Studies On Separation Of Antitumor Active Fraction Extracted From Wedelia Chinensis And Its Related Mechanisms

Introduction: Wedelia chinensis (Osbeck) Merr. is a herb that has been widely used in southern China for several hundred years. Although W.chinensis was found to have a wide range of functions, such as anti-inflammatory, antiviral and antimicrobial activities, the effects of W.chinensis on cancers and its related molecular mechanisms have largely remained unexplored. In the present study, W.chinensis plants were sequentially extracted by ethanol (E), petroleum ether (PE), ethyl acetate (EA) and butyl alcohol (BA) and each extract was examined for bioactivity by MTT assay in cancer cell lines to identify the most active fraction and the most sensitive cell line. The effect of the active fraction on cell cycle and apoptosis was determinded by flow cytometry. Using RNAi,Real-time PCR and RT-PCR, we investigated the molecular mechanism of G2/M cell cycle arrest and apoptosis induced by the active fraction in the sensitive cell line.Objective:The objectives of the present investigation were to determine whether W.chinensis extract has efficacy in suppressing proliferation of various cancer cell lines derived from different tissues and to identify novel targets for W.chinensis extract in cancer cells and to explor the molecular mechanism that W.chinensis extract inhibits cancer cell growth in vitro.Methods: 1. Plant extracts preparation: Dry whole plants of W. chinensis were homogenized in water,70% ethanol and 95% ethanol with a blender。The ethanol extract and the water extract were concentrated by evaporator and then got the water extract,70% ethanol extract and 95% ethanol extract.2. Assessment of cell growth inhibition by MTT assay: MTT was used to detect which extract (water extract,70% ethanol extract and 95% ethanol extract) exhibited the highest degree of cytotoxicity to various cancer cell lines.3. The 70% ethanol extract was filtered through qualitative filter papers and the filtrate was concentrated by evaporator and then washed with hot water. The water was further partitioned with equal volume of petroleum ether (PE) and divided into PE and H₂O fractions through liquid–liquid partition. The H₂O fraction was further partitioned with equal volume of ethyl acetate (EA) and divided into EA and H₂O subfractions. The H₂O subfraction was further partitioned with equal volume of butyl alcohol (BA) and divided into BA and H₂O subfractions. The EA subfraction was fractionated by silica gel column chromatography, resulting in the collection of other EA subfractions 1-6 (EA1-6). Each extract was examined for bioactivity by MTT assay in CNE-1 cells.4. Flow cytometry analysis of cell cycle and apoptosis: Flow cytometry were used to detect cell cycle distribution and apoptosis in CNE-1 cells treated with different doses of EA6.5. CNE-1 cells were treated with different doses of EA6 for 24 and 48 h and then subjected to Hoechst staining to evaluate apoptosis.6. Transfection of short-interfering RNA (siRNA): The siRNA sequences targeted human Chk1and Chk2 transcripts were designated as si-Chk1 and si-Chk2, respectively. Cells were transfected with siRNA to inhibit the expression of Chk1and Chk2.7. After transfection with si-Chk1or si-Chk2, CNE-1 cells were incubated with EA6 and then detected the cell proliferation by MTT and the cell cycle distribution and apoptosis by flow cytometry.8. Inhibit the expression of Chk1and Chk2 then RT-PCR was used to detect the expression of c-myc, cyclin B1 and cyclin D1 after cell were treated with EA6.Result:1. To determine the effect of three W.chinensis extract (water extract,70% ethanol extract and 95% ethanol extract ) on the proliferation of various cancer cell lines. The MTT assay results showed that 70% ethanol extract exhibited the highest degree of cytotoxicity than others. Our results also showed that CNE-1 is the cell line most sensitive to 70% ethanol extract-induced cytotoxicity (IC₅₀ =1.27±0.19 mg/ml ) and then chosen for further study.2. The 70% ethanol extract was sequentially extracted by petroleum ether (PE), ethyl acetate (EA) and butyl alcohol (BA) and each extract was examined for bioactivity by MTT assay in CNE-1 cells. Results showed that EA had the most potent antiproliferatory activity than others (IC₅₀ =489.56±22.78μg/ml).3. The EA was fractionated by silica gel column chromatography, resulting in the collection of other EA subfractions 1-6 (EA1-6). Among the six EA subfractions, EA6 showed the most potent activity (IC₅₀ =96.59±19.37μg/ml).4. The variations of cell cycle profile and apoptosis upon treatment of CNE-1 cells with different concentrations of EA6 were investigated by flow cytometry. We observed significantly increased G2/M cell cycle arrest of CNE-1 cells following treatment with 150μg/ml EA6 for 24 and 48 h. The percentage of cells in G2/M phase was increased from (17.14±0.85)% to (44.83±6.43)% in 24 h timepoint and from (18.96±3.78)% to (39.57±1.58)% in 48 h timepoint. Apoptosis was induced by EA6 (150μg/ml) in CNE cells for 48 h and the percentage of apoptotic cells was increased from (0.6±0.1)% to (12.3±2.15)%.5. RNA interfering technique was used to silence the expression of Chk1 and Chk2 genes. Real-time PCR and RT-PCR analysis showed that both Chk1 and Chk2 siRNAs dramatically suppress the Chk1 and Chk2 mRNA expression in CNE-1 cells. Forty-eight hours after transfection of with si-Chk1 or si-Chk2, CNE-1 cells were incubated with 150μg/ml EA6 for 24 h and then cell proliferation was detected by MTT assay and cell cycle distribution was determined by flow cytometry. Results showed that treatment of EA6 resulted in notable cell growth inhibition of CNE-1 cells and Chk1 knockdown significantly reversed such effect of EA6 and EA6-induced G2/M arrest was completely attenuated (P<0.05) in CNE-1 cells transfected with si-Chk1. The percentage of cells in G2/M phase were reduced from (34.79±3.8) % to (15.45±1.63)%.6. The expression of c-myc, cyclin B1 and cyclin D1 in CNE-1 cells treated with EA6 (150μg/ml) for 24 h by RT-PCR. Results showed that treatment of CNE-1 cells with EA6 significantly reduced the mRNA level of c-myc, whereas no obvious alterations of cyclin B1 and cyclin D1 levels were observed. Inhibition of Chk1 by RNAi increased c-myc expression of EA6-treated CNE-1 cells to comparable level of control cells.Conclusion:In this study, W.chinensis plants were sequentially extracted by ethanol (E), petroleum ether (PE), ethyl acetate (EA) and butyl alcohol (BA) and each extract was examined for bioactivity by MTT assay in cancer cells. Our results showed that one subfraction of the EA extract (EA6) exhibited the highest degree of cytotoxicity to nasopharyngeal carcinoma (NPC) CNE-1 cells. Further investigations revealed that treatment of EA6 mainly caused G2/M cell cycle arrest and partially induced apoptosis in CNE-1 cells. Inhibition of Chk1 by RNA interfering (RNAi) significantly abrogated EA6-mediated G2/M arrest and abolished EA6-induced cytotoxicity. EA6 treatment resulted in significant reduction of c-myc, while silencing Chk1 notably increased c-myc expression to control level. Our results indicate that Chk1 as a novel molecular target of EA6 in NPC cells and also suggest an intervention strategy for NPC by EA6 exploring its molecular mechanisms of action.