The Effects Of Propolis Lfavonoid Pinobanksin-3-acetate On Cell Proliferation, Apoptosis And Gene Expression Of Human Colorectal Cancer Cell Lines

Objective:Study the effects of Propolis flavonoid Pinobanksin-3-acetate (PB3A) on the colorectal cancer (CRC) cell lines HCT116and SW480proliferation, morphology and apoptotic rate, and further analyze the PB3A additive or synergistic effect with common clinical anti-cancer drug, investigate the PB3A anti-CRC mechanism and clinical application. On the base of above results, uncover the gene expression regulation network between PB3A treated and untreated group on SW480cells in vitro by using Human Genome Gene Chip Array U133_Plus₂(HG-U133_Plus₂), and further identify target genes or genogroups, and may provide some scientific databases for developing PB3A as an anti-tumor drug.Methods:1. The effect of different concentrations (5,25,50,100and150μg/mL) of PB3A,(25and50μg/mL)5-fluorouracil (5-FU) and a combination of PB3A and5-FU for different durations on proliferation of cell lines HCT116and SW480was analyzed by MTT assay. Morphological changes of HCT116and SW480cells were observed by inverted photo microscopy after treatment with different concentrations of PB3A for different durations.2. The24h apoptotic rate of HCT116and SW480cells induced with PB3A and5-FU were detected by FCM with Annexin V-FITC/PI double labeling.3. Total RNA was extracted from the SW480cell control groups and100μg/mL PB3A intervention groups after24h incubation time with Trizol one-step method. The gene expression profiles were analyzed by HG-U133_Plus₂and the differential expression genes compared between the control groups and PB3A intervention group cells were classified according to their function using the Molecule Annotation System3.0(MAS3.0). Scanned photography of microarray chips was digitalized and further analyzed by photo analysis in private facilities. Differentially expressed genes between the control and treated group were classified with (Gene Ontology) Mapping and make Gene-Pathway Network by MAS3.0.4. Statistic methods:Statistic analysis was performed with SPSS17.0general linear model univariate ANOVA and regression analysis where experimental data was indicated as mean±SD and50%inhibited concentration (IC50). The mean difference is statistically significant at the p-value<0.05and p-value<0.01level.Results:1. PB3A exerted a significant inhibitory effect on HCT116and SW480cell proliferation in a time-and dose-dependent manner; in all the groups5-FU indicated a stronger inhibitory effect, in some groups no significant difference was observed compared with the same concentration of the5-FU anti-tumor effect. The PB3A and5-FU combination group result indicated an additive or synergistic antitumor effect. PB3A could induce the typical changes of cell morphology in a time (24,48and72h)-and dose (50,100and150μg/mL)-dependent manner.2. FCM analysis showed that (0,50and100μg/mL) PB3A could significantly increase the apoptotic rate, suggesting a dose-dependent manner.3. PB3A intervention induced wide range of changes of the gene expression profile of SW480cells. A total of552genes were up-regulated and848genes were down-regulated by more than3-fold compared to control group, in which21genes were up-regulated and13genes were down-regulated by more than10-fold. The differentially regulated by more than3-fold genes were closely correlated with many kinds of cell signal transduction or gene expression regulation pathway, which includes cell cycle controlling, cell apoptosis, cytokinetics, colorectal carcinogenesis, p53, DNA polymerase, Wnt, and Calcium signaling. Some of them belonged to important signaling pathways related with cell proliferation, apoptosis, such as ROCK2, WNT5A, FZD2, TBL1XR1, WNT6, AXIN2, WNT3, VANGL1,FOSL1and FZD10in Wnt signaling pathway, TNFSF10, BIRC3, ATM,MAP3K14,CASP7,TNFRSF10B in apoptosis signaling pathway, SERPINE1, GADD45A, GADD45G, GTSE1, GADD45B in p53signaling pathway.Conclusions:1. These results suggest that PB3A is a potent inhibitor of CRC cell lines HCT116and SW480proliferation and it could induce apoptosis; furthermore, the PB3A dosage may have an additive or synergistic effect with chemotherapeutic drug5-FU when analyzed in this in vitro system.2. PB3A anti-cancer effect may closely correlated with a wide array of pivotal cell signal transduction or gene expression regulation pathway, in which may co-exist a sort of multitarget, multipath and multieffect of anti-cancer mechanism.