From Taiwanofungus Camphoratus
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Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2011, Article ID 750230, 13 pages doi:10.1155/2011/750230 Research Article Apoptotic Cell Death and Inhibition of Wnt/β-Catenin Signaling Pathway in Human Colon Cancer Cells by an Active Fraction (HS7) from Taiwanofungus camphoratus Chi-Tai Yeh,1, 2, 3 Chih-Jung Yao,2, 4 Jiann-Long Yan,5 Shuang-En Chuang,5 Liang-Ming Lee,4 Chien-Ming Chen,1 Chuan-Feng Yeh,5 Chi-Han Li,5 and Gi-Ming Lai1, 2, 4, 5 1 Cancer Center, Shuang Ho Hospital, Taipei Medical University, Taipei 235, Taiwan 2 Center of Excellence for Cancer Research, Taipei Medical University, Taipei 110, Taiwan 3 Graduate Institute of Clinical Medicine, Taipei Medical University, Taipei 110, Taiwan 4 Cancer Center, Wan Fang Hospital, Taipei Medical University, Taipei 116, Taiwan 5 National Institute of Cancer Research, National Health Research Institutes, Miaoli 350, Taiwan Correspondence should be addressed to Gi-Ming Lai, [email protected] Received 27 September 2010; Revised 6 January 2011; Accepted 11 January 2011 Copyright © 2011 Chi-Tai Yeh et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Aberrant activation of Wnt/β-catenin signaling plays an important role in the development of colon cancer. HS7 is an active fraction extracted from Taiwanofungus camphoratus, which had been widely used as complementary medicine for Taiwan cancer patients in the past decades. In this study, we demonstrated the effects of HS7 on the growth inhibition, apoptosis induction, and Wnt/β-catenin signaling suppression in human colon cancer cells. HS7 significantly inhibited proliferation of HT29, HCT116, and SW480 colon cancer cells in a dose- and time-dependent manner. The apoptosis induction was evidenced by DNA fragmentation and subG1 accumulation, which was associated with increased Bax/Bcl-2 ratio, activation of caspase-3 and cleavage of PARP. By using Tcf-dependent luciferase activity assay, HS7 was found to inhibit the β-catenin/Tcf transcriptional activities. In addition, HS7 strongly suppressed the binding of Tcf complexes to its DNA-binding site shown in electrophoretic mobility shift assay. This inhibition was further confirmed by the decreased protein levels of Tcf-4 and β-catenin. The β-catenin/Tcf downstream target genes, such as survivin, c-myc, cyclin D1, MMP7,andMT1-MMP involved in apoptosis, invasion, and angiogenesis were also diminished as well. These results indicate that Taiwanofungus camphoratus may provide a benefit as integrative medicine for the treatment of colon cancer. 1. Introduction cancer therapy. However, most of the advanced patients encountered inevitable relapse. Searching for new therapeu- As the diet and life styles of the industrialized Western tics targeting on other molecular pathway to improve the countries are adopted globally, the incidence of colorectal clinical outcome is urgently needed. cancer (CRC) is increasing worldwide [1]. It becomes The Wnt/β-catenin signaling pathway plays an impor- a second most common cancer and the third leading tant role in both embryogenesis and tumorigenesis. Most cause of cancer death in Taiwan. Fifty percent of patients sporadic colorectal cancer (CRC) and familial adenomatous diagnosed with CRC will eventually die from the disease, polyposis (FAP) have mutated adenomatous polyposis coli and less than 10% of patients with metastatic CRC survived (APC) gene. Normal APC protein could downregulate the more than 5 years [2]. Lots of studies have been carried Wnt signaling pathway through its binding to β-catenin and on to search for effective therapeutics targeting on the Axin, but most mutated APC proteins in colorectal tumors molecular pathogenesis of CRC. Nowadays, several effective fail to inhibit Wnt signaling, leading to the overproliferation chemotherapeutics and molecular targeting agents, such of tumor cells [3]. Recent researches have implicated that as bevacizumab and cetuximab, which target VEGFR and the aberrant Wnt/β-catenin signaling pathway plays an EGFR, respectively, had been introduced into current colon important role in several common cancers including liver, 2 Evidence-Based Complementary and Alternative Medicine lung, and colorectal cancers [4]. The current signaling model reagent, fetal bovine serum (FBS), and trypsin-EDTA (T/E) of Wnt/β-catenin indicates that the binding of Wnt protein were obtained from Gibco BRL Life Technologies (Grand to its transmembrane-frizzled receptor could stabilize β- Island, NY). The QIAGEN One-tube RT-PCR system and catenin by inhibiting the activity of the serine/threonine Annexin-V/FITC Apoptosis Kits were obtained from Qiagen kinase GSK-3β [5]. The GSK-3β kinase associated with β- Inc. (Valencia, CA) and R&D Systems, Inc. (Minneapolis, catenin in a multiprotein complex, which is composed of MN), respectively. Lipofectamine was purchased from Invit- the adenomatous polyposis coli tumor suppressor protein rogen (Rockville, MD). An enhanced chemiluminescence APC, axin, conductin, and protein phosphatase 2A. GSK- (ECL) kit was purchased from Amersham Corp. (Arlington 3β phosphorylates the NH2 terminus of β-catenin and Heights, IL). Anti-Tcf-4 and anti-active-β-catenin anti- thereby induces its degradation via the ubiquitin-proteasome bodies were purchased from Chemicon International, Inc. pathway [6]. In response to Wnt signaling, GSK-3β is (Temecula, CA). Anti-Bcl2, anti-Bax, anti-β-catenin, and inhibited by disheveled proteins, and, therefore, β-catenin anti-survivin antibodies were purchased from Santa Cruz could escape from phosphorylation. The unphosphorylated Biotechnology, Inc. (Santa Cruz, CA), and anticleaved β-catenin accumulates in the cytoplasm and translocates into caspase-3 and anti-PARP antibodies were purchased from the nucleus. In the nucleus, β-cateninplaysasatranscrip- Cell Signaling Technology, Inc. (Beverly, MA). The plas- tional activator in association with T-cell factor/lymphocyte mids TOPflash and FOPflash were purchased from Upstate enhancer factor (Tcf/Lef) family and other transcriptional Biotechnology, Inc. (Lake Placid, NY). NE-PER nuclear cofactors [7], by which a variety of target genes including extraction reagent was purchased from Thermo Fisher c-myc, cyclin D1,andsurvivin were subsequently activated Scientific Inc. (Rockford, IL). The electrophoretic-mobility [8, 9]. Dysregulation of β-catenin signaling is believed to shift assay (EMSA) kits were purchased from Panomics, Inc. be critical to the early stages of human sporadic colorectal (Fremont, CA). carcinogenesis [10]. The Wnt/β-catenin signaling pathway had been proposed as a target for drug discovery [11]. Several 2.1. Cell Lines and Cultures. The HT-29, HCT116, and substances had been studied for the ability to inhibit this SW480 human colorectal cancer cell lines were obtained β aberrantly activated Wnt/ -catenin signaling in colorectal from American Type Culture Collection (ATCC). All of cancer [12, 13]. To find clinically feasible natural substances the cell lines were maintained in RPMI-1640 culture with this activity but less toxicity is warranted. medium supplemented with 10% fetal bovine serum and 1% T. camphoratus (syn. Antrodia camphorata)isaGano- Penicillin-Streptomycin-Glutamine (100x) liquid (Gibco). derma-like fungus, which belongs to family of Polyporaceae, Cells were cultured in an incubator with humidified air at Basidiomycota, and grows in a unique host, the endemic ◦ 37 Cwith5%CO2. The cells were regularly seeded into T- perennial tree Cinnamomun kanehirae (Bull camphor tree) 75 flasks with media changes every 3 days. For experiments, in Taiwan. In the past fifteen years, T. camphoratus has been cells were grown to 70% confluence, trypsinized with 0.25% shown to exert antioxidant [14], anti-inflammatory [15], and trypsin-EDTA, and then replated at a density of 105 per ml immunomodulatory activities [16] in a variety of experi- in cultured media. As an untreated solvent control, cells were ments. On the other hand, many studies have demonstrated treated with DMSO at a final concentration of <0.05%. that T. camphoratus exerted multiple biological activities against various cancer cells in the aspects of antiproliferation, apoptosis induction, anti-invasion, and antimetastasis [17– 2.2. Preparation of Active Fraction HS7 from T. camphoratus. 19]. Recently, we have isolated the most potent fraction The fruiting body-like T. camphoratus (voucher number TC- HS7 from the n-hexane extract of T. camphoratus by silica 2004-09-001) was cultivated and provided by Well Shine gel chromatography, which appears to possess significant Biotechnology Development Co. (Taipei, Taiwan). Briefly, antiproliferative activities against a panel of human cancer air-dried ground powder of the cultivated T. camphoratus cell lines, including lung adenocarcinoma cells (CL1-0), was extracted exhaustively with n-hexane and then separated prostate cancer cells (PC3), and hepatocellular carcinoma by silica gel chromatography. Eight fractions (HS1 to HS8) cells (Hep3B & Huh7). However, the anticancer effects of were obtained, and the seventh (HS7) appeared to be the HS7 on colon cancer cells and the underlying molecular most active fraction on growth inhibition against a screening mechanisms remain unclear. panel of cancer cell lines. The aim of this study was to address whether the active fraction HS7 isolated from T. camphoratus could inhibit