Hedyotis Diffusa Willd Regulates Wnt Signaling Pathway Activity In Human Colon Rectal Cancer Cells HCT116

Objective:Based on the research foundation that the vital important of Wnt signal pathway in the development and progression of tumor and the remarkable effect of HDW in tumor treatment,we observated the effect of HDW treat tumor and discussed the molecular mechanism of the regulation of Wnt signaling pathway, The aim is, for the first time,to uncover the mechanism of regulation of intestinal tumors shown in the Wnt signaling pathway, to provide the experimental basis of science and new therapy for treatment of tumor theory of HDW.Methods:With rutin and quercetin as control product quality control of the medicinal materials fingerprint chromatography making Hedyotis diffusa by high performance liquid. Human colorectal cancer HCT1 16 cells by Herba Hedyotis diffusae in different concentration and different time. After that, testing its effect on the proliferation of HCT116 cells by MTT. The effect of HCT116 cancer cells of Hedyotis diffusa Willd node to human form by microscopy. Hoechst 33342 fluorescent staining and detection of Caspase-3 and Caspase-9 to observe its effect on the apoptosis of HCT116 cells. Effect of scratch test and Transwell assay on HCT116 cells migration and invasion. Soft agar colony formation effect of Herba Hedyotis diffusae testing experiments on the growth of HCT116 cell transformation. With the level of gene expression and protein detection related to Wnt signal pathway by real-time fluorescence quantitative PCR.Results:(1) The results of MTT show that, with the increase concentration and reaction time HDW have remarkable inhibitory effect on the proliferation of HCT116 cells, 500μg/mL HDW treated cells 48h inhibitory rate of HCT116 reached 82.9%, the inhibitory effect was concentration and time dependent manner.(2) Cell morphology was observed after treatment with inverted microscope,the control group of HCT116 cell growth active, morphology, adherentfirmly, almost no cell death. The cells of HDW and oleanolic acid group almost stopped dividing, cell morphology is not complete, the floating cells more, The above phenomenon more clearly with the increase of concentration.(3) Hoechst 33342 staining showed that HDW group and oleanolic acid group appeared more strong bright blue fluorescence in nuclei. In addition compared with the solvent control group, HDW ethanol extracts of each concentration group to some extent enhance enzyme activity of Caspase-3 of Caspase-9 (P<0.01).(4) The scratch test shows that, compared with the control group, the rate of migration of 24h to 48h was significantly lower, that of HDW can effectively inhibit two-dimensional migration of HCT116.(5) Transwell assay results showed that compared with the control group,different concentration of HDW has obvious inhibitory effect on the invasion ability of HCT116 cells, the difference was statistically significant(P<0.01).(6)Soft agar colony formation assay, compared with the control group, with the increase of the concentration of HDW,has obvious inhibitory effect on HCT116 cell growth transformation ability, the difference was statistically significant (P<0.01).(7) RT-PCR results showed that compared with the control group, HDW ethanol extract significantly up-regulated the expression level of GSK-3β, down-regulated the expression level of β-catenin, TCF4, LEF1, c-Myc, cyclinD1 mRNA(P<0.01 or P<0.05).Conclusion:HDW significantly inhibited the proliferation of HCT116, can induce apoptosis,migration and invasion and can inhibit HCT116 cells, and its mechanism maybe related to the regulation of the Wnt signaling pathway.