Tocotrienols Inhibited Growth and Induced Apoptosis in Human HeLa Cells Through the Cell Cycle Signaling Pathway

Abstract

Tocotrienols of palm oil have been shown to possess potent neuroprotective, antioxidative, anticancer, and cholesterol-lowering activities. In this study, the authors examined the antiproliferative effects of α-, γ- and δ-tocotrienols (αT3, γT3, and δT3), and α-tocopherol (αT) in human cervical carcinoma (HeLa) cells. Their mechanism(s) of action on cell cycle signaling pathway were also investigated. Results showed that the antiproliferative effect of αT3 (IC₅₀: 3.19 ± 0.05 µM) and γT3 (IC₅₀: 2.85 ± 0.07 µM) was more potent than δT3 (IC₅₀: >100 µM) and αT (IC₅₀: 69.46 ± 3.01 µM). Both αT3 and γT3 also demonstrated a dose-dependent and time-dependent induction of cell death.They caused cell cycle arrest at G2/M phase and triggered apoptosis as displayed by the externalization of annexin V—targeted phosphatidylserine and accumulation of sub-G1 peak. At a concentration of 3 µM, αT3 downregulated the expression of cyclin D3, p16, and CDK6, while having no effect on cyclin D1, p15, p21, p27, and CDK4 expression. However, γT3 showed no effect on these proteins. The induction of HeLa cell apoptosis by αT3 and γT3 appeared to be associated with the expression of IL-6, but not the other cytokines (IFN-γ, IL-2, and IL-10).Taken together, the results suggest that αT3 and γT3 are more effective than δT3 and αT in inhibiting HeLa cell proliferation, and their mode of action could be through the upregulation of IL-6, and the downregulation of cyclin D3, p16, and CDK6 expression in the cell cycle signaling pathway.

Keywords

tocotrienols antiproliferative apoptosis cell cycle HeLa cells

References

Bartek J. , Bartkova J. , Lukas J. The retinoblastoma protein pathway and the restriction point. Curr Opin Cell Biol. 1996; 8:805-814.

Mittnacht S. Control of pRB phosphorylation. Curr Opin Genet Dev. 1998;8:21-27.

Peters G. The D-type cyclins and their role in tumorigenesis. J Cell Sci. (Suppl). 1994;18:89-96.

Sherr CJ D-type cyclins. Trends Biochem Sci. 1995;20: 187-190.

Vermeulen K. , Van Bockstaele DR , Berneman ZN The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Prolif. 2003;36:131-149.

Schwartz GK , Shah MA Targeting the cell cycle: a new approach to cancer therapy. J Clin Oncol. 2005;23: 9408-9421.

Arber N. , Hibshoosh H. , Moss SF , et al. Increased expression of cyclin D1 is an early event in multistage colorectal carcinogenesis. Gastroenterology. 1996;110:669-674.

Sutter T. , Doi S. , Carnevale KA , Arber N. , Weinstein IB Expression of cyclin D1 and E in human colon. J Med. 1997;28: 285-309.

Baldassarre G. , Belletti B. , Bruni P. , et al. Overexpressed cyclin D3 contributes to retaining the growth inhibitor p27 in the cytoplasm of thyroid tumor cells . J Clin Invest. 1999;104: 865-874.

10.

Sutherland RL , Musgrove EA Cyclins and breast cancer. J Mammary Gland Biol Neoplasia. 2004;9:95-104.

11.

Bosserhoff AK Novel biomarkers in malignant melanoma. Clin Chim Acta. 2006;367:28-35.

12.

Grant S. , Roberts JD The use of cyclin-dependent kinase inhibitors alone or in combination with established cytotoxic drugs in cancer chemotherapy. Drug Resist Updates. 2003; 6:15-26.

13.

Park MT , Lee SJ Cell cycle and cancer. J Biochem Mol Biol. 2003;23:60-65.

14.

Pinion SB , Kennedy JH , Miller RW , MacLean AB Oncogene expression in cervical intraepithelial neoplasia and invasive cancer of cervix. Lancet. 1991;337:819-820.

15.

Tartour E. , Fossiez F. , Joyeux I. , et al. Interleukin 17, a T-cell-derived cytokine, promotes tumorigenicity of human cervical tumors in nude mice. Cancer Res. 1999;59: 3698-3704.

16.

Tartour E. , Gey A. , Sastre-Garau X. , et al. Analysis of interleukin 6 gene expression in cervical neoplasia using a quantitative polymerase chain reaction assay: evidence for enhanced interleukin 6 gene expression in invasive carcinoma. Cancer Res. 1994;54:6243-6248.

17.

Richter HE , Holley RL , Andrews WW , Owen J. , Miller KB The association of interleukin 6 with clinical and laboratory parameters of acute pelvic inflammatory disease. Am J Obstet Gynecol. 1999;181:940-944.

18.

HiranoT, Ishihara K. , Hibi M. Roles of STAT3 in mediating the cell growth, differentiation and survival signals relayed through the IL-6, family of cytokine receptors. Oncogene. 2000;19: 2548-2556.

19.

McIntyre BS , Briski KP , Gapor A. , Sylvester PW Antiproliferative and apoptotic effects of tocopherols and tocotrienols on preneoplastic and neoplastic mouse mammary epithelial cells. Proc Soc Exp Biol Med. 2000 ;224:292-301.

20.

Shun MC , Yu W. , Gapor A. , et al. Pro-apoptotic mechanisms of action of a novel vitamin E analog (alpha-TEA) and a naturally occurring form of vitamin E (delta tocotrienol) in MDA-MB-435 human breast cancer cells. Nutr Cancer. 2004; 48:95-105.

21.

Shah SJ , Sylvester PW Gamma-tocotrienol inhibits neoplastic mammary epithelial cell proliferation by decreasing Akt and nuclear factor kappaB activity. Exp Biol Med (Maywood). 2005;230:235-241.

22.

Agarwal MK , Agarwal ML , Athar M. , Gupta S. Tocotrienol-rich fraction of palm oil activates p53, modulates Bax/Bcl2 ratio and induces apoptosis independent of cell cycle association. Cell Cycle. 2004;3:205-211.

23.

Janmejai KS , Sanjay G. Tocotrienol-rich fraction of palm oil induces cell cycle arrest and apoptosis selectively in human prostate cancer cells . Biochem Biophys Res Commun. 2006; 346:447-453.

24.

Sakai M. , Okabe M. , Tachibana H. , Yamada K. Apoptosis induction by gamma-tocotrienol in human hepatoma Hep3B cells. J Nutr Biochem. 2006;17:672-676.

25.

Sun W. , Wang Q. , Chen B. , Liu J. , Liu H. , Xu W. γ-Tocotrienol-induced apoptosis in human gastric cancer SGC-7901 cells is associated with a suppression in MAPK signalling. Br J Nutr. 2008;99:1247-1254.

26.

Kamal-Eldin A. , Appelqvist LA The chemistry and antioxidant properties of tocopherols and tocotrienols. Lipids. 1996 ; 31:671-701.

27.

Qureshi AA , Sami SA , Salser WA , Khan FA Dose-dependent suppression of serum cholesterol by tocotrienol-rich fraction (TRF25) of rice bran in hypercholesterolemic humans. Atherosclerosis . 2002;161:199-207.

28.

Khanna S. , Roy S. , Ryu H. , et al. Molecular basis of vitamin E action: tocotrienol modulates 12-lipoxygenase, a key mediator of glutamate-induced neurodegeneration. J Biol Chem. 2003;278:43508-43515.

29.

Mantena SK , Sharma SD , Katiyar SK Berberine, a natural product, induces G1-phase cell cycle arrest and caspase-3-dependent apoptosis in human prostate carcinoma cells. Mol Cancer Ther . 2006;5:296-308.

30.

Ortega S. , Malumbres M. , Barbacid M. Cyclin D-dependent kinases, INK4 inhibitors and cancer. Biochim Biophys Acta. 2002;1602:73-87.

31.

Hoque A. , Chen H. , Xu XC Statin induces apoptosis and cell growth arrest in prostate cancer cells. Cancer Epidemiol Biomarkers Prev. 2008;17:88-94.

32.

Tsai SL , Tsai SL , Suk FM , et al. Anti-tumor potential of 15,16-dihydrotanshinone I against breast adenocarcinoma through inducing G1 arrest and apoptosis. Biochem. Pharmacol. 2007;74:1575-1586.

33.

Aggarwal BB , Ichikawa H. Molecular targets and anticancer potential of indole-3-carbinol and its derivatives. Cell Cycle. 2005;4:1201-1215.

34.

Sun W. , Xu W. , Liu H. , et al. gamma-Tocotrienol induces mitochondria-mediated apoptosis in human gastric adenocarcinoma SGC-7901 cells. J Nutr Biochem . 2009;20:276-284.

35.

Wei LH , Kuo ML , Chen CA , et al. The anti-apoptotic role of interleukin-6 in human cervical cancer is mediated by upregulation of Mcl-1 through a PI 3-K/Akt pathway. Oncogene. 2001;20:5799-5809.

36.

Strömbeck L. , Sandros J. , Holst E. , et al. Prevotella bivia can invade human cervix epithelial (HeLa) cells. APMIS. 2007 ; 115:241-251.