/jnci/83.20.1450
  33. Schulte-Hermann, R., Bursch, W., Low-Baselli, A., Wagner, A., and Grasl-Kraupp, B., Apoptosis in the liver and its role in hepatocarcinogenesis. Cell Biol. Toxicol., 13, 339-348 (1997) https://doi.org/10.1023/A:1007495626864
  34. Shibasaki, F., Kondo, E., Akagi, E., and McKeon, F., Suppresion of signaling through NF-AT by interactions between calcineurin and BCL-2. Nature, 386, 728-731 (1997) https://doi.org/10.1038/386728a0
  35. Squier, M. K. T. and Cohen, J. J., Calpain, an upstream regulator of thymocyte apoptosis. J. Immunol., 158, 3690- 3697 (1997)
  36. Squier, M. K. T., Miller, A. C. K., Malkinson, A. M., and Cohen, J. J., Calpain activation in apoptosis. J. Cell Physiol., 159, 229- 237 (1994) https://doi.org/10.1002/jcp.1041590206
  37. Surh, Y., Molecular mechanisms of chemopreventive effects of selected dietary and medicinal phenolic substances. Mutat. Res., 428, 305-327 (1999) https://doi.org/10.1016/S1383-5742(99)00057-5
  38. Szallasi, A. and Blumberg, P. M., Vanilloid (Capsaicin) receptors and mechanisms. Pharmacol. Rev., 51, 159-211 (1999)
  39. Takahata, K., Chen, X., Monobe, K., and Tada, M., Growth inhibition of capsaicin on HeLa cells is not mediated by intracellular calcium mobilization. Life Sci., 64, PL165-171 (1999) https://doi.org/10.1016/S0024-3205(99)00046-6
  40. Toth, B., Rogan, E., and Walker, B., Tumorigenicity and mutagenicity studies with capsaicin of hot peppers. Anticancer Res., 4, 117-119 (1984)
  41. Vermes, I., Haanen, C., Steffens-Nakken, H., and Reutelingsperger, C., A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immunol. Methods, 184, 39-51 (1995) https://doi.org/10.1016/0022-1759(95)00072-I
  42. Wolvetang, E. J., Larm, J. A., Moutsoulas, P., and Lawen, A., Apoptosis induced by inhibitors of the plasma membrane NADH-oxidase involves Bcl-2 and calcineurin. Cell Growth Differ., 7, 1315-1325 (1996)
  43. Wyllie, A. H., Morris, R. G., Smith, A. L., and Dunlop, D., Chromatin cleavage in apoptosis: Association with condensed chromatin morphology and dependence onmacromolecular synthesis. J. Pathol., 142, 67-67 (1984) https://doi.org/10.1002/path.1711420112
  44. Zhang, J., Nagasaki, M., Tanaka, Y., and Morikawa, S., Capsaicin inhibits growth of adult T-cell leukemia cells. Leuk. Res., 27, 275-283 (2003) https://doi.org/10.1016/S0145-2126(02)00164-9