| ObjectiveCancer stem cells(CSCs)are the main cause of tumor recurrence,metastasis and heterogeneity.According to the biological characteristics of cancer stem cells and their related signaling pathways,it is found that the regulation of stem cell therapy targets and the targeted elimination of tumor stem cells have become the research hotspot of new antitumor strategies.Hippo-YAP protein is the core molecule that maintains stem cell dryness,which connects Hippo signaling pathway with other stem cell regulatory pathways such as Wnt,hedgehod and Notch.Its loss or activity inhibition of its downstream effector protein YAP / TAZ can lead to stem cell loss of stemness.Quyu detoxification soup is the first Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine Cancer Center for the treatment of intestinal cancer side(patent application),has good clinical efficacy,strong inhibition of colorectal cancer cell proliferation.Previous studies have confirmed that in normal colorectal tissue,nuclear YAP is negatively expressed in the cytoplasm,but YAP in cancer tissue is positive in nucleus and cytoplasm,and YAP is consistent with colorectal cancer stem cell marker CD133.And preliminary clinical studies confirmed that YAP and recurrence and metastasis of colorectal cancer have a certain correlation.In addition,preliminary basic research has been done by in vitro stem cell isolation and culture,obtaining and identifying stem cell-specific colon cancer stem cells.We speculate Quyu Jiedu Decoction can interfere with colon cancer metastasis by regulating the expression of core protein YAP in Hippo signaling pathway.In this study,the effect of Quyu Jiedu Decoction on the proliferation,migration and invasion of human colon cancer HCT116 stem cells was observed in vitro,and the expression of YAP protein in Hippo signaling pathway was detected to explore whether Quyu Jiedu Decoction could regulate Ypp Protein to interfere with the mechanism of colon cancer stem cell metastasis in order to reveal the mechanism of traditional Chinese medicine anticancer metastasis to provide theoretical support and experimental evidence.MethodsHuman colon cancer HCT116 primary cells were cultured in serum-free medium and cultured for 12-14 days to form HCT116 stem cell clone pellets that were visible to the naked eye.CCK8 method was used to observe the inhibitory effect of simple Quyu Jiedu Decoction,simple chemotherapy drugs and Quyu Jiedu Decoction combined with chemotherapy drugs on cell proliferation of human colon cancer HCT116 cells.Flow cytometry analysis of colon cancer stem cell markers CD44,CD133 expression rate.The effects of different drug groups on migration and invasion of human colon cancer HCT116 stem cells were examined by in vitro matrigel migration and invasion assays.Immunofluorescence,western-mot method to explore Quyu Jiedu Decoction of colon cancer HCT116 stem cells YAP protein expression.Results1.CCK-8 experiment,compared with the blank control group,different experimental groups(75mg/ml Quyu Jiedu Decoction group,125mg/ml Quyu Jiedu Decoction group,2mg/ml 5-fu group and 75mg/ml cured The results showed that the inhibitory effect of various experimental groups on the proliferation ability of HCT116 cells in a concentration-dependent manner was the same as that of the other two groups in the treatment of HCT116 colorectal cancer cells at 24 h,48h and 72 h Time,With the increase of drug concentration,cell survival rate decreased gradually,the difference was statistically significant(p <0.01).At the same time,it was also observed that with the prolongation of drug action,the cell viability decreased gradually However,there was no obvious time dependence.In other words,there was no significant difference in cell viability between experimental groups with the same concentration of different time periods(P> 0.05).(2)Compared with the positive experimental control group(2mg/ml 5-fu group),75mg/ml Quyu Jiedu Decoction group,125 mg /ml Quyu Jiedu Decoction group and 75mg/ml Quyu Jiedu Decoction+2 mg/ml 5-fu group)intervention of HCT116 colorectal cancer stem cells 24 h,48h,72 h,found that different time periods under the simple Chinese medicine group(75mg/ml Quyu Jiedu Decoction group,125mg/ml Quyu Jiedu Decoction group)and Chinese medicine combined chemotherapy group 75mg/ml Quyu Jiedu Decoction + 2mg/ml 5-fu group)on the proliferation of human colon cancer stem cell HCT116 cells were stronger than the chemotherapy alone group(2mg/ml 5-fu group),the difference was statistically significant Significance,(p<0.01).(3)The intervention of traditional Chinese medicine group(75mg/ml Quyu Jiedu Decoction group,125mg/ml Quyu Jiedu Decoction group)and traditional Chinese medicine combined chemotherapy group(75mg/ml Quyu Jiedu Decoction + 2 mg/ ml 5-fu group)HCT116 stem cells 24 h,48h,72 h,the results showed that 24 h,48h,72 h different periods 75mg/ml Quyu Jiedu Decoction group and 75mg/ ml Quyu Jiedu Decoction + 2mg/ml 5-fu group on cell viability The difference was statistically significant(P<0.01,125mg/ml Quyu Jiedu Decoction group and 75mg/ml Quyu Jiedu Decoction +2 mg/ml 5-fu group on cell viability was no significant difference,P>0.01.2.Flow cytometer to detect the effect of different experimental groups on the expression of CD133CD44,a marker of HCT116 stem cell of colon cancer.After 75mg/ml Quyu Jiedu Decoction,125mg/ml Quyu Jiedu Decoction,2mg/ml 5-fu and 75mg/ml Quyu Jiedu Decoction +2 mg/ml 5-fu treatment of HCT116 stem cell markers were significantly reduced compared with the blank control group.Compared with the positive control group(2 mg/ml 5-fu),the stem cell markers in the traditional Chinese medicine group and the combination chemotherapy group were all decreased,with statistical significance(P<0.01).Compared with traditional Chinese medicine and chemotherapy alone group,the effect of 75 concentration group and 75 + fu concentration group on stem cell markers had significant difference,P<0.01.125 concentration group and 75 + fu concentration group had no significant difference on stem cell marker The difference was not statistically significant(P>0.01).3.In vitro Transwell migration experiments,the 75mg/ml Quyu Jiedu Decoction,125mg/ml Quyu Jiedu Decoction,2mg/ml 5-fu and 75mg/ml Quyu Jiedu Decoction +2 mg/ml 5-fu treated intestine The number of HCT stem cells and the number of cells that migrated to the lower compartment under high magnification were significantly decreased compared with the blank control group(P<0.01).75mg/ml Quyu Jiedu Decoction group,125mg/ml Quyu Jiedu Decoction group and 75mg/ml Quyu Jiedu Decoction + 2 mg/ml 5-fu group compared with 2mg/ml 5-fu group,75mg/ml stasis There was no significant difference between Jiedu Decoction group and 2-mg/ml 5-fu group,P> 0.01,p<0.01,125 mg /ml Quyu Jiedu Decoction group compared with 2mg / ml 5-fu group,the difference was not statistically significant p>0.01,75mg/ml Quyu Jiedu Decoction +2 mg /ml 5-fu group compared with 2mg/ml 5-fu group,the difference was statistically significant,p<0.01.4.Transwell invasion assay in vitro,the 75mg/ml Quyu Jiedu Decoction,125mg/ml Quyu Jiedu Decoction,2 mg/ml 5-fu and 75mg/ml Quyu Jiedu Decoction +2 mg/ml 5-fu treated intestine The number of HCT stem cells and cells passing through the lower chamber under high magnification were significantly decreased compared with the blank control group.75mg/ml Quyu Jiedu Decoction group,125mg/ml Quyu Jiedu Decoction group(and 75mg/ml Quyu Jiedu Decoction +2 mg/ml 5-fu group compared with 2mg/ml 5-fu group,75mg/ml cured Compared with 2-mg/ml 5-fu group,there was no significant difference in Yu-Xu Jie Du Decoction group,P>0.01,125mg/ml p>0.01,75mg/ml Quyu Jiedu Decoction +2 mg /ml 5-fu group compared with 2mg/ml 5-fu group,the difference was statistically significant,p<0.01.5.Flow cytometry analysis of different experimental groups on the expression of E-cadherin marker protein in the experiment,the 75mg/ml Quyu Jiedu Decoction,125mg/ml Quyu Jiedu Decoction,2 mg/ml 5-fu and 75mg/ml stasis Jiedu decoction +2 mg/ml 5-fu EMT marker protein E-cadherin marker significantly decreased compared with the blank control group.Compared with the positive control group(2 mg/ml 5-fu),EMT marker E-ca markers in the traditional Chinese medicine group and traditional Chinese medicine combination chemotherapy group were all decreased,the difference was statistically significant,P<0.01.Compared with traditional Chinese medicine and chemotherapy alone group,the effect of EMT marker E-cadherin was significantly different between 75 concentration group and 75 concentration group,P<0.01.125 concentration group and 75 + fu concentration group There was no significant difference in the EMT marker E-cadherin marker(P>0.01).6.The result of immunofluorescence showed that the YAP protein of colon cancer stem cell HCT116 in the blank control group showed bright green fluorescence in the cytoplasm,cell membrane and nucleus,showing strong expression.However,the YAP protein of the colon cancer stem cell HCT116 in the blank control group was stained by 75mg/ml Quyu Jiedu Decoction and 125 mg /ml After treatment with detoxification decoction,2 mg/ml 5-fu and 75 mg/ ml Quyu Jiedu Decoction + 2mg/ml 5-fu for HCT116 colon cancer cells,compared with the blank control group,ml group,2 mg/ml 5-fu group and 75 mg/ml Quyu Jiedu Decoction + 2 mg/ml 5-fu group,the cytoplasm,plasma membrane and nucleus of the low concentration group showed more blank control group Weak green fluorescence,of which 125mg/ml Quyu Jiedu Decoction 75mg/ml Quyu Jiedu Decoction + 2mg/ml 5-fu group compared with the control group immunofluorescence was significantly weakened.This shows that,YAP expression in colon cancer HCT116 stem cells located in the cell membrane,cytoplasm and nucleus,after the treatment of different groups of drugs,whether membrane,cytoplasm or nucleus were some extent weakened.7.Western-Blot results showed that after treated with 75mg/ml Quyu Jiedu Decoction,125mg/ml Quyu Jiedu Decoction,2mg/ml 5-fu and 75mg/ml Quyu Jiedu Decoction + 2mg/ml 5-fu treatment of colon cancer HCT116 stem cells Compared with the blank control group,the expression of YAP protein,which is closely related to HIPPO signaling pathway,was significantly decreased.Compared with the positive control group 2mg/ml 5-fu,YAP protein expression in 125mg/ml Quyu Jiedu Decoction group and 75mg/ml Quyu Jiedu Decoction+2mg/ml 5-fu group was significantly less.Conclusion1.In vitro study,we found that Quyu Jiedu Decoction can significantly inhibit the growth and proliferation of colon cancer HCT116 stem cells in a concentration-dependent manner,and also found that Quyu Jiedu Decoction can significantly inhibit colon cancer stem cell migration and invasion ability.2.Quyu Jiedu Decoction may interfere with the metastasis of colon cancer by inhibiting the proliferation,migration and invasion of colon cancer stem cells by inhibiting the expression of core protein YAP in hippo signaling pathway.3.We also found that Quyu Jiedu Decoction combined with chemotherapy drugs can achieve a better inhibitory effect on the proliferation,migration and invasion of colon cancer stem cells. |
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