68 วารสารวิจัย มข. (บศ.) 15 (3) : ก.ค. - ก.ย. 2558 Anticancer Effects of Cepharanthine on Human Colon Cancer Cells ฤทธิ์ต้านมะเร็งของเซฟราเรนทีนต่อเซลล์มะเร็งล�าไส้ใหญ่ของมนุษย์ Arkornnut Rattanawong (อากรณัท รัตนวงษ์)* Dr.Wacharee Limpanasithikul (ดร.วัชรี ลิมปนสิทธิกุล)** Dr.Piyanuch Wonganan (ดร.ปิยนุช วงศ์อนันต์)1*** ABSTRACT Cyclooxygenase (COX-2) has been found to be overexpressed in various tumors including colorectal cancer. Cepharanthine (CEP), a biscoclaurine alkaloid isolated from Stephania cepharantha Hayata, exhibits anticancer activity against several different types of cancer including oropharynx cancer, leukemia, hepatocarcinoma and cholangiocarcinoma. In this study, we investigated the anticancer effects of diclofenac, celecoxib, berberine, CEP and 5-fluorouracil against a COX-2 positive human colon cancer cell line, HT-29. Of all compounds tested, CEP was the most effective agents for controlling the growth of the cancer cells. CEP could significantly induce colon cancer cell apoptosis and effectively inhibit COX-2 mRNA expression. These findings demonstrated that CEP could potentially be used as a novel anticancer agent for COX-2-positive colon cancer cells. บทคัดย่อ มะเร็งหลายชนิดรวมทั้งมะเร็งล�าไส้ใหญ่มีการแสดงออกของ COX-2 ที่มากผิดปกติ จากการศึกษา ก่อนหน้านี้พบว่า cepharanthine (CEP) ซึ่งเป็นสารในกลุ่ม biscoclaurine alkaloid ที่พบได้ในราก ของต้น Stephania cepharantha Hayata มีฤทธิ์ต้านมะเร็งหลายชนิดเช่น มะเร็งช่องปากและล�าคอ มะเร็งเม็ดเลือดขาว มะเร็งตับ และมะเร็งท่อน�้าดี วัตถุประสงค์ของการทดลองนี้เพื่อศึกษาฤทธิ์ต้านมะเร็ง ของ diclofenac, celecoxib, berberine, CEP และ 5-fluorouracil ต่อเซลล์ HT-29 ซึ่งเป็นเซลล์มะเร็ง ล�าไส้ใหญ่ของมนุษย์ที่มีการแสดงออกของ COX-2 จากผลการทดลองแสดงให้เห็นว่า CEP สามารถยับยั้ง การเจริญของเซลล์มะเร็งได้ดีกว่า diclofenac, celecoxib, berberine, และ 5-fluolouracil โดย CEP สามารถชักน�าให้เซลล์มะเร็งตายแบบอะพอพโทซิสและสามารถยับยั้งการแสดงออกของยีน COX-2 การ ทดลองนี้แสดงให้เห็นว่า CEP อาจจะเป็นยาที่มีประสิทธิภาพในการรักษาโรคมะเร็งล�าไส้ใหญ่ที่มีการแสดงออก ของ COX -2 Keywords: Cepharanthine (CEP), Apoptosis, Colon cancer ค�าส�าคัญ: เซฟราเรนทีน อะพอพโทซิส มะเร็งล�าไส้ใหญ่ 1Correspondent author: [email protected] * Student, Master of Pharmacy Program in Pharmacology, Graduate School, Chulalongkorn University ** Assistant Professor, Department of Pharmacology, Faculty of Medicine, Chulalongkorn University *** Lecturer, Department of Pharmacology, Faculty of Medicine, Chulalongkorn University KKU Res J (GS) 15 (3) : July - September 2015 69 Introduction particularly, PGE2 which promotes colorectal Colorectal cancer is one of the most tumor development by stimulating common cancers in both man and woman angiogenesis, cell proliferation and apoptosis [1]. The majority of colorectal carcinomas evasion. Previous study showed that HT-29 are adenocarcinomas, which originate from cells overexpressing COX-2 were resistant the epithelial cells of the colorectal mucosa. to apoptotic cell death [5]. Epidemiological The most common treatment for colorectal studies have been suggested that long-term cancer is surgical resection, followed by treatment with aspirin, nonsteroidal adjuvant therapy with 5-fluorouracil, anti-inflammatory drugs (NSAIDs), or oxaliplatin, leucovorin or radiation. Although selective cyclooxygenase 2 (COX-2) inhibitors chemotherapy has been widely used, its use may reduce the risk of colorectal cancers [6], often limited due to drug resistance and suggesting that COX-2 is an important target serious side effects. Therefore, a novel in the treatment of colorectal cancer. compound that has potent anticancer activity Cepharanthine (CEP), a natural and minimal side effects is urgently needed. compound isolated from Stephania Evidence has shown that patients with cepharantha Hayata possess many pharma- inflammatory bowel diseases including cological effects such as anti-inflammation, ulcerative colitis (UC) and Crohn’s disease anti-retrovirus, anti-oxidant and anti- (CD) have a significant higher risk of cancer [7]. CEP could inhibit production of developing colorectal cancer [2]. pro-inflmmatory cytokines (tumor necrosis Cyclooxygenase (COX) enzyme factor (TNF)-α, interleukin (IL)-1β, has played a key role in the biosynthesis of IL-6) and nitric oxide via suppressing prostaglandins and thromboxane from NF-κB [8]. Many studies also showed that arachidonic acid. There are two major COX CEP has anticancer activity against several isoforms, including COX-1 and COX-2. types of cancer such as oropharynx cancer, COX-1 is constitutively expressed in many leukemia, hepatocarcinoma and cholan- tissues and plays an important role in tissue giocarcinoma [9-11]. It has been reported homeostasis, while COX-2 is induced by that CEP inhibit tumor growth through inflammatory stimuli and involved in multiple mechanisms, including increasing pathological processes [3]. COX-2 overex- host immune response [12], inducing cancer pression has been detected in 84.9% of colon cell to undergo apoptosis [13-14] and carcinoma and 57.9% of adenomas, suggesting stimulating cell cycle arrest [15]. Although a critical role of COX-2 in colorectal cancer there were reports regarding to cytotoxicity of development [4]. Several studies have found CEP against several tumors, the anticancer that COX-2 overexpression could increase activity of this compound has not been accumulation of prostaglandins (PGs) evaluated in COX-2- positive colon cancer cells. 70 วารสารวิจัย มข. (บศ.) 15 (3) : ก.ค. - ก.ย. 2558 Objective of the study nm using a microplate reader (Thermo). The aim of this study were to The percent of cell viability was calculated determine anticancer effect of diclofenac, using the following equation: (Abs. sample/ celecoxib, berberine, cepharanthine, and Abs. control) x100. The values of half 5-fluorouracil against COX-2-positive inhibitory concentration (IC50) were HT-29 human colon cancer cells and calculated using the fitted line by GraphPad investigate the mechanism(s) underlying prism software. the anticancer effect of cepharanthine. Quantitative real time PCR HT-29 cells were seeded in a 6-well plate at a density of 5 x105 cell/well and Methodology incubated overnight. The culture medium Cell culture was then replaced with fresh complete Human colorectal cancer cell line medium containing 2.5, 5, 10 or 20 µM CEP HT-29 was obtained from American Type and incubated for 6 or 24 additional hours. Culture Collection (ATCC) (Rockville, MD). At the end of treatment, the total RNA was The cells were maintained in complete extracted using TRIzol (Invitrogen, USA) Dulbecco’s modified Eagle’s medium (DMEM) and mRNA was reversed transcribed using medium supplemented with 10% fetal bovine Improm-IITM Reverse Transcription system serum (FBS), 100 U/mL penicillin and 100 (Promega, USA) according to the µg/mL streptomycin at 37°C in 5% CO 2 manufactuers’ instructions. Real-time PCR incubator. was carried out using SYBR Green qPCR Cytotoxicity assay super mix universal (Invitrogen, USA) with Cell viability was evaluated by the the following primers specific for COX-2.: ability of mitochondrial reductase enzyme in 5’-CCCTG AGCATC TACGGTTTG-3’ (for- living cells to reduce resazurin into resorufin ward), 5’-TCGCATACTCTGTTGTGTTCC-3’ [16]. Cells were seeded in a 96-well plate at a (reverse) and for GAPDH: 5’-AAGG TCG- density 5x103 cell/well and incubate overnight GAGTCAACGGATTTGGT-3’ (forward) and at 37°C, 5% CO . Cells were then treated 2 5’-ATGGCATGGACTGTGGTCATGAGT-3’ with berberine, celecoxib, cepharanthine, (reverse). GAPDH was used as an internal diclofenac, 5-fluorouracil or 0.2% DMSO control. DNA amplifications were carried out (vehicle control) in complete DMEM medium using a StepOnePlus™ Real-Time PCR with at a concentration of 0.01, 0.1, 1, 10, 50 or 100 the following cycling conditions: 50 �C for 2 µM for 48 hours. Then, 15 µL of resazurin min, 95 �C for 2 min, and 40 cycles of 95 �C solution (0.05 mg/ml) was added to each for 30s, 60 �C for 30s, and 72 �C for 30s. The well and incubated at 37 �C for another 5 fold change in COX-2 gene expression after hours. The colorimetric was quantified by CEP treatment normalized to GAPDH and measuring the absorbance at 570 and 600 KKU Res J (GS) 15 (3) : July - September 2015 71 relative to the expression in vehicle treatment Results -ΔΔCT was calculated using the 2 method. Effect of cepharanthine on the Apoptosis assay COX-2 mRNA expression in HT-29 cells HT-29 cells were seeded in a 6-well COX-2 has been found to be 5 plate at density of 3x10 cell/well and overexpressed in various tumors including incubated overnight. The cells were then colorectal cancer [17]. Several studies have treated with CEP at different concentrations shown that cepharanthine (CEP) possess (2.5, 5, 10 or 20 µM) in culture medium for anti-inflammatory activity by inhibiting 24 hours. At the end of treatment, the cells production of pro-inflammatory cytokines were washed with PBS and harvested by (tumor necrosis factor (TNF)-α, interleukin trypsinization and centrifuged at 1500 rpm (IL)-1β, IL-6) and nitric oxide [18, 19]. for 5 min. The cell pellets were washed twice Moreover, it was recently reported that with cold PBS and re-suspended with 100 µl inhibition of COX-2 expression is involved of assay buffer. The cells were then stained in radio sensitization of CEP in cervical with 1 µl Annexin V FIT-C (Invitrogen, adenocarcinoma cell line [20]. Therefore, the USA) and 1 µl of 0.05 µg/ml PI (Santa Cruz effect of CEP on COX-2 mRNA expression Biotechnology, USA) for 15 min at room in HT-29 cells