Effect Of Tetrahydrocurcumin On Proliferation、apoptosis And Metastasis Of MCF-7/CT26 Tumor Cells

Objective:In this paper,^A we^B sc^Sree^Tne^Rd th^Ae a^Cntit^Tumor effect of THC in vitro,and identified that THC showed tumor suppresion effect to MCF-7 cells of breast cancer and CT26 cells of colon cancer in vitro,and investigated the effect and mechanism of THC in breast cancer cells（MCF-7）and colon cancer cells（CT26）on the proliferation,apoptosis and metastasis,laying the experimental foundation of THC for the development and clinical research of anti-tumor drugs.Methods:The first part was about the effect of THC on proliferation,apoptosis and metastasis in breast cancer cells.The anti-tumor effect of THC on breast cancer cells in vitro was studied by CCK8 and clone formation experiment,and we screened the experimental cells,and determined the drug administration experiment and concentration by calculating IC50.THC in vitro intervention was performed to observe the morphological changes of breast cancer cells.Using Hoechst staining and flow cytometry observed the apoptotic morphology of breast cancer cells and calcutated the number of apoptotic cells.The expression levels of apoptosis-related proteins Bcl-2 and Bax in cells treated with THC were detected by Western Biotting.Scratch test was used to investigate the effect of breast cancer cells in inhibiting metastasis,matrigel invasion experiment crystal violet staining,taking pictures and counting conducted to observe the invasion and metastasis of breast cancer cells.After THC intervention,the protein expression levels of metastasis-related proteins MMP2,MMP9,E-cadherin,N-cadherin,and Vimentin were detected by Western Blotting.The second part was the effect of Tetrahydrocurcuminoids（THC）on the proliferation,apoptosis and metastasis in colon cancer cells.The anti-tumor effect of THC on colon cancer cells in vitro was studied by CCK8 and clone formation experiment,and we screened the experimental cells,and determined the drug administration experiment and concentration by calculating IC50.THC in vitro intervention was performed to observe the morphological changes of colon cancer cells.Using Hoechst staining and flow cytometry observed the apoptotic morphology of colon cancer cells and calcutated the number of apoptotic cells.The expression levels of apoptosis-related proteins Bcl-2 and Bax in cells treated with THC were detected by Western Biotting.Scratch test was used to investigate the effect of colon cancer cells in inhibiting metastasis,matrigel invasion experiment crystal violet staining,taking pictures and counting conducted to observe the invasion and metastasis of colon cancer cells.After THC intervention,the protein expression levels of metastasis-related proteins MMP2,MMP9,E-cadherin,N-cadherin,and Vimentin were detected by Western Blotting..The inhibition of THC on tumor growth was studied in Balb/c mice by heterotopic inoculation of CT26 cells from colon cancer.Results:The first part was about the effect of THC on proliferation,apoptosis and metastasis function in breast cancer cell.Contrasted with blank control group,THC groups showed stronger inhibitory effect on MCF-7 cells in cell proliferation experiment,which different concentrations of THC dose groups decreased cell survival rate,the clone formation inhibition rate compared with the blank control group difference with statistically significant.apoptosis related experiments show that THC dose groups showed that after 48h cells growthed slowly,cells morphological changeed and the cells decreased,intracellular appeared cavitation,Large amounts of cells debried float on the surface of the culture medium.Hoechst 33254 staining test showed that the nuclear fluorescence intensity of THC dose groups was significantly stronger than that the normal cells.Contrasted with blank control group,there were obvious apoptotic characteristics:nuclear fragmented and showed dotted blue fluorescence,nuclear chromatin was aggregated and marginalized,the apoptotic bodies appeared,the difference with statistically significant.Flow cytometry assay showed that THC dose groups could promote the apoptosis of MCF-7 breast cancer cells,and the difference with statistically significant.Western blotting（WB）assay showed that THC dose groups could up-regulate the expression of Bax and down-regulate the expression of Bcl-2 protein,compared with blank control group,the difference with statistically significant.In the metastasis-related experiment,the scratch experiment showed that after THC dose groups treatment for 24h,compared with the control group,the cell migration rate at 24h decreased significantly.Compared with the control group,MCF-7 breast cancer cells migrated to the edge of the scratch area toward the middle of the scratch area.,the migration rate of cells on THC dose groups at 48 h with significantly decreased.The migration and invasion experiments showed that after THC dose groups treatment for 48 h,the number of cells were significantly reduced which penetrated the septum to the bottom of the upper compartment,compared with the blank control group,the difference was statistically significant.Western blotting assay showed that:Contrasted with blank control group THC dose groups could significantly inhibited the expression of MMP2,MMP-9,Vimentin,N-cadherin.the THC dose groups promoted the expression of E-cadherin,the difference with statistically significantThe second part was about the effect of THC on proliferation,apoptosis and metastasis function in colon cancer cell.Contrasted with blank control group,THC groups showed stronger inhibitory effect on CT26 cells in cell proliferation experiment,which different concentrations of THC dose groups decreased cell survival rate with statistically significant.The plate clone formation experiment showed that different concentrations of THC dose groups had inhibitory effect on CT26 colon cancer cells,compared with the blank control group,the difference was statistically significant,.Apoptosis results showed that after THC dose groups for48 h,the cells growthed slowly,CT26 cells decreased,the cells changed those form,and theintracellular vacuoles appeared,and a large number of cell debris floated on the surface of the culture medium.Hoechst 33254 staining test showed that the nuclear fluorescence intensity of THC dose groups was significantly stronger than the blank control group.Apperaed obvious apoptotic features:nuclear chromatin aggregated and gradually marginalized,nuclear rupture showed round dot blue fluorescence and apoptotic corpuscle,as the concentration changes,there are statistical differences.Annexin V-FITC/DAPI THC dose group could promote the apoptosis of CT26 cells,contrasted with blank control group,the difference with statistically significant.Western blotting（WB）assay showed that THC could up-regulate the expression of Bax and down-regulate the expression of Bcl-2 on CT26colon cancer cells,Contrasted with blank control group,the difference with statistically significant.On the migration related experiments.At 48 h,the migration distance of cells in THC groups were significantly shortened and the scratch gap became wider.Compared with the control group,which showed significantly decreased.According to the migration and invasion experiments,THC dose groups treated after 48h,compared with the blank control,group reduced the number of cells penetrated the septum arrived the bottom,and the difference with statistically significant.Western blotting（WB）showed that THC dose groups was significantly different from the blank control group.THC inhibited the expression of MMP2 and MMP9,indicating that THC could inhibit the metastasis of CT26 cells.The expression of Vimentin was inhibited by THC,have statistically significant.THC could also inhibit the expression of N-cadherin,whice apperaed statistical significance between THC and blank control group.THC promoted the expression of E-cadherin,and there was statistical significancewith the dose range of 25.00、50.00 and 100.00μmol·L^-1.All of experiments showed that THC could inhibit CT26 cell metastasis.The results of the THC in vivo on CT26 cell transplantation tumor Balb/c condition and body tumor quality,.compared with model group,THC（800 mg/kg）group Balb/c weight lower after 9 days which had treatment,12 days appeared significantly difference.According to the result with tumor volume when 6 days after the first dose,compared with the model group,THC（800,400 mg/kg）groups’toumor volume significantly decreased,12 daystoumor volume was decreased at THC（200mg/kg）,Compared with model group,the difference with statistically significant.,THC（800mg/kg,400mg/kg）groups’tumor mass significantly decreased,Compared with model group.Conclusions:THC can inhibit the proliferation and metastasis of MCF-7、CT26cancer cells,and promote the apoptosis of MCF-7、CT26 cancer cells.The mechanism of THC may be to inhibit the migration of MCF-7、CT26 cancer cells by inhibiting the expression of EMT.The results of this study may as a experimental basis for further studies about prevention and/or treatment of metastasis of colon cancer.