Biomolecules & Therapeutics

The Korean Society of Applied Pharmacology (한국응용약물학회)
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Lovastatin Induces Apoptotic Cell Death by Activation of Intracellular Ca2+ Signal in HepG2 Human Hepatoma Cells

  • Published : 2007.09.30
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Abstract

Although lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase, has been shown to have anti-cancer actions, the effect on human hepatoma cells was not investigated. Moreover, the exact mechanism of this action is not fully understood. In this study we investigated the mechanism by which lovastatin induces apoptosis using HepG2 human hepatoblastoma cells. Lovastatin induced apoptotic cell death in a dose-dependent manner in the cells, assessed by the flow cytometric analysis. Treatment with mevalonic acid, a precursor of cholesterol, did not significantly suppress the lovastatin-induced apoptosis. Lovastatin induced a rapid and sustained increase in intracellular $Ca^{2+}$ concentration. Treatment with EGTA, an extracellular $Ca^{2+}$ chelator did not significantly alter the lovastatin-induced intracellular $Ca^{2+}$ increase and apoptosis, whereas intracellular $Ca^{2+}$ reduction with BAPTA/AM and intracellular $Ca^{2+}$ release blockers (dantrolene and TMB-8) completely blocked these actions of lovastatin. In addition, the lovastatin-induced apoptosis was significantly reduced by a calpain inhibitor, a broad spectrum caspase inhibitor z-VAD-fmk and inhibitors specific for caspase-9 and caspase-3 (z-LEHD-fmk and z-DEVD-fmk, respectively), but not by an inhibitor specific for caspase-8 (z-IETD-fmk). Collectively, these results suggest that lovastatin induced apoptosis of HepG2 hepatoma cells through intracellular $Ca^{2+}$ release and calpain activation, leading to triggering mitochondrial apoptotic pathway. These results further suggest that lovastatin may be valuable for the therapeutic management of human hepatoma.

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References

  1. Adams, J. M. and Cory, S. (1998). The Bcl-2 protein family: arbiters of cell survival. Science 281, 1322-1326 https://doi.org/10.1126/science.281.5381.1322
  2. Alegret. M. and Silvestre, J. S. (2006). Pleiotropic effects of statins and related pharmacological experimental approaches. Methods Find. Exp. Clin. Pharmacol. 28, 627-656 https://doi.org/10.1358/mf.2006.28.9.1003573
  3. Alonso, D. F., Farina, H. G., Skilton, G., Gabri, M. R., de Lorenzo, M. S. and Gomez, D. E. (1998). Reduction of mouse mammary tumor formation and metastasis by lovastatin, an inhibitor of the mevalonate pathway of cholesterol synthesis. Breast Cancer Res. Treat. 50, 83-93 https://doi.org/10.1023/A:1006058409974
  4. Bombeli, T., Karsan, A., Tait, J. F. and Harlan, J. M. (1997). Apoptotic vascular endothelial cells become procoagulant. Blood 89, 2429-2442
  5. Borner, M. M., Myers, C. E., Sartor, O., Sei, Y., Toko, T., Trepel, J. B. and Schneider, E. (1995). Drug-induced apoptosis is not necessarily dependent on macromolecular synthesis or proliferation in the p53-negative human prostate cancer cell line PC-3. Cancer Res. 55, 2122-2128
  6. Broitman, S. A., Wilkinson, J. T., Cerda, S and Branch, S. K. (1996). Effects of monoterpenes and mevinolin on murine colon tumor CT-26 in vitro and its hepatic 'metastases' in vivo. Adv. Exp. Med. Biol. 401, 111-130 https://doi.org/10.1007/978-1-4613-0399-2_9
  7. Brower, V. (2003). Of cancer and cholesterol: studies elucidate anticancer mechanisms of statins. J. Natl. Cancer Inst. 95, 844-846 https://doi.org/10.1093/jnci/95.12.844
  8. Buhaescu, I. and Izzedine, H. (2007). Mevalonate pathway: a review of clinical and therapeutical implications. Clin. Biochem. 40, 575-584 https://doi.org/10.1016/j.clinbiochem.2007.03.016
  9. Chan, K. K., Oza, A. M. and Siu, L. L. (2003). The statins as anticancer agents. Clin. Cancer Res. 9, 10-19
  10. Crompton, N. E. (1998) Programmed cellular response in radiation oncology. Acta Oncol. 37, 1-4 https://doi.org/10.1080/028418698430656
  11. Dalenc, F., Giamarchi, C., Petit, M., Poirot, M., Favre, G. and Faye, J. C. (2005). Farnesyl-transferase inhibitor R115,777 enhances tamoxifen inhibition of MCF-7 cell growth through estrogen receptor dependent and independent pathways. Breast Cancer Res. 7, R1159-R1167 https://doi.org/10.1186/bcr1357
  12. Ehrlich, B. E., Kaftan, E., Bezprozvannaya, S., and Bezprozvanny, I. (1994). The pharmacology of intracellular $Ca^{2+}$-release channels. Trends Pharmacol. Sci. 15, 145-149 https://doi.org/10.1016/0165-6147(94)90074-4
  13. Farina, H. G., Bublik, D. R., Alonso, D. F. and Gomez, D. E. (2002). Lovastatin alters cytoskeleton organization and inhibits experimental metastasis of mammary carcinoma cells. Clin. Exp. Metastasis 19, 551-559 https://doi.org/10.1023/A:1020355621043
  14. Fernndez, C., Lobo, M., Gmez-Coronado, D. and Lasuncin, M. A. (2004). Cholesterol is essential for mitosis progression and its deficiency induces polyploid cell formation. Exp. Cell Res. 300, 109-120 https://doi.org/10.1016/j.yexcr.2004.06.029
  15. Gogvadze, V. and Orrenius S. (2006). Mitochondrial regulation of apoptotic cell death. Chem. Biol. Interact. 163, 4-14 https://doi.org/10.1016/j.cbi.2006.04.010
  16. Goldstein, J. L. and Brown, M. S. (1990). Regulation of the mevalonate pathway. Nature 343, 425-430 https://doi.org/10.1038/343425a0
  17. Goll, D. E., Thompson, V. F., Li, H., Wei. W. and Cong, J. (2003). The calpain system. Physiol. Rev. 83, 731-801 https://doi.org/10.1152/physrev.00029.2002
  18. Gray-Bablin, J., Rao, S. and Keyomarsi, K. (1997). Lovastatin induction of cyclin-dependent kinase inhibitors in human breast cells occurs in a cell cycle-independent fashion. Cancer Res. 57, 604-609
  19. Grynkiewicz, G., Poene, M. and Tsien, R. Y. (1985). A new generation of $Ca^{2+}$ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440-3450
  20. Hengartner, M. O. (2000). The biochemistry of apoptosis. Nature 407, 770-776 https://doi.org/10.1038/35037710
  21. Hindler, K., Cleeland, C. S, Rivera, E. and Collard, C. D. (2006). The role of statins in cancer therapy. Oncologist 11, 306-315 https://doi.org/10.1634/theoncologist.11-3-306
  22. Inano, H., Suzuki, K., Onoda, M. and Wakabayashi, K. (1997). Anti-carcinogenic activity of simvastatin during the promotion phase of radiation-induced mammary tumorigenesis of rats. Carcinogenesis 18, 1723-1727 https://doi.org/10.1093/carcin/18.9.1723
  23. Jani, J. P., Specht, S., Stemmler, N., Singh, S. V., Gupta, V. and Katoh, A. (1993). Metastasis of B16F10 mouse melanoma inhibited by lovastatin, an inhibitor of cholesterol biosynthesis. Invasion Metastasis 13, 314-324
  24. Jurgensmeier, J., Xie, Z,. Deveraux, Q., Ellerby, L., Bredesen, D. and Reed, J. (1998). Bax directly induces release of cytochrome c from isolated mitochondria. Proc. Natl Acad. Sci. USA 95, 4997-5002 https://doi.org/10.1073/pnas.95.9.4997
  25. Kamesaki, H. (1998). Mechanisms involved in chemotherapyinduced apoptosis and their implications in cancer chemotherapy. Int. J. Hematol. 68, 29-43 https://doi.org/10.1016/S0925-5710(98)00038-3
  26. Kastan, M. B., Canman, C. E. and Leonard, C. J. (1995). P53, cell cycle control and apoptosis: implications for cancer. Cancer Metastasis Rev. 14, 3-15 https://doi.org/10.1007/BF00690207
  27. Keyomarsi, K., Sandoval, L., Band, V. and Pardee, B. (1991). Synchronization of tumor and normal cells from G1 to multiple cell cycles by lovastatin. Cancer Res. 51, 3602-3609
  28. Kidd, V. J. (1998). Proteolytic activities that mediate apoptosis. Annu. Rev. Physiol. 60, 533-573 https://doi.org/10.1146/annurev.physiol.60.1.533
  29. Kornblau, S. M. (1998) The role of apoptosis in the pathogenesis, prognosis, and therapy of hematologic malignancies. Leukemia 12, S41-46
  30. Kozar, K., Kaminski, R., Legat, M., Kopec, M., Nowis, D., Skierski, J. S., Koronkiewicz, M., Jakobisiak, M. and Golab, J. (2004). Cerivastatin demonstrates enhanced antitumor activity against human breast cancer cell lines when used in combination with doxorubicin or cisplatin. Int. J. Oncol. 24, 1149-1
  31. Koyuturk, M., Ersoz, M. and Altiok, N. (2004). Simvastatin induces proliferation inhibition and apoptosis in C6 glioma cells via c-jun N-terminal kinase. Neurosci. Lett. 370, 212-217 https://doi.org/10.1016/j.neulet.2004.08.020
  32. Luo, X., Budihardjo, I., Zuo, H., Slaughter, C. and Wang, X. (1998). Bid, a $Bcl_{2}$ interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptor. Cell 94, 481-490 https://doi.org/10.1016/S0092-8674(00)81589-5
  33. Marcelli, M., Cunningham, G. R., Haidacher, S. J., Padayatty, S. J., Sturgis, L., Kagan, C. and Denner, L. (1998). Caspase-7 is activated during lovastatin-induced apoptosis of the prostate cancer cell line LNCaP. Cancer Res. 58, 76-83
  34. Matar, P., Rozados, V. R., Roggero, EA. and Scharovsky, O. G. (1998). Lovastatin inhibits tumor growth and metastasis development of a rat fibrosarcoma. Cancer Biother. Radiopharm. 13, 387-393 https://doi.org/10.1089/cbr.1998.13.387
  35. McCollum, A. T., Nasr, P. and Estus, S. (2002). Calpain activates caspase-3 during UV-induced neuronal death but only calpain is necessary for death. J. Neurochem. 82, 1208-1220 https://doi.org/10.1046/j.1471-4159.2002.01057.x
  36. McConkey, D. J. and Orrenius, S. (1996). The role of calcium in the regulation of apoptosis. J. Leukoc. Biol. 59, 775-783 https://doi.org/10.1002/jlb.59.6.775
  37. McConkey, DJ. and Orrenius, S. (1997). The role of calcium in the regulation of apoptosis. Biochem. Biophys. Res. Commun. 239, 357-366 https://doi.org/10.1006/bbrc.1997.7409
  38. McTaggart, S. J. (2006). Isoprenylated proteins. Cell. Mol. Life Sci. 63, 255-267 https://doi.org/10.1007/s00018-005-5298-6
  39. Mo, H. and Elson, C. E. (2004). Studies of the isoprenoid-mediated inhibition of mevalonate synthesis applied to cancer chemotherapy and chemoprevention. Exp. Biol. Med. 229, 567-585 https://doi.org/10.1177/153537020422900701
  40. Molinari, M. and Carafoli, E. (1997). Calpain: a cytosolic proteinase active at the membranes. J. Membr. Biol. 156, 1-8 https://doi.org/10.1007/s002329900181
  41. Muck, A. O, Seeger, H. and Wallwiener, D. (2004). Inhibitory effect of statins on the proliferation of human breast cancer cells. Int. J. Clin. Pharmacol. Ther. 42, 695-700 https://doi.org/10.5414/CPP42695
  42. Munaron, L., Antoniotti, S., Pla, A. F. and Lovisolo, D. (2004). Blocking $Ca^{2+}$ entry: a way to control cell proliferation. Curr. Med. Chem. 12, 1533-1543
  43. Mutoh, T., Kumano, T., Nakagawa, H. and Kuriyama, M. (1999). Role of tyrosine phosphorylation of phospholipase C $\gamma$1 in the signaling pathway of HMG-CoA reductase inhibitor-induced cell death of L6 myoblasts. FEBS Lett. 446, 91-94 https://doi.org/10.1016/S0014-5793(99)00188-X
  44. Nath, R., Raser, K. J., Stafford, D., Hajimohammadreza, I., Posner, A., Allen, H., Talanian, R. V., Yuen, P., Gilbertsen. R. B. and Wang, K. K. (1996). Non-erythroid $\alpha$-spectrin breakdown by calpain and interleukin 1$\beta$-converting-enzyme-like protease( s) in apoptotic cells: contributory roles of both protease families in neuronal apoptosis. Biochem. J. 319, 683-690 https://doi.org/10.1042/bj3190683
  45. Poynter, J. N., Gruber, S. B., Higgins, P. D., Almog, R., Bonner, J. D. and Rennert, H. S. (2005) Statins and the risk of colorectal cancer. N. Engl. J. Med. 352, 2184-2192 https://doi.org/10.1056/NEJMoa043792
  46. Rao, S., Lowe, M., Herliczek, T. W. and Keyomarsi, K. (1998). Lovastatin-mediated G1 arrest in normal and tumor breast cells is through inhibition of CDK2 activity and redistribution of p21 and p27, independent of p53. Oncogene 17, 2393-2402 https://doi.org/10.1038/sj.onc.1202322
  47. Saito, M., Korsmeyer, S. J. and Schlesinger, P. H. (2000). BAXdependent transport of cytochrome c reconstituted in pure liposomes. Nature Cell Biol. 2, 553-555 https://doi.org/10.1038/35019596
  48. Schulte-Hermann, R., Bursch, W., Low-Baselli, A., Wagner, A. and Grasl-Kraupp, B. (1997). Apoptosis in the liver and its role in hepatocarcinogenesis. Cell. Biol. Toxicol. 13, 339-348 https://doi.org/10.1023/A:1007495626864
  49. Shellman, Y. G., Ribble, D., Miller, L., Gendall, J., Vanbuskirk, K., Kelly, D., Norris, D. A. and Dellavalle, R. P. (2005). Lovastatin-induced apoptosis in human melanoma cell lines. Melanoma Res. 15, 83-89 https://doi.org/10.1097/00008390-200504000-00001
  50. Shibata, M. A., Ito, Y., Morimoto, J. and Otsuki, Y. (2004). Lovastatin inhibits tumor growth and lung metastasis in mouse mammary carcinoma model: a p53-independent mitochondrialmediated apoptotic mechanism. Carcinogenesis 25, 1887-1898 https://doi.org/10.1093/carcin/bgh201
  51. Shibata, M. A., Kavanaugh, C., Shibata, E., Abe, H., Nguyen, P., Otsuki, Y., Trepel, J. B. and Green, J. E. (2003). Comparative effects of lovastatin on mammary and prostate oncogenesis in transgenic mouse models. Carcinogenesis 24, 453-459 https://doi.org/10.1093/carcin/24.3.453
  52. Shimizu, S., Narita, M. and Tsujimoto, Y. (1999). Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature 399, 483-487 https://doi.org/10.1038/20959
  53. Squier, M. K., Miller, A. C., Malkinson, A. M. and Cohen, J, J. (1994). Calpain activation in apoptosis. J. Cell Physiol. 159, 229-237 https://doi.org/10.1002/jcp.1041590206
  54. Taylor, J. M. and Simpson, R. U. (1992). Inhibition of cancer cell growth by calcium channel antagonists in the athymic mouse. Cancer Res. 52, 2413-2418
  55. van de Donk, N. W., Bloem, A. C., van der Spek, E. and Lokhorst, H. M. (2006). New treatment strategies for multiple myeloma by targeting BCL-2 and the mevalonate pathway. Curr. Pharm. Des. 12, 327-340 https://doi.org/10.2174/138161206775201974
  56. van de Loosdrecht, A. A., Nennie, E., Ossenkoppele, G. J., Beelen, R. H. and Langenhuijsen, M. M. (1991). Cell mediated cytotoxicity against U 937 cells by human monocytes and macrophages in a modified colorimetric MTT assay. A methodological study. J. Immunol. Methods 141, 15-22 https://doi.org/10.1016/0022-1759(91)90205-T
  57. Wang, K., Nath, R., Raser, K. J. and Hajimohammadreza, I. (1996). Maitotoxin induces calpain activation in SH-SY5Y neuroblastoma cells and cerebrocortical cultures. Biochem. Biophys. 331, 208-214 https://doi.org/10.1006/abbi.1996.0300
  58. Wood, D. E. and Newcomb, E.W. (1999). Caspase-dependent activation of calpain during drug-induced apoptosis. J. Biol. Chem. 274, 8309-8315 https://doi.org/10.1074/jbc.274.12.8309
  59. Wood, D. E., Thomas, A., Devi, L. A., Berman, Y., Beavis, R. C., Reed, J. C. and Newcomb, E. W. (1998). Bax cleavage is mediated by calpain during drug-induced apoptosis. Oncogene 17, 1069-1078 https://doi.org/10.1038/sj.onc.1202034
  60. Yoshida, J., Ishibashi, T. and Nishio, M. (2003). Antiproliferative effect of $Ca^{2+}$ channel blockers on human epidermoid carcinoma A431 cells. Eur. J. Pharmacol. 472, 23-31 https://doi.org/10.1016/S0014-2999(03)01831-4