| [Objective]:Many studies have found that artemether (ARE) and its derivatives not only have antitumor activity, but also increase tumor radiosensitivity. However, the specific mechanism of action remains unclear in many aspects. By studying the effect of ARE on radiotherapy sensitive human colorectal cancer HCT116 cells and radiotherapy outcome of chemoradioresistant HCT116-CRR cells in nude mouse transplantation tumor of human colorectal cancer, the present research was to discuss whether the potential mechanism of action is associated with the regulation of epithelial mesenchymal transition (EMT) and provide experimental evidence for using ARE as a drug of increasing colorectal cancer radiosensitivity.[Methods]:1. Establishing models of nude mouse transplantation tumor of human colorectal cancer: radiotherapy sensitive human colorectal cancer HCT116 cells and chemoradioresistant HCT116-CRR cells of human colorectal cancer were cultured.BALB/c-nu nude mouse subcutaneous transplantation tumor model of human colorectal cancer was built respectively. After the tumors grew up to about 5×5×5mm3,these nude mice were randomly divided into four groups: control, ARE, RT and RT+ARE. Treatment was given to each group;2.The tumor formation rate, survival condition and change in weight of each group were observed;3. These tumors were monitored in terms of long radius and short radius every other day to calculate the tumor volume inhibition rate and sensitization efficiency factor(EF) and draw a tumor volume change curve;4. The nude mice of each group were sacrificed to remove tumor masses before weighing them to calculate the tumor inhibition rate;5. The apoptosis rate and necrosis rate of tumor tissue of each group were determined by flow cytometry;6. The tumor mass of each group was fixed with 10% formaldehyde, embedded with conventional paraffin, sectioned and stained with HE for observation under light microscope;7.The expression of EMT-associated factors (E-cadherin, N-cadherin, Vimentin, Snail,Slug,Twist andβ-Catenin) in tumor tissue in each group was determined by immunohistochemical detection;8.The expression of E-cadherin, N-cadherin, Vimentin, Snail, Slug,Twist andβ-Catenin gene in tumor tissue in each group was determined by RT-PCR detection;9.The expression of E-cadherin, N-cadherin, Vimentin, Snail, Slug,Twist andβ-Catenin protein in tumor tissue in each group was determined by Western blot10. Statistical analysis was made by using SPSS22.0 software. The data were expressed in mean ± standard deviation (x±s). ANOVA analysis was carried on those data meeting the requirements of homogeneity test of variance, and LSD-t detection was used to make post hoc comparison; Brown-Forsythe analysis was performed on those data that did not meet the requirements of homogeneity test of variance, and Dunnett’s T3 test conducted to make post hoc comparison.[Results]:1. The tumor formation rates of HCT116 and HCT116-CRR cells in nude mice were 100%; there was no obvious difference in nude mouse weight between different groups. During treatment, nude mouse activities were obvious decreased in RT group and RT+ARE group, and their spirit was slightly poor.2. Different groups showed no obvious difference in nude mouse tumor volume of HCT116 cells before treatment, but the nude mouse tumor volume of RT+ARE was smaller than RT after treatment. The tumor inhibition rate of RT+ARE was 91.66%,obviously higher than 88.13 of RT group. The EF of ARE for HCT116 cells in nude mouse transplantation tumor was 3.20. The nude mouse tumor volume of HCT116-CRR cells showed no difference in different groups before treatment.However, after treatment, the nude mouse tumor volume of RT+ARE was smaller than those of RT, the tumor volume inhibition rate of RT+ARE was 80.84%, which was much higher than 65.04% of RT, and the EF of ARE for HCT116 cells in nude mouse transplantation tumor 2.43.3. The mean tumor weight of HCT116 cells of RT++ARE was smaller than that of RT(P<0.05), and the tumor inhibition rate of RT+ARE was 91.20%,which was much higher than 88.83%; the mean tumor weight of HCT116-CRR cells of RT+ARE was smaller than that of RT (P<0.05), and the tumor inhibition rate of RT+ARE was 83.84%, which was much higher than 67.24%.4.The apoptosis rate of HCT116 cells of RT+ARE was (41.353±2.628)%, higher than(30.47±0.738)% of RT (P<0.05), and there was no obvious difference in necrosis rate of nude mouse tumor tissue between different groups; The apoptosis rate of HCT116-CRR cells of RT+ARE was (24.280±1.883)%, higher than (12.803±0.788)%of RT (P<0.05), and there was no obvious difference in necrosis rate of nude mouse tumor tissue between different groups.5. The tumor tissue of each group was observed under light microscope after HE staining. An obvious large bleeding liquefactive necrosis area was seen in the center of nude mouse tumor tissue in the control group of HCT116 cells. There was hemocyte infiltration in the necrosis area. The necrosis area of nude mouse tissue was obviously enlarged in each group, which was the most significant in RT+ARE group.Some tumor necrosis was noted in nude mouse tumor tissue in the control group of HCT116-CRR cells and tumor mesenchymal proliferation was obvious. The necrosis area of nude mouse tissue was enlarged in different treatment groups, which was the most significant in RT+ARE group.6. Expression of EMT-associated factors in nude mouse tumor tissue in each group by IHC: In the nude tumor tissue of HCT116 cells in each group, epithelial marker E-cadherin and β-Catenin were strongly positively expressed, and the expression of the two in RT+ARE was up-regulated compared with RT group; mesenchymal marker N-cadherin was negatively expressed in each group, Vimentin, Snail, Slug and Twist were weakly positively expressed,and the expression of the above in RT+ARE was down-regulated compared with RT group. In the nude tumor tissue of HCT116-CRR cells in each group, epithelial marker E-cadherin was negatively expressed in RT and weakly positive expressed in RT+ARE, β-Catenin was weakly positive expressed in each group, andβ-Catenin in RT group was upregulated compared with that in RT+ARE; mesenchymal marker N-cadherin and Slug were weakly positive expressed in each group and Vimentin, Snail and Twist were strongly positively expressed in each group, and the above levels in RT+ARE was down-regulated compared with RT.7. Relative gene expression of EMT-associated factors in nude mouse tumor tissue in each group by RT-PCR: Compared with RT, the gene expression of β-Catenin in nude mouse tumor tissue of HCT116 cells in RT+ARE was increased than RT (P<0.05),while the gene expression of Vimentin in RT+ARE was decreased than RT (P<0.05).Compared with RT, the gene expressions of E-cadherin and β-Catenin in nude mouse tumor tissue of HCT116 cells in RT+ARE were increased than RT (P<0.05), while those of N-cadherin, Vimentin, Snail, Slug and Twist were decreased than RT(P<0.05).8. Protein expression of EMT-associated factors in nude mouse tumor tissue in each group by Western blot: Compared with RT, the protein expression of β-Catenin in nude mouse tumor tissue of HCT116 cells in RT+ARE was increased than RT(P<0.05), while that of Vimentin in RT+ARE was decreased than RT (P<0.05).Compared with RT, the protein expression of E-cadherin and β-Catenin in nude mouse tumor tissue of HHCT116-CRR cells in RT+ARE were increased than RT(P<0.05), while those of N-cadherin, Vimentin, Snail, Slug and Twist were decreased than RT (P<0.05).[Conclusion]:1. ARE and radiotherapy can obviously inhibit the growth of HCT116 cells in human colorectal cancer and HCT116-CRR cells in nude mouse transplantation tumor, thus enhancing its radiotherapy effect; ARE and radiotherapy can obviously inhibit the growth of HCT116-CRR cells in nude mouse transplantation tumor, thus enhancing its radiotherapy effect and reversing its chemoradiotherapy resistance.2. ARE and radiotherapy can promote the apoptosis and necrosis of HCT116 cells in human colorectal cancer and HCT116-CRR cells in nude mouse transplantation tumor,thus enhancing. its radiotherapy effect; ARE and radiotherapy can promote the apoptosis and necrosis of HCT116 cells in nude mouse transplantation tumor, thus enhancing its radiotherapy effect and reversing its chemoradiotherapy resistance.3. ARE and radiotherapy can upregulate the expression of epithelial marker P-Catenin in HCT116 cells in nude mouse transplantation tumor of human colorectal cancer and down-regulate the expression of mesenchymal marker Vimentin, suggesting that ARE may enhance the radiotherapy effect on HCT116 cells in nude mouse transplantation tumor of human colorectal cancer by blocking EMT.4. ARE and radiotherapy can upregulate the expression of epithelial marker E-cadherin and β-Catenin and down regulate the expression of N-cadherin, Vimentin,Snail, Slug and Twist in HCT116-CRR cells in nude mouse transplantation tumor of human colorectal cancer, suggesting that ARE may enhance the radiotherapy effect on HCT116-CRR cells in nude mouse transplantation tumor of human colorectal cancer and reverse its chemoradiotherapy resistance by reversing EMT.5. ARE can enhance the radiotherapy effect on nude mouse transplantation tumor of human colorectal cancer. Thus, it may become a radiation-sensitizing agent with high efficiency and low toxicity and be used in the radiotherapy of colorectal cancer. |
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