Effect And Mechanism Of Artemether On The Reversal Of Chemoradiotherapy Resistance In Colorectal Cancer

Objectives:1.To investigate the effect of artemether on the reversal of chemoradiotherapy resistance in colorectal cancer.2.To investigate the mechanism of artemether reversal of chemoradiotherapy resistance in colorectal cancer.3.To investigate the changes of biological behavior of chemoradiotherapy resistant cells in colorectal cancer compared with their parents.Methods:1.HCT116CRR and HT29CRR cells were divided into 4 groups:A.The control group;B.Artemether group;C.Chemoradiotherapy group;D.Artemether combined with chemoradiotherapy group.2.CCK8 and clone formation assay were used to determine the cell activity and clone formation ability of each group.3.Cell apoptosis and cell cycle arrest were measured by flow cytometry.4.EMT-associated proteins E-cadherin,N-cadherin,Vimentin and Snail,apoptosis related proteins Bax and Bcl-2,Wnt/β-catenin signaling pathway related proteinsβ-catenin,c-Myc and cyclinDl were measured by Western Bolt in each group after treatment.5.HCT116CRR/HT29CRR cells were injected into the right back of the nude mice to establish the subcutaneous tumor model.After the tumor volume reached about 100mm3,the nude mice were randomly divided into four groups:A.The control group;B.Artemether group;C.Radiotherapy and chemotherapy group;D.Artemether combined with chemoradiotherapy group.6.The body weight and tumor length and diameter of the nude mice were measured every 3 days after grouping treatment,and the tumor volume was calculated,and the curve of tumor weight and volume growth was plotted.At the end of treatment(21 days later),the cervical vertebra dislocation of nude mice in each group was sacrificed,the tumor tissue was removed,the tumor weight was weighed,and the tumor inhibition rate was calculated.7.Tumor tissue apoptosis was measured by flow cytometry.8.EMT-associated proteins E-cadherin,N-cadherin,Vimentin,Snail,apoptosis related proteins Bax and Bcl-2,β-catenin,a key factor in the Wnt/β-catenin signaling pathway,and downstream targets C-MYC and CyclinD1 were measured by Western Bolt and immunohistochemical in each group after treatment.9.HCT116-Luc and HCT116CRR-Luc cells labeled with luciferase were constructed.HCT116-Luc and HCT116CRR-Luc cells were injected through the tail vein to establish the lung metastasis model of colorectal cancer.Changes in body weight of nude mice were observed and recorded.In vivo imaging was used to compare lung metastases of each group,and the number of lung metastatic nodules of different cells was counted after death of nude mice.10.Hepatic metastasis model of colorectal cancer was established by injecting HCT116-Luc and HCT116CRR-Luc cells into the spleen.Weight changes of nude mice were observed and recorded.In vivo imaging was used to observe the luminescence intensity and compare the liver metastasis of different tumor cells.Results:1.Effect and mechanism of artemether on in vitro reversal of chemoradiotherapy resistance and sensitization in colorectal cancer.(1)The results of CCK8 and clone formation showed that artemether had a certain inhibitory effect on the proliferation of chemoradiotherapy resistant cells in colorectal cancer.The combination of artemether and chemoradiotherapy could significantly enhance the sensitivity of cells to chemoradiotherapy,showing a higher inhibition of cell proliferation.(2)The apoptosis rate of HCT116CRR cells was as follows:control group(1.97±0.06)%,artemether group(6.39±0.26)%,chemoradiotherapy group(8.56±0.38)%,and artemether+chemoradiotherapy group(22.93±1.20)%,respectively.The apoptosis rate of HT29CRR cells in each group was(1.92±0.09)%in the control group,(5.75±0.10)%in the artemether group,(8.21±0.22)%in the chemoradiotherapy group,and(16.39±0.71)%in the artemether+chemoradiotherapy group.Compared with the control group,the apoptosis rate of HCT116CRR and HT29CRR cells was increased in the artemether group,the chemoradiotherapy group and the artemether combined chemoradiotherapy group,and the apoptosis rate of the artemether combined chemoradiotherapy group was the highest(P<0.05).(3)Flow cytometry showed that the ratio of G2/M phase in HCT116CRR cells was(17.41±2.53)%in control group,respectively.Artemether group(6.53±2.61)%;Radiotherapy and chemotherapy group(31.09±3.50)%;Artemether+chemoradiotherapy group(9.71±2.34)%.The proportion of HT29CRR cells in G2/M phase was(17.68±1.47)%in control group,respectively.Artemether group(9.35±0.78)%;Radiotherapy and chemotherapy group(27.88±1.93)%;Artemether+chemoradiotherapy group(27.88±1.93)%.The cells in the control group and the chemoradiotherapy group showed higher G2/M phase arrest,the artemether group was compared with the control group,the artemether combined with chemoradiotherapy group decreased the proportion of G2/M phase arrest,and reversed the G2/M phase arrest(P<0.05).(4)Western Bolt analysis of EMT-associated proteins showed that the expression of N-cadherin,Vimentin and Snail was increased in the chemoradiotherapy group compared with the control group.The expression of E-cadherin was increased in the artemether group and the artemether combined with chemoradiotherapy group,while the expression of N-cadherin,Vimentin and Snail were decreased(P<0.05).(5)Western Bolt assay of apoptosis-related proteins showed that,compared with the control group,the expression of Bax was increased in the artemether group,the chemoradiotherapy group and the artemether combined with chemoradiotherapy group,while the expression of Bcl-2 was decreased,and the effect of artemether combined with chemoradiotherapy group was the most obvious(P<0.05).(6)Western Bolt assay of Wnt/β-catenin pathway related proteins showed that the expressions of β-catenin,c-myc and cyclid 1 were significantly decreased in the artemether group compared with the control group.Compared with the chemoradiotherapy group,the artemether combined with chemoradiotherapy group also significantly inhibited the expression of β-catenin,c-myc,and cyclinD1(P<0.05).2.Effect and mechanism of artemether on internal and external reversal of chemoradiotherapy resistance in colorectal cancer.(1)There was no significant difference in body weight between the groups during treatment.In HCT116CRR cell transplantation tumor,the tumor inhibition rates of each group were as follows:artemether group:29.82%,radiotherapy and chemotherapy group:52.73%,artemether combined with radiotherapy and chemotherapy group:76.73%;In the HT29CRR cell transplantation tumor,the tumor inhibition rates of each group were:artemether group:29.92%,radiotherapy and chemotherapy group:47.94%,artemether combined with radiotherapy and chemotherapy group:66.72%.(2)Flow cytometry results showed that compared with the control group,the artemether group,the chemoradiotherapy group and the artemether combined chemoradiotherapy group all increased the rate of tumor tissue apoptosis,and the artemether combined chemoradiotherapy group had the highest rate of apoptosis(P<0.05).(3)Western Bolt and immunohistochemical measurements of EMT,apoptosis,and Wnt/β-catenin pathway related protein expression were consistent with those in vitro.Artemether can promote the expression of E-cadherin,inhibit the expression of N-cadherin,Vimentin and Snail,and inhibit EMT.Promote the expression of Bax,inhibit the expression of Bcl-2,promote apoptosis;The expression ofβ-catenin,c-myc and Cyclidl was inhibited,and the Wnt/β-catenin pathway was inactivated(P<0.05).3.The luminescence intensity of liver metastases in the two groups was as follows:HCT116-Luc(1.97± 0.68)×108 p/s/Sr,HCT116Cr-Luc(4.17± 1.11)×108 p/s/Sr,(P<0.05),the luminescence intensity of lung metastases were as follows:HCT116-Luc(1.85±0.29)×108 P/s/Sr,HCT116Cr-Luc(2.63± 0.33)×108 p/s/Sr,(P<0.05).The results showed that radio-chemoradiotherapy resistant cells of colorectal cancer showed stronger ability of liver and lung metastasis in vivo than their parent cells.Conclusions:1.Artemether can inhibit proliferation,promote apoptosis,reverse G2/M phase arrest,inhibit EMT,and reverse chemoradiotherapy resistance of colorectal cancer.2.The effect of artemether on the reversal of chemoradiotherapy resistance may be realized through the inactivation of Wnt/β-catenin signaling pathway.3.Radio-chemoradiotherapy resistant cells of colorectal cancer have stronger ability of liver metastasis and lung metastasis in vivo than their parent cells.