| The human mortality caused by cancer is becoming a serious global health issue.Recent studies have found that cancer stem cells(CSCs)were responsible for tumor progression,metastasis and cancer recurrence.CSCs,a small subset in solid tumor,possess the characteristics of migration and invasion,self-renewal,drug-resistant and strong tumorigenicity.CSCs are liable to lead to anti-radiotherapy(chemotherapy),cancer relapse and metastasis development.Compared with traditional two-dimensional monolayer culture,the three-dimensional(3D)culture can effectively improve the expression of CSCs properties and more closely recapitulate the dimensionality of the tumor microenvironment.Therefore,3D culture is one of the ideal models to research CSCs in vitro.Epigallocatechin-3-gallate(EGCG),one of the major natural active ingredients in green tea,possess the anti-carcinogenic pharmacological effects of inhibiting proliferation and inducing apoptosis in different cancer cells.Therefore,based on the 3D culture system,this study explores the effects of EGCG on the migration and self-renewal of CSCs in breast and liver cancer,and draws the following conclusions.1.3D culture system of breast cancer cell line MCF-7 was constructed by collagen scaffold material.MTT assay was used to detect the inhibitory effect of EGCG on the proliferation of breast cancer CSCs.There was not significant difference on the proliferation of breast cancer CSCs when the concentration of EGCG was less than 80μM(P>0.05).However,the proliferation of CSCs was significantly inhibited if the concentration of EGCG was higher than 80μM(P<0.05).The results of q RT-PCR assay showed that the expression of mesenchymal related genes,such as Snail,Twist1,ZEB1and so on,was down-regulated following EGCG treatment in breast cancer CSCs.Meanwhile,EGCG could promote the expression of epithelial marker gene,E-Cadherin.The wound healing and Transwell assay further showed that EGCG had a significant inhibitory effect on the migration of CSCs in breast cancer.At the same time,EGCG could down-regulate the level of Mrna expression of stem cell marker genes in breast cancer CSCs by q RT-PCR assay.And EGCG could reduce the number of tumor spheroid formation and clone formation of CSCs in breast cancer cells(P<0.05).These was indicated that EGCG could inhibit self-renewal ability of CSCs.In addition,flow cytometry results showed that EGCG significantly reduced the content of CD24~-like cells in breast cancer MCF-7(P<0.05).2.Porous 3D scaffolds were prepared from porous liver tissue.There were no cell residues in the 3D scaffolds by HE and DNA assay.The hepatoma cell Hep G2 were inoculated on three-dimensional scaffolds for three-dimensional culture.The the state of cells growth was observed by stereomicroscope and laser confocal microscopy.MTT assay was used conform that the 3D scaffolds were suitable for the growth of Hep G2cells.The results of q RT-PCR assay showed that the m RNA expression levels of migration-related genes and stem marker genes were up-regulated in 3D Hep G2.Then,the number of penetrating cells,spheroid formation and clone formation were increased significantly.These results were indicated that the 3D scaffolds was an ideal material for studying liver cancer CSCs in vitro.MTT assay was used to investigate the effect of EGCG on the proliferation of hepatocellular carcinoma CSCs.It was found that the proliferation of hepatocellular carcinoma CSCs was not significantly inhibited if the concentration of EGCG fewer 80μM(P>0.05).But,the significant inhibitory of proliferation was observed when the concentration of EGCG was higher than 80μM in Hep G2 CSCs(P<0.05).q RT-PCR assay showed that EGCG can down-regulate the expression of migratory genes in Hep G2 CSCs.The number of penetrating cells was decreased in Hep G2 CSCs following EGCG treatment,indicating that EGCG had an inhibitory effect on the migration ability of liver cancer CSCs.At the same time,the results of q RT-PCR showed that EGCG can inhibit the m RNA expression level of stem marker gene of liver cancer CSCs.In addition,EGCG was able to inhibit the number of spheroid formation and clone formation in liver cancer CSCs(P<0.05),indicating that EGCG could inhibit the self-renewal ability of CSCs.This study further determined the inhibitory effect of EGCG on CSCs in 3D by exploring the effects of EGCG on CSCs migration and self-renewal in breast cancer and liver cancer.These data laid a foundation for subsequent step in mice vivo experiments,and provided theoretical basis for the application of EGCG in clinical treatment of cancer and the development of new drugs. |
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