p53-dependent Fas expression is critical for Ginsenoside Rh2 triggered caspase-8 activation in HeLa cells

Abstract

We have recently reported that Ginsenoside Rh2 (G-Rh2) induces the activation of two initiator caspases, caspase-8 and caspase-9 in human cancer cells. However, the molecular mechanism of its death-inducing function remains unclear. Here we show that G-Rh2 stimulated the activation of both caspase-8 and caspase-9 simultaneously in HeLa cells. Under G-Rh2 treatment, membrane death receptors Fas and TNFR1 are remarkably upregulated. However, the induced expression of Fas but not TNFR1 was contributed to the apoptosis process. Moreover, significant increases in Fas expression and caspase-8 activity temporally coincided with an increase in p53 expression in p53-nonmutated HeLa and SK-HEP-1 cells upon G-Rh2 treatment. In contrast, Fas expression and caspase-8 activity remained constant with G-Rh2 treatment in p53-mutated SW480 and PC-3 cells. In addition, siRNA-mediated knockdown of p53 diminished G-Rh2-induced Fas expression and caspase-8 activation. These results indicated that G-Rh2-triggered extrinsic apoptosis relies on p53-mediated Fas over-expression. In the intrinsic apoptotic pathway, G-Rh2 induced strong and immediate translocation of cytosolic BAK and BAX to the mitochondria, mitochondrial cytochrome c release, and subsequent caspase-9 activation both in HeLa and in SW480 cells. p53-mediated Fas expression and subsequent downstream caspase-8 activation as well as p53-independent caspase-9 activation all contribute to the activation of the downstream effector caspase-3/-7, leading to tumor cell death. Taken together, we suggest that G-Rh2 induces cancer cell apoptosis in a multi-path manner and is therefore a promising candidate for antitumor drug development.

Keywords

G-Rh2 / Fas / p53 / apoptosis

References

[1] Andrews GA, Xi SC, Pomerantz RG, Lin CJ, Gooding WE, Wentzel AL, Wu L, Sidransky D, Grandis JR (2004) Mutation of p53 in head and neck squamous cell carcinoma correlates with Bcl-2 expression and increased susceptibility to cisplatin-induced apoptosis. Head Neck26: 870-877

[2] Bamford S, Dawson E, Forbes S, Clements J, Pettett R, Dogan A, Flanagan A, Teague J, Futreal PA, Stratton MR, Wooster R(2004) The COSMIC (Catalogue of Somatic Mutations in Cancer) database and website. Br J Cancer91: 355-358

[3] Brooks CL, Gu W(2003) Ubiquitination, phosphorylation and acetylation: the molecular basis for p53 regulation. Curr Opin Cell Biol15: 164-171

[4] Brown JM, Attardi LD(2005) The role of apoptosis in cancer development and treatment response. Nat Rev Cancer5: 231-237

[5] Brown JM, Wilson G(2003) Apoptosis genes and resistance to cancer therapy: what does the experimental and clinical data tell us? Cancer Biol Ther2: 477-490

[6] Cappello F, Bellafiore M, Palma A, Bucchieri F(2002) Defective apoptosis and tumorigenesis: role of p53 mutation and Fas/FasL system dysregulation. Eur J Histochem46: 199-208

[7] de la Monte SM, Sohn YK, Wands JR (1997) Correlates of p53- and Fas (CD95)-mediated apoptosis in Alzheimer’s disease. J Neurol Sci152: 73-83

[8] Debatin KM, Krammer PH(2004) Death receptors in chemotherapy and cancer. Oncogene23: 2950-2966

[9] Embree-Ku M, Venturini D, Boekelheide K (2002) Fas is involved in the p53-dependent apoptotic response to ionizing radiation in mouse testis. Biol Reprod66: 1456-1461

[10] Favaloro B, Allocati N, Graziano V, Di Ilio C, De Laurenzi V (2012) Role of apoptosis in disease. Aging4: 330-349

[11] Galmarini CM, Galmarini FC(2003) Multidrug resistance in cancer therapy: role of the microenvironment. Curr Opin Investig Drugs4: 1416-1421

[12] Gaur U, Aggarwal BB(2003) Regulation of proliferation, survival and apoptosis by members of the TNF superfamily. Biochem Pharmacol66: 1403-1408

[13] Guo XX, Guo Q, Li Y, Lee SK, Wei XN, Jin YH(2012) Ginsenoside Rh2 induces human hepatoma cell apoptosis via Bax/Bak triggered cytochrome C release and caspase-9/caspase-8 activation. Int J Mol Sci22: 15523-15535

[14] Ham YM, Chun KH, Choi JS, Kim DH, Lee SK(2003) SEK1-dependent JNK1 activation prolongs cell survival during G-Rh2-induced apoptosis. Biochem Biophys Res Commun304: 358-364

[15] Ham YM, Lim JH, Na HK, Choi JS, Park BD, Yim H, Lee SK(2006) Ginsenoside-Rh2-induced mitochondrial depolarization and apoptosis are associated with reactive oxygen species- and Ca2+-mediated c-Jun NH2-terminal kinase 1 activation in HeLa cells. J Pharmacol Exp Ther319: 1276-1285

[16] Hengartner MO(2000) The biochemistry of apoptosis. Nature407: 770-776

[17] Jin YH, Yoo KJ, Lee YH, Lee SK(2000) Caspase 3-mediated cleavage of p21WAF1/CIP1 associated with the cyclin A-cyclindependent kinase 2 complex is a prerequisite for apoptosis in SKHEP-1 cells. J Biol Chem275: 30256-30263

[18] Kawamata H, Omotehara F, Nakashiro K, Uchida D, Shinagawa Y, Tachibana M, Imai Y, Fujimori T(2007) Oncogenic mutation of the p53 gene derived from head and neck cancer prevents cells from undergoing apoptosis after DNA damage. Int JOncol30: 1089-1097

[19] Kim YS, Jin SH (2004) Ginsenoside Rh2 induces apoptosis via activation of caspase-1 and-3 and up-regulation of Bax in human neuroblastoma. Arch Pharm Res27: 834-839

[20] Kim YS, Jin SH, Lee YH, Park JD, Kim SI(2000) Differential expression of protein kinase C subtypes during Ginsenoside Rh2-induced apoptosis in SK-N-BE(2) and C6Bu-1 cells. Arch Pharm Res23: 518-524

[21] Kim SY, Kim DH, Han SJ, Hyun JW, Kim HS(2007) Repression of matrix metalloproteinase gene expression by Ginsenoside Rh2 in human astroglioma cells. Biochem Pharmacol74: 1642-1651

[22] Li Y, He K, Huang YH, Zheng DX, Gao C, Cui L, Jin YH(2010) Betulin induces mitochondrial cytochrome c release associated apoptosis in human cancer cells. Mol Carcinog49: 630-640

[23] Li B, Zhao J, Wang CZ, Searle J, He TC, Yuan CS, Du W (2011a) Ginsenoside Rh2 induces apoptosis and paraptosis-like cell death in colorectal cancer cells through activation of p53. Cancer Lett301: 185-192

[24] Li Q, Li Y, Wang XY, Fang XX, He K, Guo XX, Zhan Z, Sun C, Jin YH (2011b) Co-treatment with Ginsenoside Rh2 and betulinic acid synergistically induces apoptosis in human cancer cells in association with enhanced capsase-8 activation, Bax translocation, and cytochrome C release. Mol Carcinog50: 760-769

[25] Liu FS (2009) Mechanisms of chemotherapeutic drug resistance in cancer therapy-a quick review. Taiwan JObstet Gynecol48: 239-244

[26] Müller M, Wilder S, Bannasch D, Israeli D, Lehlbach K, Li-Weber M, Friedman SL, Galle PR, Stremmel W, Oren M, Krammer PH (1998) p53 activates the CD95 (APO-1/Fas) gene in response to DNA damage by anticancer drugs. J Exp Med188: 2033-2045

[27] Nagata S(1999) Fas ligand-induced apoptosis. Annu Rev Genet33: 29-55

[28] Nigro JM, Baker SJ, Preisinger AC, Jessup JM, Hostetter R, Cleary K, Bigner SH, Davidson N, Baylin S, Devilee P(1989) Mutations in the p53 gene occur in diverse human tumour types. Nature342: 705-708

[29] Odashima S, Ohta T, Kohno H, Matsuda T, Kitagawa I, Abe H, Arichi S(1985) Control of phenotypic expression of cultured B16 melanoma cells by plant glycosides. Cancer Res45: 2781-2784

[30] Oh JI, Chun KH, Joo SH, Oh YT, Lee SK(2005) Caspase-3-dependent protein kinase C delta activity is required for the progression of Ginsenoside-Rh2-induced apoptosis in SK-HEP-1 cells. Cancer Lett230: 228-238

[31] Owen-Schaub LB, Zhang W, Cusack JC, Angelo LS, Santee SM, Fujiwara T, Roth JA, Deisseroth AB, Zhang WW, Kruzel E (1995) Wild-type human p53 and a temperature-sensitive mutant induce Fas/APO-1 expression. Mol Cell Biol15: 3032-3040

[32] Peter ME, Legembre P, Barnhart BC(2005) Does CD95 have tumor promoting activities? Biochim Biophys Acta1755: 25-36

[33] Schilling T, Schleithoff ES, Kairat A, Melino G, Stremmel W, Oren M, Krammer PH, Muller M(2009) Active transcription of the human FAS/CD95/TNFRSF6 gene involves the p53 family. Biochem Biophys Res Commun387: 399-404

[34] Strasser A, Jost PJ, Nagata S(2009) The many roles of FAS receptor signaling in the immune system. Immunity30: 180-192

[35] Takahashi R(1997) Role of p53 tumor suppressor gene and Fas/ Apo-1 in induction of apoptosis and differentiation of cancer cells. Leukemia11: 3331-3333

[36] van Loo G, Saelens X, van Gurp M, MacFarlane M, Martin SJ, Vandenabeele P (2002) The role of mitochondrial factors in apoptosis: a Russian roulette with more than one bullet. Cell Death Differ9: 1031-1042

[37] Wong RS(2011) Apoptosis in cancer: from pathogenesis to treatment. J Exp Clin Cancer Res30: 87

[38] Xie X, Eberding A, Madera C, Fazli L, Jia W, Goldenberg L, Gleave M, Guns ES(2006) Rh2 synergistically enhances paclitaxel or mitoxantrone in prostate cancer models. J Urol175: 1926-1931

[39] Yi JS, Choo HJ, Cho BR, Kim HM, Kim YN, Ham YM, Ko YG(2009) Ginsenoside Rh2 induces ligand-independent Fas activation via lipid raft disruption. Biochem Biophys Res Commun385: 154-159

[40] Zalcenstein A, Stambolsky P, Weisz L, Muller M, Wallach D, Goncharov TM, Krammer PH, Rotter V, Oren M(2003) Mutant p53 gain of function: repression of CD95(Fas/APO-1) gene expression by tumor-associated p53 mutants. Oncogene22: 5667-5676

[41] Zhou M, Gu L, Yeager AM, Findley HW(1998) Sensitivity to Fasmediated apoptosis in pediatric acute lymphoblastic leukemia is associated with a mutant p53 phenotype and absence of Bcl-2 expression. Leukemia12: 1756-1763

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