Triptolide downregulates Rac1 and the JAK/STAT3 pathway and inhibits colitis-related colon cancer progression

EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 41, No. 10, 717-727, October 2009 Triptolide downregulates Rac1 and the JAK/STAT3 pathway and inhibits colitis-related colon cancer progression 1 2 3 Zhipeng Wang *, Haifeng Jin *, Ruodan Xu , 1,4 2,4 Qibing Mei and Daiming Fan 1 Department of Pharmacology School of Pharmacy 2 State Key Laboratory of Cancer Biology and Institute of Digestive Diseases Xijing Hospital The Fourth Military Medical University Shaanxi Province 710032, China 3 Protein Laboratory Department of Neuroscience and Pharmacology University of Copenhagen Copenhagen 2200, Denmark 4 Corresponding authors: (Q. Mei), (D. Fan) *These authors contributed equally to this work. DOI 10.3858/emm.2009.41.10.078 Accepted 28 May 2009 Abbreviations: COX, cyclooxygenase; CRC, colorectal cancer; IBD, inflammatory bowel disease; JAK, Janus kinase; STAT, signal transducers and activators of transcription Abstract Triptolide, a diterpenoid triepoxide from the traditional Chinese medicinal herb Tripterygium wilfordii Hook. f., is a potential treatment for autoimmune diseases as well a possible anti-tumor agent. It inhibits proliferation of coloretal cancer cells in vitro and in vivo. In this study, its ability to block progress of colitis to colon cancer, and its molecular mechanism of action are investigated. A mouse model for colitis-induced colorectal cancer was used to test the effect of triptolide on cancer progression. Treatment of mice with triptolide decreased the incidence of colon cancer formation, and increased survival rate. Moreover, triptolide decreased the incidence of tumors in nude mice inoculated with cultured colon cancer cells dose-dependently. In vitro, triptolide inhibited the proliferation, migration and colony formation of colon cancer cells. Secretion of IL6 and levels of JAK1, IL6R and phosphorylated STAT3 were all reduced by triptolide treatment. Triptolide prohibited Rac1 activity and blocked cyclin D1 and CDK4 expression, leading to G1 arrest. Triptolide interrupted the IL6R-JAK/STAT pathway that is crucial for cell proliferation, survival, and inflammation. This suggests that triptolide might be a candidate for prevention of colitis induced colon cancer because it reduces inflammation and prevents tumor formation and development. Keywords: colonic neoplasms; interleukin-6; rac1 GTP-binding protein; STAT3 transcription factor; triptolide Introduction Traditional medicines represent an abundant source of potential pharmaceuticals. One of the most promising compounds from a traditional medical herb is triptolide, a diterpenoid triepoxide purified from the Thundergod vine, Tripterygium wilfordii Hook. f. Triptolide has anti-inflammatory effects, and has been the subject of clinical trials as a rheumatoid arthritis treatment (Corson and Crews, 2007; Qiu and Kao, 2003; Schmidt et al., 2007). Triptolide has a complex mechanism of action, suppressing production of prostaglandin E2 via down-regulation of cyclooxygenase (COX)-2, and reducing levels of cytokines, including, among others, IL-1β, TNF and IL6 (Qiu and Kao, 2003; Lin et al., 2007). A known effect of triptolide is inhibition of the transcription factor NF-κB activation (Qiu and Kao, 2003; Wei et al., 2008), which can have manifold effects on inflammation and proliferation. In addition to its anti-inflammatory action, several reports have indicated that triptolide can inhibit the proliferation of cancer cells in vitro and reduce the growth and metastases of tumors in vivo. Results with triptolide in vivo include inhibition of the growth of cholangiocarcinoma cells in hamsters (Tengchaisri et al., 1998), as well as of xenografts from human melanoma, breast cancer, bladder cancer, and gastric and coloral carcinoma lines in nude mice (Yang et al., 2003). Triptolide reduced the growth and spread of pancreatic tumors in mice (Phillips et al., 2007), and showed weak activity against solid tumors in a mouse breast cancer model (Shamon et al., 1997). It was also efficacious as a adjunct agent in combination chemotherapy against tumors developed from human ovarian cancer cells inoculated into nude mice (Westfall et al., 2008). In addition, clinical trials in China using 718 Exp. Mol. Med. Vol. 41(10), 717-727, 2009 triptolide have shown remission rates of 71% and 87% in mononucleocytic and granulocytic leukemia, respectively (Lu et al, 1992). Although the precise mechanism of its anti-cancer effects is unknown, triptolide has pro-apoptotic effects (Carter et al., 2006; Liang and Fu, 2008; Yao et al., 2008). triptolide ameliorates chronic colitis mediated by the acquired immune response in a mouse model (Wei et al., 2008), but no previous reports has examined the effect of triptolide on colon cancer induced by chronic colitis. Inflammatory bowel disease (IBD), such as ulcerative colitis and Crohn's disease are associated with an increased risk for developing colorectal cancer and precancerous dysplastic epithelial changes (Krok and Lichtenstein, 2004). In the most recent model for progression from IBD to colorectal cancer (CRC), the pro-inflammatory cytokine IL6 has a key role as a mechanistic link between inflammation and colonic cancer (Lin and Karin, 2007, Atreya and Neurath, 2005b). Levels of both IL6 and a soluble form of the IL6 receptor (sIL6R), which is generated by limited proteolysis of the membrane-bound IL6 receptor, are elevated in IBD and CRC (Belluco et al., 2000; Mudter and Neurath, 2007). The signal generated by IL6 is transduced through Janus kinase (JAK) activation of Signal Transducers and Activators of Transcription (STAT), in particular STAT3. Aberrant regulation of STAT3regulated genes is associated with oncogenic transformation. Thus, all components of this pathway may play crucial roles in the pathogenic process that links intestinal inflammation and colonic cancer. In fact, high levels of IL6 and the soluble IL6 receptor (sIL6R) have been reported in several chronic inflammatory and autoimmune diseases as well as in cancer (Mudter and Neurath, 2007). In addition, IL6 is one part of an autocrine inflammatory loop involving Rac1, a small GTPase in the Rho family (Faruqi et al., 2001). Specifically blocking IL6-regulated signalling pathways represents a promising approach for the therapy of IBD and colorectal cancer (Mudter and Neurath, 2007). In fact, antibodies against the sIL6 receptor effectively reduced inflammation in IBD and activated apoptosis (Atreya et al., 2001). With its anti-inflammatory and anti-cancer properties, triptolide is a natural candidate for treatment of IBD leading to colorectal cancer. We used a colitis-induced colon cancer mouse model to demonstrate that triptolide has an in vivo protective effect against IBD-induced cancer. Mechanistically, experiments with cultured colon cancer cell lines demonstrated that triptolide inhibits components of the IL6 and JAK/STAT3 signal transduction pathway, as well as through the small GTPase Rac1. Results Tr (...truncated)