Icaritin Induces Apoptosis of HepG2 Cells via the JNK1 Signaling Pathway Independent of the Estrogen Receptor

Abstract

Chinese herbs have become a focus in cancer treatment. Icaritin, a prenylflavonoid derivative from Chinese herbs of the Epimedium genus, has selective estrogen receptor (ER) modulating activity. This study evaluates the effects of icaritin on the apoptosis of HepG2 hepatocellular carcinoma cells. Icaritin (at 5–50 µM) induced apoptosis of HepG2 cells. Few changes in icaritin-induced apoptosis were observed after pretreatment with ICI182780. Consistent with apoptosis induction, icaritin increased the Bax/Bcl-2 ratio and caspase-3 activation in HepG2 cells. Furthermore, icaritin was capable of stimulating the c-Jun N-terminal kinase 1 (JNK1), but not the JNK2, ERK1/2, and p38 subgroups of the mitogen-activated protein kinase (MAPK) family. Coincidently, icaritin-induced cell apoptosis was abolished by SP600125, a specific inhibitor for JNK. Collectively, our results suggest a novel pro-apoptotic activity of icaritin mediated via the JNK1 signaling pathway that is not associated with ER in HepG2 cells.

Key words

icaritin - Epimedium - Berberidaceae - apoptosis - c‐Jun N‐terminal kinase - HepG2 cells

References

• 1 Mendizabal M, Reddy K R. Current management of hepatocellular carcinoma.  Med Clin North Am. 2009;  93 885-900
• 2 Rampone B, Schiavone B, Martino A, Viviano C, Confuorto G. Current management strategy of hepatocellular carcinoma.  World J Gastroenterol. 2009;  15 3210-3216
• 3 Tamvakopoulos C, Dimas K, Sofianos Z D, Hatziantoniou S, Han Z, Liu Z L, Wyche J H, Pantazis P. Metabolism and anticancer activity of the curcumin analogue, dimethoxycurcumin.  Clin Cancer Res. 2007;  13 1269-1277
• 4 Kunnumakkara A B, Guha S, Krishnan S, Diagaradjane P, Gelovani J, Aggarwal B B. Curcumin potentiates antitumor activity of gemcitabine in an orthotopic model of pancreatic cancer through suppression of proliferation, angiogenesis, and inhibition of nuclear factor-kappaB-regulated gene products.  Cancer Res. 2007;  67 3853-3861
• 5 Hong J H, Ahn K S, Bae E, Jeon S S, Choi H Y. The effects of curcumin on the invasiveness of prostate cancer in vitro and in vivo.  Prostate Cancer Prostatic Dis. 2006;  9 147-152
• 6 Wang L, Tang Z Y, Qin L X, Wu X F, Sun H C, Xue Q, Ye S L. High-dose and long-term therapy with interferon-alfa inhibits tumor growth and recurrence in nude mice bearing human hepatocellular carcinoma xenografts with high metastatic potential.  Hepatology. 2000;  32 43-48
• 7 Cheng Y L, Chang W L, Lee S C, Liu Y G, Chen C J, Lin S Z, Tsai N M, Yu D S, Yen C Y, Harn H J. Acetone extract of Angelica sinensis inhibits proliferation of human cancer cells via inducing cell cycle arrest and apoptosis.  Life Sci. 2004;  75 1579-1594
• 8 Yano H, Mizoguchi A, Fukuda K, Haramaki M, Ogasawara S, Momosaki S, Kojiro M. The herbal medicine sho-saiko-to inhibits proliferation of cancer cell lines by inducing apoptosis and arrest at the G0/G1phase.  Cancer Res. 1994;  54 448-454
• 9 Kao S T, Yeh C C, Hsieh C C, Yang M D, Lee M R, Liu H S, Lin J G. The Chinese medicine Bu-Zhong-Yi-Qi-Tang inhibited proliferation of hepatoma cell lines by inducing apoptosis via G0/G1 arrest.  Life Sci. 2001;  69 1485-1496
• 10 Lee S M, Li M L, Tse Y C, Leung S C, Lee M M, Tsui S K, Fung K P, Lee C Y, Waye M M. Paeoniae Radix, a Chinese herbal extract, inhibits hepatoma cell growth by inducing apoptosis in a p 53 independent pathway.  Life Sci. 2002;  71 2267-2277
• 11 Wang Z, Zhang X, Wang H, Qi L, Lou Y. Neuroprotective effects of icaritin against beta amyloid-induced neurotoxicity in primary cultured rat neuronal cells via estrogen-dependent pathway.  Neuroscience. 2007;  145 911-922
• 12 Wang Z, Wang H, Wu J, Zhu D, Zhang X, Ou L, Yu Y, Lou Y. Enhanced co-expression of beta-tubulin III and choline acetyltransferase in neurons from mouse embryonic stem cells promoted by icaritin in an estrogen receptor-independent manner.  Chem Biol Interact. 2009;  179 375-385
• 13 Wo Y B, Zhu D Y, Hu Y, Wang Z Q, Liu J, Lou Y J. Reactive oxygen species involved in prenylflavonoids, icariin and icaritin, initiating cardiac differentiation of mouse embryonic stem cells.  J Cell Biochem. 2008;  103 1536-1550
• 14 Zhu D Y, Lou Y J. Inducible effects of icariin, icaritin, and desmethylicaritin on directional differentiation of embryonic stem cells into cardiomyocytes in vitro.  Acta Pharmacol Sin. 2005;  26 477-485
• 15 Zhang G, Qin L, Sheng H, Wang X L, Wang Y X, Yeung D K, Griffith J F, Yao X S, Xie X H, Li Z R, Lee K M, Leung K S. A novel semisynthesized small molecule icaritin reduces incidence of steroid-associated osteonecrosis with inhibition of both thrombosis and lipid-deposition in a dose-dependent manner.  Bone. 2009;  44 345-356
• 16 Huang J, Yuan L, Wang X, Zhang T L, Wang K. Icaritin and its glycosides enhance osteoblastic, but suppress osteoclastic, differentiation and activity in vitro.  Life Sci. 2007;  81 832-840
• 17 Huang X, Zhu D, Lou Y. A novel anticancer agent, icaritin, induced cell growth inhibition, G1 arrest and mitochondrial transmembrane potential drop in human prostate carcinoma PC-3 cells.  Eur J Pharmacol. 2007;  564 26-36
• 18 Liu J, Ye H, Lou Y. Determination of rat urinary metabolites of icariin in vivo and estrogenic activities of its metabolites on MCF-7 cells.  Pharmazie. 2005;  60 120-125
• 19 Tang S Y, Xie H, Yuan L Q, Luo X H, Huang J, Cui R R, Zhou H D, Wu X P, Liao E Y. Apelin stimulates proliferation and suppresses apoptosis of mouse osteoblastic cell line MC3T3-E1 via JNK and PI3-K/Akt signaling pathways.  Peptides. 2007;  28 708-718
• 20 Wang Q P, Xie H, Yuan L Q, Luo X H, Li H, Wang D, Meng P, Liao E Y. Effect of progesterone on apoptosis of murine MC3T3-E1 osteoblastic cells.  Amino Acids. 2009;  36 57-63
• 21 Xie H, Tang S Y, Li H, Luo X H, Yuan L Q, Wang D, Liao E Y. L-carnitine protects against apoptosis of murine MC3T3-E1 osteoblastic cells.  Amino Acids. 2008;  35 419-423
• 22 Xie H, Yuan L Q, Luo X H, Huang J, Cui R R, Guo L J, Zhou H D, Wu X P, Liao E Y. Apelin suppresses apoptosis of human osteoblasts.  Apoptosis. 2007;  12 247-254
• 23 Xie H, Tang L L, Luo X H, Wu X Y, Wu X P, Zhou H D, Yuan L Q, Liao E Y. Suppressive effect of dexamethasone on TIMP-1 production involves murine osteoblastic MC3T3-E1 cell apoptosis.  Amino Acids. 2010;  38 1145-1153
• 24 Kuo Y C, Kuo P L, Hsu Y L, Cho C Y, Lin C C. Ellipticine induces apoptosis through p 53-dependent pathway in human hepatocellular carcinoma HepG2 cells.  Life Sci. 2006;  78 2550-2557
• 25 Xie H, Tang S Y, Luo X H, Huang J, Cui R R, Yuan L Q, Zhou H D, Wu X P, Liao E Y. Insulin-like effects of visfatin on human osteoblasts.  Calcif Tissue Int. 2007;  80 201-210
• 26 Xie H, Tang S Y, Cui R R, Huang J, Ren X H, Yuan L Q, Lu Y, Yang M, Zhou H D, Wu X P, Luo X H, Liao E Y. Apelin and its receptor are expressed in human osteoblasts.  Regul Pept. 2006;  134 118-125
• 27 Xie H, Yang B, Zhou X M, Song F L, Li J M, Zhou K, Hu W, Peng Y Q, Tang S Y, Yuan L Q, Xiong S Y, Liao X B. L-carnitine and taurine synergistically inhibit the proliferation and osteoblastic differentiation of vascular smooth muscle cells.  Acta Pharmacol Sin. 2010;  31 289-296
• 28 Liao X B, Peng Y Q, Zhou X M, Yang B, Zheng Z, Liu L M, Song F L, Li J M, Zhou K, Meng J C, Yuan L Q, Xie H. Taurine restores Axl/Gas6 expression in vascular smooth muscle cell calcification model.  Amino Acids. in press; 
• 29 Li H, Xie H, Liu W, Hu R, Huang B, Tan Y F, Xu K, Sheng Z F, Zhou H D, Wu X P, Luo X H. A novel microRNA targeting HDAC5 regulates osteoblast differentiation in mice and contributes to primary osteoporosis in humans.  J Clin Invest. 2009;  119 3666-3677
• 30 Kim B R, Hu R, Keum Y S, Hebbar V, Shen G, Nair S S, Kong A N. Effects of glutathione on antioxidant response element-mediated gene expression and apoptosis elicited by sulforaphane.  Cancer Res. 2003;  63 7520-7525
• 31 Ip Y T, Davis R J. Signal transduction by the c-Jun N-terminal kinase (JNK)–from inflammation to development.  Curr Opin Cell Biol. 1998;  10 205-219
• 32 Kumagae Y, Zhang Y, Kim O J, Miller C A. Human c-Jun N-terminal kinase expression and activation in the nervous system.  Brain Res Mol Brain Res. 1999;  67 10-17
• 33 Ricchi M, Bertolotti M, Anzivino C, Carulli L, Canedi I, Bormioli M L, Tiozzo R, Croce M A, Lonardo A, Carulli N, Loria P. 17 Beta-estradiol prevents cytotoxicity from hydrophobic bile acids in HepG2 and WRL-68 cell cultures.  J Gastroenterol Hepatol. 2006;  21 894-901
• 34 Adams J M, Cory S. The Bcl-2 protein family: arbiters of cell survival.  Science. 1998;  281 1322-1326
• 35 Li X, Darzynkiewicz Z. Cleavage of poly (ADP-ribose) polymerase measured in situ in individual cells: relationship to DNA fragmentation and cell cycle position during apoptosis.  Exp Cell Res. 1998;  25 125-132
• 36 Orth K, Chinnaiyan A M, Garth M, Froelich C J, Dixit V M. The CED-3/ICE-like protease Mch2 is activated during apoptosis and cleaves the death substrate lamin A.  J Biol Chem. 1996;  271 16443-16446
• 37 Gustafsson J A. Estrogen receptor beta – a new dimension in estrogen mechanism of action.  J Endocrinol. 1999;  163 379-383
• 38 Solakidi S, Psarra A M, Sekeris C E. Differential subcellular distribution of estrogen receptor isoforms: localization of ERalpha in the nucleoli and ERbeta in the mitochondria of human osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cell lines.  Biochim Biophys Acta. 2005;  1745 382-392
• 39 Wakeling A E, Dukes M, Bowler J. A potent specific pure antiestrogen with clinical potential.  Cancer Res. 1991;  51 3867-3873
• 40 Blüthgen N, Legewie S. Systems analysis of MAPK signal transduction.  Essays Biochem. 2008;  45 95-107
• 41 Chang L, Karin M. Mammalian MAP kinase signalling cascades.  Nature. 2001;  410 37-40
• 42 Ma F Y, Flanc R S, Tesch G H, Han Y, Atkins R C, Bennett B L, Friedman G C, Fan J H, Nikolic-Paterson D J. A pathogenic role for c-Jun amino-terminal kinase signaling in renal fibrosis and tubular cell apoptosis.  J Am Soc Nephrol. 2007;  18 472-484
• 43 Liu J, Minemoto Y, Lin A. c-Jun N-terminal protein kinase 1 (JNK1), but not JNK2, is essential for tumor necrosis factor alpha-induced c-Jun kinase activation and apoptosis.  Mol Cell Biol. 2004;  24 10844-10856
• 44 Wong S P, Shen P, Lee L, Li J, Yong E L. Pharmacokinetics of prenylflavonoids and correlations with the dynamics of estrogen action in sera following ingestion of a standardized Epimedium extract.  J Pharm Biomed Anal. 2009;  50 216-223