| Objective1.To investigate whether Hep G2 stem cell-like cells are resistant to sorafenib.2.To explore whether the combination of metformin and sorafenib at a low concentration can inhibit the proliferation of Hep G2 stem cell-like cells and study the mechanism in it.Methods1.To enrich the spheroids of hepatocellular carcinoma cell line Hep G2 stem cell-like cells with serum-free stem cell culture medium from human hepatocellular carcinoma cell line Hep G2.The morphological changes of the Hep G2 stem cell-like cells were observed under inverted microscope,and the diameter of the spheroids was measured as well as the number of the spheroids were counted after the spheroids formation.Collecting Hep G2 stem cell-like cells that have grown for 6 days after passage of 3 times and then detecting the expression of CD133 and CD90 which are markers of cancer stem cell by FACS.2.The colony forming ability of Hep G2 stem cell-like cells and Hep G2 cells was verified by soft agar assay with 0.4% upper agar gel and1% lower agar gel.The two groups of cells were planted in the upper agar gel at the concentration of 1000 cell per well,and 500?L culture medium needed for the two group was added every 3 days.To compare the colony formation ability of the two groups after culture for 13 days.3.Hep G2 cells and Hep G2 stem cell-like cells were treated with 5mM,10 mM,15mM,20mM and 25mM sorafenib for 24 h,48 h and 72 h,respectively.The cell viability was detected by CCK8 assay,and the optimum time point for drug treatment was selected according to the results.The half maximal inhibitory concentration(IC50)was calculated at the optimum time point,the none-treated group was used as the control group.And the sensitivity of the two groups to sorafenib was compared.At the same time,the morphological changes of suspension spheres of Hep G2 stem cell-like cells treated with sorafenib were observed,and three typical visual fields were selected,and the diameter and number of suspension balls were recorded under microscope.4.Hep G2 stem cell-like cells were treated with 1m M,3m M and 5m M metformin combined with 5mM sorafenib.The cell viability was detected by CCK8 assay.And the optimum time point was selected according to the results.None treatment group was regarded as control group,the cell growth inhibition rate of metformin combined with sorafenib on Hep G2 stem cell-like cells was calculated and statistically analyzed.The optimum medicine concentration and time point were selected for subsequent experiments.5.The expression of EMT related genes such as Vimentin,Twist1,Snail and E-cadherin in Hep G2 stem cell-like cells and Hep G2 cells were detected by Western blot.Hep G2 stem cell-like cells were treated with1 m M,5m M metformin for 48 hours,then the expression of EMT related genes such as Vimentin,Twist1,Snail and E-cadherin were detected by Western blot assay.The results were analyzed statistically.Results1.We could observe spheroids formation after 3 days of enriching hepatocellular carcinoma cell line Hep G2 stem cell-like cells.The diameter and number of spheroids were increasing following culture.But the number of spheroids did not increased until cultured for 6 days.The positive expression rate of CD133 molecular on the surface of Hep G2 stem cell-like cells was(5.59+0.31)%,but the positive expression rate of CD133 molecular on the surface of Hep G2 cell was only(0.33+0.32)%.And the positive expression rate of CD90 molecular on the surface of Hep G2 stem cell-like cells was(9.57+0.29)%,but the positive expression rate of CD90 molecular on the surface of Hep G2 cell was only(0.8+0.26)%,which were detected by FACS.The different expression level of CD133 and CD90 between Hep G2 stem cell-like cells and Hep G2 cells was statistically significant(t=165.3,P <0.001;t=27.99,P <0.05).2.Hep G2 stem cell-like cells and Hep G2 cells were cultured 13 days in soft agar gel.The clone formation rate of Hep G2 stem cell-like cells was(35.5~39.5)%,and the clone formation rate of Hep G2 cells was(17.2~19.0)%.The difference between the two groups was statistically significant(t=25.58,P<0.01).3.The optimum time point for sorafenib treatment of HepG2 stem cell-like cells and Hep G2 cells was 48 h.The half maximal inhibitory concentration(IC50)of sorafenib to Hep G2 cells and Hep G2 stem cell-like cells are 10.40?M and 15.10?M respectively.4.The growth inhibition rates of Hep G2 stem cell-like cells treated with 1m M?3m M?5m M metformin combined with 5?M sorafenib for 48 h were(27.73+5.52)%?(32.20+4.25)% and(35.11+4.06)% respectively.And the growth inhibition rate of Hep G2 stem cell-like cells treated with5?M sorafenib for 48 h was(22.07+2.17)%.The inhibitory rate between3 m M ? 5m M metformin combined with 5?M sorafenib and 5?M sorafenib along on Hep G2 stem cell-like cells were significantly different.(t=4.514,P<0.05;t=5.812,P<0.01).5.Compared with Hep G2 cells,the expression of EMT related gene such as Vimentin,Twist1 and Snail in Hep G2 stem cell-like cells was increased,and the expression of E-cadherin in Hep G2 stem cell-like cells was decreased,which was detected by Western blot assay.The differences were statistically significant(t=4.205,P<0.05;t=5.588,P<0.01;t=17.70,P<0.001;t=10.87,P<0.001).However,compared with the control group,treated Hep G2 stem cell-like cells with 1m M,5m M metformin,the expression of EMT related gene such as Twist1 and Snail in Hep G2 stem cell-like cells were decreased and the expression of E-cadherin in Hep G2 stem cell-like cells was increased.The expression of E-cadherin ? Vimentin were statistically different between 5m M metformin and control group.(t=6.421,P<0.01;t=6.793,P<0.01).The expression of Twist1 and Snail in Hep G2 stem cell-like cells treated with metformin at different concentrations were statistically different compared with control group.(t=4.810,P<0.05,t=7.923,P<0.01;t=6.828,P<0.01,t=17.7,P<0.001).Conclusion1.The sensitivity of Hep G2 stem cell-like cells to sorafenib is decreased.2.Metformin could promote sorafenib to inhibit Hep G2 stem cell-like cells at a low concentration.EMT may be related with it. |
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