The Regulatory Mechanism Of Propolis Flavonoid Pinobanksin-3-acetate On Gene Expression Transcription Level In Human Colorectal Cancer Cell Line

The Propolis flavonoid (Pinobanksin-3-acetate, PB3A) has significant inhibition ofproliferation and induction of apoptosis effects on colorectal cancer cells HCT116andSW480, but its anti-tumor mechanism remains unclear yet. In our previous study, wehave investigated the role of PB3A (100μg/mL) on SW480cells by using HumanGenome Gene Chip Array, the gene with more than10times of differential expressionfor quantitative criteria, and we have found a variety of down regulated andupregulated candidate genes including down-regulated gene PDE4D and11up-regulated genes HSPA6, MMP1, HMOX1, SNAI2, GEM, IL11, RGS2, GADD45GDNAJA4, FOS, KLHL20as the important candidate genes. Currently the studies ofPB3A on signal transduction pathway related gene transcription level of colorectalcancer have not been reported Currently.Objective:In this study, to verify the accuracy and reliability of previous study for HumanGenome Gene Chip Array results, the transcription level of differential expressedcandidate genes by using real-time quantitative PCR have been detected. To furtherexplore the mechanism of propolis PB3A on colorectal cancer cells SW480and cellsto drug response through correlation and functional analysis of expressed genes,provide a theoretical databases for developing and clinical treatment programs ofPB3A as the new anti-cancer drug.Methods：1. After24h incubation total cellular RNA was extracted from control groups and100μg/mL PB3A intervention groups of SW480cell by using Trizol one-step reagentand cDNA was synthesized by using cDNA synthesis kit.2. Based on the different genes mRNA sequence, the QRT-PCR primers of differentcandidate genes were designed and synthesised relying on the TaKaRa. Real-timePCR was performed according to SYBR Green Real-time PCR, and the PCR productwas analyzed using Bio-RAD-IQ5Multicolor-Real-Time quantitative PCR system and β-actin as an internal control.3. Statistical Methods: The results of△Ct were expressed as mean士SD，Statisticsoftware SPSS11.0was used to△Ct values of target genes both of drug treatmentgroup and control group were analyzed by two independent samples t-test (two-sided)analysis. The accepted level of significance was p<0.05and P <0.01.Results：1. During colon cancer SW480cells were treated with100μg/ml of PB3A for arequired time24h, compared with control group there was a reduction in the mRNAexpression of PDE4D gene due to treatment with highly significant difference (△Ctcomparison, P<0.01); Whereas, the mRNA expression of HSPA6, MMP1, GEM, IL11,HMOX1, RGS2, SNAI2, DNAJA4, FOS, GADD45G were increased due to thePB3A treatment, and also have very significant significant different compared withthe control group (△Ct comparison, P <0.01), the trends of fold change in expressionlevel were consistent with the microarray results. Though there were no significantdifference of mRNA expression of KLHL20gene in the treated group compared withthe control group (△CT comparison, P>0.05), the expression level (0.88) of thisgene was opposite to its gene chip result (15.62), but its fold change in expressionconsistent with the other two gene probe of previous gene chip result.2. Correlation analysis of mRNA expression of differentially expressed genes showedthat, there were negative correlation between downregulated gene PDE4D andupregulated gene SNAI2(r=-0.833, p <0.05), but no correlation have been found withthe other gene (p>0.05). And there were also have high positive correlation between10upregulated genes such as HSPA6, HMOX1, MMP1, GEM, FOS, IL11, DNAJA4,GADD45G, SNAI2and RGS2each other (0.738<r <0.95, p <0.05).Conclusions：1. In this study, we have verify the accuracy and reliability of the results of previousstudy for Human Genome Gene Chip Array through analysis of gene fold change in mRNA expression level due to the treatment with100μg/mL PB3A by usingReal-time quantitative PCR method.2. The anti-tumor effects of Propolis flavonoids PB3A is closely related to theregulation of gene transcriptional expression participated in the cell proliferation,differentiation, regulator genes of apoptosis, and metabolic enzyme genes.3. In the anti-tumor effect of PB3A, the increased mRNA expression of HSPA6、HMOX1may play a protective effect on colorectal cancer, and upregulated genesMMP1, SNAI2, IL11have resistance to drug treatment; while upregulated genesGADD45G, FOS, DNAJA4, RGS2, GEM may promote the drug anti-cancer effect.4. PB3A promote its apoptotic and anti-proliferative activity by down-regulation oftumor cell proliferation-promoting factor-PDE4D expression, these results can clarifythe anti-tumor mechanism of PB3A on colorectal cancer cells as well as the responseof cells to drug treatment.