Piper betle leaf extract enhances the cytotoxicity effect of 5-fluorouracil in inhibiting the growth of HT29 and HCT116 colon cancer cells

Piper betle leaf extract enhances the cytotoxicity effect of 5-fluorouracil in inhibiting the growth of HT29 and HCT116 colon cancer cells 蒌叶提取物增强5-氟尿嘧啶对结肠癌细胞HT29和HCT116的生长抑制作用 1 first-author 1 2 3 4 2 4 5 6 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:MeeLee.Looi@taylors.edu.my †E-mail:MeeLee.Looi@taylors.edu.my MeeLee.Looi@taylors.edu.my Department of Biomedical Sciences, Faculty of Allied Health, Universiti Kebangsaan Malaysia (UKM), Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia Department of Biomedical Sciences, Faculty of Allied Health, Universiti Kebangsaan Malaysia (UKM), Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan Malaysia (UKM), Jalan Yaacob Latiff, Bandar Tun Razak, Cheras, 56000 Kuala Lumpur, Malaysia UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan Malaysia (UKM), Jalan Yaacob Latiff, Bandar Tun Razak, Cheras, 56000 Kuala Lumpur, Malaysia School of Biomedical Science, University of Nottingham Malaysia Campus, 43500 Semenyih, Selangor, Malaysia School of Biomedical Science, University of Nottingham Malaysia Campus, 43500 Semenyih, Selangor, Malaysia Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia (UKM), Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia (UKM), Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia Forest Research Institute Malaysia (FRIM), 52109 Kuala Lumpur, Kepong, Malaysia Forest Research Institute Malaysia (FRIM), 52109 Kuala Lumpur, Kepong, Malaysia School of Biosciences, Taylor’s University, Lakeside Campus, 47500 Subang Jaya, Selangor, Malaysia School of Biosciences, Taylor’s University, Lakeside Campus, 47500 Subang Jaya, Selangor, Malaysia Objective: 2 The combination effect of Piper betle (PB) and 5-fluorouracil (5-FU) in enhancing the cytotoxic potential of 5-FU in inhibiting the growth of colon cancer cells was investigated. Methods: 2 HT29 and HCT116 cells were subjected to 5-FU or PB treatment. 5-FU and PB were then combined and their effects on both cell lines were observed after 24 h of treatment. PB-5-FU interaction was elucidated by isobologram analysis. Apoptosis features of the treated cells were revealed by annexin V/PI stain. High-performance liquid chromatography (HPLC) was performed to exclude any possible chemical interaction between the compounds. Results: 2 In the presence of PB extract, the cytotoxicity of 5-FU was observed at a lower dose (IC 50 12.5 μmol/L) and a shorter time (24 h) in both cell lines. Both cell lines treated with 5-FU or PB alone induced a greater apoptosis effect compared with the combination treatment. Isobologram analysis indicated that PB and 5-FU interacted synergistically and antagonistically in inhibiting the growth of HT29 and HCT116 cells, respectively. Conclusions: 2 In the presence of PB, a lower dosage of 5-FU is required to achieve the maximum drug effect in inhibiting the growth of HT29 cells. However, PB did not significantly reduce 5-FU dosage in HCT116 cells. Our result showed that this interaction may not solely contribute to the apoptosis pathway. 研究目的 2 探讨蒌叶（PB）提取物对5-氟尿嘧啶（5-FU）抑制结肠癌细胞HT29和HCT116生长的影响。 研究方法 2 HT29和HCT116细胞分别给予PB、5-FU以及两种药物联合治疗24小时，应用等效线图法分析PB和5-FU的药效学相互作用，AnnexinV/PI染色法检测HT29和HCT116细胞的凋亡情况，高效液相色谱法排除PB和5-FU间任何可能的相互化学作用。 重要结论 2 联合PB，低剂量5-FU可以在短时间内起到细胞毒作用，而单独应用PB或5-FU治疗较联合治疗可以诱导更多细胞发生凋亡。进一步采用等效线图法分析显示PB和5-FU的联合作用在抑制结肠癌细胞HT29和HCT116的生长中分别体现出协同和拮抗作用。因此可以认为在HT29细胞中，PB使得较低剂量5-FU发挥最大抑制结肠癌细胞生长效果，然而在HCT116细胞中，PB没有显著降低5-FU的药物浓度，说明PB和5-FU的相互作用不仅仅体现在诱导细胞凋亡方面。 Piperaceae Piper betle L. 5-Fluorouracil Isobologram analysis Herb-drug interaction 胡椒科 蒌叶 5-氟尿嘧啶 等效线图法 草药-药物相互作用 Introduction 1 5-Fluorouracil (5-FU) is an antimetabolite-based chemotherapeutic drug commonly used in treating solid human tumors, especially colorectal cancer. Cytotoxicity of 5-FU is attributed to its ability to inhibit thymidylate synthase activity and interrupt DNA and RNA syntheses (Kinsella et al., 1997 1997 ; Noordhuis et al., 2004 2004 ). However, its use is limited because of the rapid development of acquired resistance and its short half-life (Mader et al., 1998 1998 ; Jin et al., 2002 2002 ). Different treatment approaches have been designed to increase the efficacy of 5-FU such as optimizing the administration schedule (Pizzorno et al., 2009 2009 ) and using a combination treatment with leucovorin and oxaliplatin (FOLFOX) (Klampfer et al., 2005 2005 ). Nevertheless, drug resistance has also been noted with increasing 5-FU dosage (Mader et al., 1998 1998 ; Pizzorno et al., 2009 2009 ) and in combination therapy (Pizzorno et al., 2009 2009 ). 5-FU resistance is conferred by the alteration in drug metabolism, drug transport mechanism, and expression of targeted proteins such as p53; as a result, cancer cells fail to undergo cell cycle arrest and apoptosis (Mader et al., 1998 1998 ). p53 is the tumor-suppresser protein that helps in coordinating the response towards 5-FU treatment. About 30%–50% of colorectal cancer cases have been found to have mutated p53 protein and hence loss of sensitivity towards 5-FU (Mader et al., 1998 1998 ; Bunz et al., 1999 1999 ). Thus, there is a need for strategies to overcome drug resistance and hence enhance drug delivery to the target site of cancer. Combination anticancer treatments using the active compounds from plant extracts are becoming a new focus of drug discovery. Natural plant extracts enriched with flavanoids, phenolic compounds, and other phytochemicals have fewer side-effects compared with chemotherapy drugs, and thus play important roles as anticancer agents (Pourcel et al., 2007 2007 ). There are numerous studies (Tang et al., 2007 2007 ; Wang and Yuan, 2008 2008 ; Fishbein et al., 2009 2009 ) showing synergistic drug-herb interactions in enhancing anticancer treatment with minimum cytotoxicity to the host cells. Fishbein et al. ( 2009 2009 ) showed that an anti-proliferative effect of Red ginsenoside enhanced 5-FU activity in human colon carcinoma, HCT116 cells. Mice treated with combined triptolide and 5-FU showed a greater tumor growth inhibition in primary tumor xenografts (Tang et al., 2007 2007 ). Piper betle (PB) is a well-known ethnomedicinal plant in the Asian region (Kumar et al., 2010 2010 ). PB leaves have been known for centuries for their curative properties in, for example, preventing halitosis, and in the treatment of boils, abscesses, as well as throat and lung diseases (Guha, 2006 2006 ). An active compound of PB, hydroxychavicol (HC), has been recognized as an antioxidant, anti-proliferative, and anticancer agent (Young et al., 2006 2006 ; Fathilah et al., 2010 2010 ). Previous research has revealed that pre-treatment of oral KB carcinoma cells with HC inhibited the attachment of KB cells to type I collagen and fibronectin, and subsequently resulted in cell cycle arrest in S and G2/M phases (Chang et al., 2002 2002 ). Chakraborty et al. ( 2012 2012 ) showed that HC-induced reactive oxygen species (ROS)-mediated phosphorylation of c-Jun N-terminal kinase (JNK), which, in turn, phosphorylates endothelial nitric oxide synthase to produce nitric oxide and mediate cell death in chronic myeloid leukaemia without depleting glutathione levels. Chakraborty et al. ( 2012 2012 ) also showed that multiple compounds present in crude extracts of PB may exert better anticancer effects compared with a single active compound. Combination treatment of the anticancer drug, cisplatin, with PB increased the sensitivity of liver cancer cells to cisplatin (Young et al., 2006 2006 ). In this present study, we attempt to investigate the effect of PB on colon cancer cells and its interaction with the anticancer drug, 5-FU. Drugs when given in combination may produce a greater or lesser effect compared with a single drug. Drug interaction occurs when the pharmacokinetics and/or pharmacodynamics of a drug is altered by the presence of another drug, food, or herbs (Hussin, 2001 2001 ; Renuka et al., 2011 2011 ). Pharmacodynamic interactions are usually analyzed by the isobologram, which can distinguish the additive interaction from synergistic and antagonistic interactions (Tallarida and Raffa, 1996 1996 ; Chou, 2006 2006 ). Synergism is achieved when either or both respective drugs require a lower concentration to reach the same effect as in the individual treatment respectively with combination index (CI) < 1. Antagonism is defined when a higher concentration of either or both drugs achieves the same effect as the individual treatment (Chou, 2006 2006 ). In this study, we found a synergistic interaction of 5-FU and PB on HT29 cells. This justified further investigation of the major active compound of PB, HC, on HT29 cells. Apart from causing a systemic drug interaction effect, drug-drug or herb-drug interaction could reduce or enhance the bioavailability of co-administered drug (Cabrera et al., 2011 2011 ). This is known as ‘pharmaceutical interaction’, in which two substances interact by direct chemical reaction, either before ingestion or while mixed together in the stomach and intestine (Renuka et al., 2011 2011 ). For example, combination treatment of ampicillin with tannins forms an insoluble complex; this bioavailability impairment reduces the gastrointestinal absorption of ampicillin (Esimone, 2011 2011 ). In our current study, we also attempt to identify any pharmaceutical interaction between PB and 5-FU. Materials and methods 1 Aqueous extraction of PB leaves 2 PB leaves were collected from Bentong, Pahang, Malaysia. The fresh specimen was sent to the Botany Department Herbarium, Universiti Kebangsaan Malaysia (UKM), Bangi, Malaysia for identification. The specimen was then certified with a voucher number Herbarium UKMB-29852. The leaves were dried and ground prior to the extraction. Extraction was conducted using a Soxhlet extractor coupled with a cooling system. The extraction method was standardized in collaboration with the Forest Research Institute of Malaysia, according to the method of Pin et al. ( 2011 2011 ). The ratio of solvent (distilled water) to raw material was 10 L/kg and the mixture was stirred at 60 °C for 2 h. The aqueous extract was filtered with a muslin cloth and filter paper, and then dried in a Virtis freeze-drier (SP Scientific, USA). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) total free radical scavenging assay 2 Total free radical scavenging activities of PB leaf aqueous extract and 4-allylpyrocatechol were determined by performing DPPH radical assay as previously described (Molyneux, 2004 2004 ). Ascorbic acid (Sigma Chemical, USA) was used as a positive control. Samples were mixed with methanolic 0.5 mmol/L DPPH solution in a 1:1 ratio for 30 min in the dark. A total of 200 μl of the reaction mixture was then transferred to a 96-well plate and the absorbance was read at 517 nm. The scavenging activity was calculated with the equation: radical scavenging activity=[1−(absorbance of sample/absorbance of positive control)]×100%. The antioxidant activity of the sample is expressed as IC 50 , which is defined as the concentration of sample required to inhibit the formation of DPPH radicals by 50%. Cell cultures and treatments 2 Human colon cancer cell lines, HT29 and HCT116 cells (American Tissue Culture Center), were grown in complete McCoy’s 5A medium with L -glutamine (Invitrogen, USA). The medium was supplemented with 10% heat-inactivated (30 min, 56 °C), filter-sterilized fetal bovine serum (Gibco, USA). The cells were maintained in a 37 °C humid incubator with 5% CO 2 . The experiments consisted of four groups for each cell line: (1) control (cancer cells without treatment); (2) cancer cells treated with 5-FU; (3) cancer cells treated with PB leaf extract (range 100.0 to 500.0 μg/ml); (4) cancer cells treated with 5-FU and PB leaf extract. HT29 cells were also treated with (1) 4-allylpyrocatechol (synthetic HC) (62.5 to 1000.0 μmol/L) and (2) with 5-FU in combination with 4-allylpyrocatechol. Serial concentrations of PB or 4-allylpyrocatechol, which gave 10% to 90% cell growth inhibition, were combined with different concentrations of 5-FU. Cell proliferation assay 2 Cells were seeded 24 h prior to treatment in a 96-well plate at plating densities of 10 000 cells/well in order to obtain semi-confluent culture. After respective treatments at every 12-h interval from 12 to 72 h, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeth-oxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) solution (20 μl) was added to each well and the plates were incubated at 37 °C for 1 h. MTS product was measured at absorbance 490 nm and cell viability was calculated using the equation: cell viability=(absorbance of sample/absorbance of control)×100%. Combination index (CI) analysis 2 Drug combination effect was determined by the Chou and Talalay equation (Chou, 2006 2006 ): CI=( d A / D A )+( d B / D B ). d A and d B are the doses of individual drugs alone, i.e., the concentration of 5-FU or PB/4-allylpyrocatechol, respectively, that gives 50% inhibition. D A and D B are the doses of 5-FU and PB/4-allylpyrocatechol in combination that inhibits 50% of cell growth. CI > 1, CI=1, and CI < 1 indicate antagonistic, additive, and synergistic effects, respectively. Cell apoptosis analysis 2 Control and treated cancer cells were trypsinized and cell pellets collected. Pellets were resuspended in 100 μl of 1× annexin binding buffer (100 mmol/L 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), pH 7.5, 1.4 mol/L NaCl, and 25 mmol/L CaCl 2 ). Five microlitres of annexin V conjugates (Invitrogen, USA) and 1 μl of 100 μg/ml propidium iodide (Sigma, USA) were then added to the mixture and incubated at room temperature for 15 min. At the end of incubation, 400 μl of annexin binding buffer was added and the cells were analyzed within 1 h using a Facs Arial II flow cytometer (BD Biosciences, Canada). High-performance liquid chromatography (HPLC) analysis 2 HPLC analysis was performed on a Prominence LC (Shimadzu Scientific Intruments, Japan) coupled with SPD-20A/20AV, UV-VIS detector. A C18 100A column (250 mm×4.6 mm, 5 μm particle size) was used as stationary phase. Elution was performed using a gradient that consisted of a mixture of 0.1% orthophosphoric acid in Milli-Q water and 100% acetonitrile, in accordance with the method of Pin et al. ( 2011 2011 ). The flow rate of the mobile phase was 1 ml/min at room temperature. Chromatograms were acquired at 200 and 260 nm for the detection of PB/4-allylpyrocatechol and 5-FU, respectively (Ahmad et al., 2011 2011 ; Pin et al., 2011 2011 ). Results 1 A higher concentration (200.0 μg/ml) of PB extract was needed to reduce DPPH radicals by 50% compared with ascorbic acid (60.0 μg/ml). The IC 50 values of 5-FU-treated HT29 and HCT116 cells were 130.0 and 12.5 μmol/L, respectively, at 72 h. The IC 50 values of PB-treated HT29 and HCT116 cells were 200.0 and 187.5 μg/ml, respectively, after 36 h of treatment. HT29 cells treated with 4-allylpyrocatechol showed a lower IC 50 (15.0 μg/ml) after 24 h of treatment. In the presence of PB extract or 4-allylpyrocatechol, the cytotoxic effect of 5-FU against HT29 cells was observed at a lower dose ( < 12.5 μmol/L) and at a shorter time (24 h) (Figs. 1a–1c 1a–1c ). HT29 and HCT116 cells treated with 5-FU and PB alone induced greater apoptosis effects compared with the combination treatment (Figs. 2a and 2b 2a and 2b ). (a, b) HT29 and HCT116 cells were treated with the indicated concentrations of 5-FU for 24 h in the presence of PB extract, respectively. (c) HT29 cells were also treated with 5-FU for 24 h in the presence 4-allylpyrocatechol Data are expressed as mean±standard deviation ( n =3). * P < 0.05, compared with control/untreated cells Annexin V-positive/PI negative cells are in early stages of apoptosis and double positive cells are in late stages of apoptosis. Annexin V-negative/PI positive cells are necrotic CI analysis indicated PB and 5-FU interacted synergistically (as shown by CI < 1) in HT29 cells, but antagonistically in HCT116 cells (CI > 1) (Fig. 3a 3a ). Combination treatment of 4-allylpyrocatechol and 5-FU interacted antagonistically in HT29 cells (Fig. 3b 3b ). The solid line (CI=1) indicates the alignment of theoretical values of an additive interaction between two substances. Values above the solid line represent antagonistic interaction; values below the line represent synergism. (a) Concurrent 24 h exposure of HT29 cells and HCT116 cells to 5-FU and PB exhibits synergistic and antagonistic treatment effect respectively. (b) Simultaneous treatment of 5-FU and 4-allylpyrocatechol treatment inhibits HT29 cell growth antagonistically Interaction of 5-FU and PB/4-allylpyrocatechol was evaluated by HPLC. Retention time of PB and 5-FU was observed at 4.0 min (200 nm) and 3.0 min (260 nm), respectively, with a noticeable shift of peak-height as the concentration varied. The mixture was analyzed at 200 and 260 nm, respectively. Peak intensities of 5-FU and PB/4-allylpyrocatechol from the mixture were the same as when they were analyzed individually. The mixture of 5-FU and PB/4-allylpyrocatechol did not show additional compounds by HPLC separation. Discussion 1 In this study, we utilized two different colon cancer cell lines, i.e., HT29 and HCT116, each with specific characteristics, to test whether these cancer cells were resistant to the treatment of PB leaf extract and the chemotherapy drug 5-FU alone and whether the combination therapy would enhance the inhibitory effect of 5-FU. HT29 and HCT116 cells have different gene profiles. HT29 cells are known to have the mutated tumor-suppressor gene, p53 . This is a determinant factor for the success of 5-FU treatment (Bunz et al., 1999 1999 ). Our results showed that HCT116 cells with a functional p53 are more sensitive to 5-FU than HT29 cells. Lack of functional p53 protein in HT29 cells leads to reduced expression of pro-apoptosis protein, which then renders the cells resistant to apoptosis (Boyer et al., 2004 2004 ). Thus, higher dosage of 5-FU was needed to induce cell death in HT29 cells. Previous studies suggested that combination treatment is more effective in treating cancer; it targets different mechanisms and reduces the chances of developing resistance (Hiss et al., 2007 2007 ; Felth et al., 2009 2009 ; Majumdar et al., 2009 2009 ; Tong et al., 2011 2011 ; Hörmann et al., 2012 2012 ; Kumar et al., 2012 2012 ; Slovin, 2012 2012 ). Tunicamycin is an antibiotic which can reduce the effective dosage of doxorubicin, epirubicin, vincristine, and cis -diamine dichloroplatinum in multi-resistant UWOV2 ovarian cancer cells, thus enhancing their cytotoxic effect (Hiss et al., 2007 2007 ). Majumdar et al. ( 2009 2009 ) found the combination of curcumin and resveratrol was effective in inhibiting the growth of colon cancer cells. Low doses of genistein from soybean increased the sensitivity of prostate cancer initiating cells to drugs such as docetaxel and cyclopamine (Hörmann et al., 2012 2012 ; Kumar et al., 2012 2012 ). Earlier studies showed that 5-FU interacts synergistically with either triptolide or dichloroacetate in killing HT29 cells (Tang et al., 2007 2007 ; Tong et al., 2011 2011 ). Combination treatment of 5-FU with dichloroacetate enhanced the 5-FU cytotoxicity at reducing 5-FU concentration from 798.4 to 80.0 μmol/L (Tang et al., 2007 2007 ). PB exhibited potent anticancer properties (Pin et al., 2010 2010 ), but it is seldom tested in the combination treatment. Here, we investigated its combination treatment effect with 5-FU in colorectal cancer cells. In our study, PB extract enhanced the cytotoxicity of 5-FU by reducing its dosage and treatment time in HT29 cells (Fig. 1a 1a ). However, combination treatment did not show an obvious apoptosis hallmark in HT29 cells. Therefore, the cell death observed in the combination treatment group is unlikely to be triggered by the apoptosis pathway and we suggest that PB causes cell death by mediating cell cycle arrest. On the other hand, we observed that the combination of 5-FU and PB did not mitigate the 5-FU dosage in HCT116 cells, but necrosis was prominent when HCT116 cells were treated by combination. This may be due to the excessive cytotoxic induction by the treatment, which leads to early massive apoptosis followed by failure of complete phagocytosis as previously described (Zong and Thompson, 2006 2006 ). The synergistic interaction of 5-FU and PB on HT29 cells justified further investigation of the major active compound of PB, HC, on HT29 cells. HC is a major phenolic compound found in PB leaves (Hemamalini et al., 2012 2012 ). The presence of two hydroxyl (OH) groups in HC increases the antioxidant and anticancer properties of PB (Pin et al., 2010 2010 ). Our DPPH result revealed that PB at a concentration of 250 μg/ml had the highest scavenging activity while HC showed lower scavenging activity compared with the crude PB extract. The potent anti-oxidant capacity exhibited by the crude PB extract may be due to the presence of other phenolic compounds such as vitamins C and E (Hemamalini et al., 2012 2012 ). Our study demonstrated the potential anti-cancer effects of PB and HC since they exerted a cytotoxic effect in both colorectal cancer cells at concentrations of more than 100 μg/ml. The cytotoxic effect of HC was through the formation of electrophilic metabolites—quinone, quinone methide, and imine methide—via the oxidative metabolism of HC (Chang et al., 2002 2002 ; Jeng et al., 2002 2002 ). The formation of electrophile metabolite conjugates with the reduced glutathione and reduced glutathione content sensitized the cells towards ROS (Nakagawa et al., 2009 2009 ). Our results showed that 4-allylpyrocatechol (synthetic HC) is more potent compared with the crude PB extract in killing HT29 cells. However, combination treatment of 4-allylpyrocatechol and 5-FU on HT29 cells showed an antagonistic interaction. This observation suggests that the synergistic effect of PB extract and 5-FU on HT29 cells may not solely be due to its major active compound, HC, since PB extract also contains other phytochemicals such as chavibetol, chavicol, eugenol, and ursolic acid, which may be involved in mediating apoptosis in cancer cells (Chang et al., 2002 2002 ; Jeng et al., 2004 2004 ; Arambewela et al., 2006 2006 ; Young et al., 2006 2006 ; Yamai et al., 2009 2009 ). These other bioactive compounds may be present and may have triggered the synergistic effect observed in our current study. Our finding is in accordance with the study of Young et al. ( 2006 2006 ), who found that the crude extract of PB exhibited a stronger cytotoxicity than the bioactive compounds, HC and eugenol, when acting in combination with cisplatin. Incompatibility interaction is an undesired reaction that occurs between a drug and another drug or solution (Newton, 2009 2009 ). There are two types of incompatibility interaction. Physical reaction usually refers to either phase separation or precipitation due to a change of relation between ionization, non-ionization, and solubility (Newton, 2009 2009 ). In chemical incompatibility, drugs are chemically degraded and can manifest themselves through turbidity, precipitation, and color change. Chemical incompatibility can probably be identified with HPLC (Newton, 2009 2009 ; Cabrera et al., 2011 2011 ). In our study, a simple visual examination of the 5-FU and PB mixture did not show the presence of any precipitation in solution, suggesting that the interaction of 5-FU/PB and 5-FU/4-allylpyrocatechol is physically compatible. Based on our HPLC results, no additional compound was resolved from the 5-FU and PB/4-allylpyrocatechol mixture, indicating that there is a compatible and stable interaction between 5-FU and PB/4-allylpyrocatechol prior to treatment of the cells. 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This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1300303 Project supported by the Ministry of Higher Education, Malaysia (No. UKM-JJ-03-FRGS0044-2010) and Universiti Kebangsaan Malaysia (No. UKM-DPP-2014-131) 2013-11-26 2014-07-11 2014-04-15 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2014 2014 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 08 15