Anticancer Effects of Cepharanthine on Human Colon Cancer Cells ฤทธิ์ต้านมะเร็งของเซฟราเรนทีนต่อเซลล์มะเร็งล�ำไส้ใหญ่ของมนุษย์

68 วารสารวิจัย มข. (บศ.) 15 (3) : ก.ค. - ก.ย. 2558

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�ำไส้ใหญ่มีการแสดงออกของ COX-2 ที่มากผิดปกติ จากการศึกษา ก่อนหน้านี้พบว่า cepharanthine (CEP) ซึ่งเป็นสารในกลุ่ม biscoclaurine alkaloid ที่พบได้ในราก ของต้น Stephania cepharantha Hayata มีฤทธิ์ต้านมะเร็งหลายชนิดเช่น มะเร็งช่องปากและล�ำคอ มะเร็งเม็ดเลือดขาว มะเร็งตับ และมะเร็งท่อน�้ำดี วัตถุประสงค์ของการทดลองนี้เพื่อศึกษาฤทธิ์ต้านมะเร็ง ของ diclofenac, celecoxib, berberine, CEP และ 5-fluorouracil ต่อเซลล์ HT-29 ซึ่งเป็นเซลล์มะเร็ง ล�ำไส้ใหญ่ของมนุษย์ที่มีการแสดงออกของ COX-2 จากผลการทดลองแสดงให้เห็นว่า CEP สามารถยับยั้ง การเจริญของเซลล์มะเร็งได้ดีกว่า diclofenac, celecoxib, berberine, และ 5-fluolouracil โดย CEP สามารถชักน�ำให้เซลล์มะเร็งตายแบบอะพอพโทซิสและสามารถยับยั้งการแสดงออกของยีน COX-2 การ ทดลองนี้แสดงให้เห็นว่า CEP อาจจะเป็นยาที่มีประสิทธิภาพในการรักษาโรคมะเร็งล�ำไส้ใหญ่ที่มีการแสดงออก ของ COX -2

Keywords: Cepharanthine (CEP), Apoptosis, Colon cancer ค�ำส�ำคัญ: เซฟราเรนทีน อะพอพโทซิส มะเร็งล�ำไส้ใหญ่ 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 was evaluated in this study. temperature in dark. The stained cells were We initially determined the basal level of analyzed using flow cytometry (BD LSR II, COX-2 mRNA in two human colon cancer cell Biosciences). Four populations of cells can be line, HT-29 and Colo-205 cells. As shown in distinguished, including viable cells (annexin Figure 1A, COX-2 mRNA was detected only V- , PI -), early apoptotic cells (annexin V in HT-29 cells. Quantitative real time PCR +, PI -), late apoptotic cells (annexin V +, analysis showed that COX-2 mRNA level in PI +) and necrotic cell (annexin V -, PI +), HT-29 cells was 2000 fold higher than that which are located in the lower left, lower right, in Colo-205. Treatment with 5 or 10 µM upper right, and upper left quandrants of the CEP significantly decreased COX-2 mRNA cytograms, respectively. expression level only at 6 h after incubation Statistical analysis (p<0.05) (Figure 1B). We however did not All data are presented as mean ± observe any change in COX-2 mRNA level standard error of mean (SEM). Statistical after treatment with CEP at 24 h after analysis of data was performed by one-way incubation (Figure 1B) analysis of variance (ANOVA) followed by Effect of dilofenac, celecoxib, LSD post hoc test using SPSS statistics 21 berberine, cepharanthine, and 5-Fluo- software. Difference is considered significant rouracil on cell viability of HT-29 cells if P ≤ 0.05. We then evaluated anticancer effects of diclofenac (a NSAID), celecoxib (a selective 72 วารสารวิจัย มข. (บศ.) 15 (3) : ก.ค. - ก.ย. 2558

COX-2 inhibitor), berberine and CEP (al- cells are greater than early apoptotic cells. kaloids exhibiting both anti-inflammatory Treatment of CEP at 5, 10 and 20 µM could and anticancer activities) and 5-fluorouracil induce cells to undergo late apoptotic about (5-FU) (an anticancer drug) against 11.25±2.66, 30.47±1.68 and 30.36±2.80%, COX-2-positive HT-29 cells using resazurin respectively. These results suggest that CEP assay. At 48 h of treatment, diclofenac at 50 effectively induced COX-2-positive HT-29 and 100 µg/ml and celecoxib and berberine cell death via apoptosis induction. at 50 and 100 µM significantly inhibited the growth of HT-29 cells (Figure 2A-C) while Discussion the cytotoxic effects of CEP and 5-FU were It has been that cyclooxygenase detected at 10-100 µM in a concentration- (COX)-2 and its product, PGE2, are involved dependent manner (p< 0.05) (Figure 2D in cell proliferation, angiogenesis, and and E). The IC50 value of diclofenac was 71.88 inflammation, leading to development of ± 5.04 µg/ml while they were73.39± 3.58, many cancers including colorectal cancer 6.21± 1.68, and 54.73± 3.86 µM for celecoxib, [21]. It has been shown that COX-2 mRNA is CEP and 5-FU, respectively, suggesting that over-expressed in almost 80% of the colorectal CEP exhibits the potent anticancer effect tumors, compared with normal colorectal against COX-2-positive human colon cancer mucosa [22]. Previous study showed that cells. HT-29 cells overexpressing COX-2 were Effect of cepharanthine on resistant to apoptotic cell death [5]. In the apoptosis induction of HT-29 cells present study, we therefore investigated the Most of the currently chemothera- effect of cepharanthine (CEP) on COX-2 peutic drugs induce cancer cells to undergo expression in HT-29, a COX-2 positive human apoptosis. We therefore examined the colon cancer cell line. CEP could significantly apoptosis induction effect of cepharanthine inhibit COX-2 mRNA expression (Figure 1). in HT-29 cells using annexin V-FITC and We then evaluated the cytotoxic effects of PI double staining. At 24 h after incubation, diclofenac, celecoxib, as well as berberine CEP at 2.5, 5, 10 or 20 µM could significantly and CEP against the HT-29 cells. The induce apoptotic cell death in a concentration- cytotoxicity of 5-FU, a commonly chemo- dependent manner (Figure 3). Treatment of therapeutic drug used for colon cancer, was 20 µM of CEP reduced cell viability about 3 also tested. We found that all test agents could times with respect to the vehicle control group. effectively inhibit the growth of HT-29 cell in The number of viable cells were 94.54±1.43% a concentration dependent manner, however, for the vehicle control group and 35.95±5.72% with different potencies (Figure 2). Of five for the 20 µM CEP group. It however should compounds tested, CEP was the most effective be noted that the percentage of late apoptotic anticancer agents against HT-29 cells. KKU Res J (GS) 15 (3) : July - September 2015 73

The IC50 value of CEP was approximately 6 laboratories. CEP has also shown to induce µM which was much more potent than 5-FU, cancer cells to undergo apoptosis through the anticancer agent commonly used for colon several mechanisms including i) activating cancer. Previously, the anticancer activities of caspase-3 and 9 [11, 14-15], ii) stimulating diclofenac and celecoxib, at high doses, were pro-apoptotic signaling pathways such as also observed in colon cancer cells. Mechanis- JNK, ERK and p38 MAPK [14, 28], and iii) tic studies indicated that diclofenac exhibited inhibiting expression of anti-apoptotic gene its tumor suppression effect in HCT-116 Bcl-xl [15]. Therefore, mechanisms other cell, a colon cancer cell line, by inhibiting than down-regulating of COX-2 may likely Wnt/β-catenine signaling pathway [23] while be responsible for the anticancer activity of celecoxib could inhibit COX-2 expression, CEP in HT-29 cells as well. leading to nuclear accumulation of p53 in HCT-116 and HT-29 cells [24], suggesting Conclusion different mechanisms underlying anticancer The results in the present study effects of anti-inflammatory drugs. It has clearly demonstrated that cepharanthine been reported that 5-FU and berberine induce (CEP) has a potent anticancer activity against cancer cells to undergo apoptosis in a p53- HT-29 cells, a p53-mutant and COX2-positive dependent manner [25, 26]. Therefore, it is human colon cancer cell line. Mechanistic likely that p53 plays a key role in 5-FU and studies indicated that CEP effectively berberine resistance of HT-29 cells. Besides induced HT-29 cells to undergo apoptosis as p53, it was shown that COX-2-derived PGE2 well as inhibited COX-2 expression. These contributes to acquired resistance of colon findings suggest that CEP could potentially cancer cells, SNU-C5, to 5-FU [27]. Thus, it be used as a novel anticancer agent for is possible that overexpression of COX-2 may p53-mutant or COX-2-positive colon cancer also be involved in resistance of HT-29 cells cells which are commonly resistant to to 5-FU and the potent anticancer activity of currently chemotherapeutic agent. CEP against the HT-29 cells may be associ- ated with COX-2 down-regulation. Moreover, constitutive up-regulation of COX-2 has been Acknowledgement This study was supported by the found to be related to apoptosis resistance 90th Anniversary of Chulalongkorn University [27]. .In the present study, we found that Fund (Ratchadaphisekso- mpot Endowment CEP effectively induced apoptotic cell death Fund) (AR) and Grants for Development of in HT-29 cells (Figure3). Additional studies New Faculty Staff, Ratchadaphiseksompot to determine whether inhibition of COX-2 Endowment Fund, Chulalongkorn University is related to anticancer effect of CEP are (PW). currently under active investigation in our 74 วารสารวิจัย มข. (บศ.) 15 (3) : ก.ค. - ก.ย. 2558

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B) A) A) B)

FigureFigure 1 1Effect Effect of of CEP CEP on COXon COX-2-2 mRNA mRNAexpression expression in HT-29 cells. in BasalHT-29 level cells. of COX Basal-2 mRNA level inof HT COX-2-29 and Figure 1 EffectmRNA of CEP in on HT-29 COX-2 and mRNA Colo-205 expression were in determinedHT-29 cells. Basalusing level RT-PCR. of COX B)-2 mRNA COX-2 in mRNAHT-29 and Colo-205 were determined using RT-PCR. B) COX-2 mRNA expression in HT-29 cells after treatment Colo-expression205 were determined in HT-29 using cells RT after-PCR. treatmentB) COX-2 withmRNA CEP expression at 2.5, in5, HT 10- 29and cells 20 after µM treatmentCEP with CEP at 2.5, 5, 10 and 20 µM CEP for 6 and 24 h.* P<0.05, **P  0.01 with respect to vehicle control for 6 and 24 h.* P<0.05, **P < 0.01 with respect to vehicle control (0.2% DMSO). with CEP(0.2% at 2.5,DMSO). 5, 10 and 20 µM CEP for 6 and 24 h.* P<0.05, **P 0.01 with respect to vehicle control (0.2% DMSO).

A) B) C)

D) E)

Figure 2 Cytotoxic effect of diclofenac, celecoxib, berberine, cepharanthine and 5-fluo- Figure 2 Cytotoxic effect of diclofenac, celecoxib, berberine, cepharanthine and 5-fluorouracilagainst COX2-positive rouracilagainst COX2-positive HT-29 human colon cancer cells. The cells were treatedHT-29 human with colon0.01, cancer 0.1, 1,cells. 10, The 50 cells and were 100 treated µM withor µg/ml 0.01, 0.1, (for 1, 10, diclofenac) 50 and 100 µMfor or 48 µg/ml h. Cell (for Figure 2 Cytotoxicviabilitydiclofenac) effect was of for diclofenac, determined48 h. Cell viability celecoxib, using was berberine,resazurin determined cepharanthine usingassay. resazurin Each and assay. value 5- fluorouracilagainst Each is expressed value is expressed as COX2 the as mean the-positive  HT-29± mehumanSEM.an ± SEM. colon(n=3). (n=3). cancer *** *** Pcells.

78 วารสารวิจัย มข. (บศ.) 15 (3) : ก.ค. - ก.ย. 2558

A) 0.2 % DMSO CEP 2.5 µM CEP 5 µM

1.91% 1.80% 11.25% 94.54% 91.84% 77.67%

2.50% 1.97% 6.33%

CEP 10 µM CEP 20 µM B)

30.36% 30.47% 44.18% 35.95% 7.54% 14.47%

FigureFigure 3 Apoptotic 3 Apoptotic induction inductioneffect of CEP effect in HT- 29of cells. CEP A) in Representative HT-29 cells. cytograms A) Representative of cell apoptosis analysiscytograms of of cell apoptosis analysis of HT-29 cells after treatment with CEP (2.5-20 µM) HT-29 cells after treatment with CEP (2.5-20 µM) for 24 h. B) The percentage of different HT-29 cell for 24 h. B) The percentage of different HT-29 cell populations (alive, early and populationslate apoptotic(alive, early cells)and late after apoptotic treatment cells) after with treatment CEP. with Each CEP. value Each value is expressed is expressed as as mean ### ± SEM.± (n=3).SEM. ** (n=3).P<0.01, *****P <0.01,P<0.001 *** compared P<0.001 to th comparede control late to apoptotic the control cells, lateP<0.001 apoptotic compared cells, to the control###P <0.001viable cells. compared to the control viable cells.