| Objective:Hepatocellular carcinoma(HCC)is the third main cancer in the world,most patients will require several forms of chemotherapy,but HCC is resistant to chemotherapy drugs,which is one of the main causes of chemotherapy failure in patients.In this study,we examined the expression of BCAT1 in HCC tissues and HCC cell lines(HepG2 and 97H),and further investigated the role of BCAT1 in HCC chemoresistance to cisplatin(CDDP)and its underlying regulatory mechanism for CDDP resistance,which may provide a new theoretical basis for the use of ATOcombined with CDDP and targeted therapy for HCCMethods:(1)The mRNA and protein levels of BCAT1 in HCC tissues and HCC cell lines(HepG2 and 97H)and normal hepatic cells(L02)were detected by western blot analysis and qRT-PCR analysis,respectively.Student’s t-tests were used to analyze the difference of several groups.(2)HCC cells were often resistant to CDDP treatment.To verify whether high level of BCAT1 acts some role in chemoresistance to CDDP,we first use small interfering RNA to silence BCAT1 in HCC cell lines(HepG2 and 97H).And the effect of BCAT1 silencing on the cell viability of CDDP-treated HCC cells was examined by MTT assay.At the same time,the effect of BCAT1 silencing on the apoptosis of HCC cells treated with CDDP was detected by flow cytometry analysis.(3)Arsenic trioxide has a good effect in tumor therapy,so we used western blot to determine whether ATO could inhibit the expression of BCAT1 in HCC cells,and then to detect the protein level of BCAT1 in HCC cells co-treated with ATO and CDDP and its potential expression mechanism.(4)The effect of ATO combined with CDDP on the viability of HCC cells(HepG2 and 97H)cells was detected by MTT assay,the effects of BCAT1 overexpression on the cell viability of ATO combined with CDDP-treated HCC cells(HepG2 and 97H)were also examined.The effect of ATO combined with CDDP on HCC cell apoptosis was detected by flow cytometry,and the effect of BCAT1 overexpression on the apoptosis of HCC cells treated with ATO and CDDP was examined.Results:(1)Real-time PCR results showed that the mRNA level of BCAT1 in HCC tissues was significantly higher than that in para-tumor tissues,the results of immunoblotting also showed that the protein level of BCAT1 in HCC tissues was also significantly higher than that in para-tumor tissues.Then,we used detect mRNA and protein levels of BCAT1 in HCC cell lines(HepG2 and 97H)and normal hepatocytes(LO2)by real-time quantitative PCR and immunoblotting.Compared with L02 cells,the mRNA and protein levels of BCAT1 in HepG2 and 97H cells were significantly higher.(2)HepG2 cells were treated with different concentrations of CDDP for 48 hours,BCAT1 siRNA-treated cells exhibited more sensitivity to CDDP and a lower proliferation or a higher apoptosis rate than control cells.Similarly,the suppression of BCAT1 expression in 97H cells enhanced their chemosensitivity to CDDP,reduced their proliferation rate and increased their apoptosis.(3)To determine whether the BCAT1 expression could be inhibited by ATO,we treated HCC cells with 2.5,5,and 10 μM of ATO,and examined the protein expression of BCAT1 at 48 h by western blotting assay.The results showed that cells with ATO treatment significantly decreased the BCAT1 protein expression in a dose-dependent manner.Besides,5 μM of ATO treatment repressed the BCAT1 expression in time-dependent manner.It has been described previously that ATO treatment decreased c-Myc expression,which is the trans-acting factor of BCAT1 gene.So HepG2 cells were treated with 5 μM of ATO or/and 20 μM of CDDP for 48 h.Western blotting assay was conducted to measure c-myc and BCAT1 expression.The results showed that 5 μM of ATO inhibited the c-Myc and BCAT1 expression.(4)When HepG2 and 97H cells were treated with 5 μM of ATO and 20 μM of CDDP for 48 h.ATO-treated cells show a lower proliferation and less sensitivity to CDDP or a higher apoptosis rate than normal cells.However,exogenous BCAT1-overexpressing cells with ATO treatment showed a higher proliferation and less sensitivity to CDDP or a lower apoptosis rate than ATO-treated cells.Conclusion:(1)The mRNA and protein levels of BCAT1 were significantly increased in HCC tissues and HCC cell lines(HepG2 and 97H).(2)BCAT1 silencing can induce HCC cells to be more sensitive to CDDP,indicating that decreased BCATI expression can inhibit the chemosensitivity to CDDP in HCC cells.(3)The decreased BCAT1 expression by ATO treatment is partially mediated by the down-regulation of the c-myc level.(4)BCAT1-inducing chemoresistance to CDDP was partially suppressed by ATO in HCC cells,this suppression can be partially reversed by BCATI overexpression,indicating that BCATI can play a role in CDDP resistance of HCC cells.Objective:The incidence of intrahepatic cholangiocarcinoma(ICC)is after hepatocellular carcinoma(HCC),which accounting for about 10-15%of primary liver cancer。In this study,we examined the effect of ATO combined with CDDP on ICC cells,and further explore the role of BCAT1 in ICC CDDP resistance and its potential regulatory mechanism,which provide some clinical reference for ATO combined with CDDP in the treatment of ICC.Methods:(1)In order to assess the sensitivity of chemotherapeutic drugs,we treated RBE cells with 20 μM CDDP alone or in combination with 5 μM ATO for 48 hours,then the effect of arsenic trioxide combined with CDDP on the viability of ICC cells(RBE and HUCCT1 cells)was examined by MTT assay.(2)In order to verify whether BCAT1 expression has an effect on CDDP resistance in ICC,we used detect the expression of H2AX and BCAT1 expression by immunoblotting,H2AX were used to detect DNA damage in ICC cells after drug treatment.(3)To further validate the role of BCAT1 in the chemotherapy of ATO combined with CDDP,we first examined the effect of ATO combined with CDDP on HUCCT1 cell apoptosis by comet assay,then the BCAT1 of HUCCT1 cells was silenced by small interfering RNA;finally the effect of BCAT1 silencing on the apoptosis of ICC cells treated with ATO and CDDP was detected by comet assay.Results:(1)The cell viability of RBE cells treated with CDDP alone did not show a significant reduction,However,RBE cells treated by ATO combined with CDDP showed greater sensitivity to CDDP,proliferative dysfunction and cell significant decreased viability.Moreover,a similar phenomenon was found in another HUCCT1 cell,and ATO combined with CDDP treatment significantly reduced the viability of HUCCT1 cells.(2)There was no significant effect on the expression of BCAT1 and H2AX in RBE cells treated with CDD.P alone.However,ATO combined with CDDP treatment significantly induced the expression of H2AX,but also decreased the expression of BCAT1.We also used another cholangiocarcinoma cell line HUCCT1 to verify the effect of ATO combined with CDDP,the experimental results are similar to RBE cells that ATO combined with CDDP treatment significantly induced the expression of H2AX,but also decreased the expression of BCAT1.(3)In comet experiments,there are more the DNA fragments in the tail of the comet when severe DNA damage are more,the DNA damage and the degree of apoptosis can be quantitatively analyzed by measuring the length of the comet tail and the intensity of the fluorescence.The experimental results show that after treated with ATO alone or CDDP alone,The DNA damage of HUCCT cells was slightly increased compared with the control,which has no significant difference,While ATO combined with CDDP treatment can induce significant DNA damage in HUCCT1 cells.Then we silence BCAT1 by little interference to verify whether BCAT1 is involved in the DNA damage induced by ATO combined with CDDP,As shown in the figure,BCAT1 silencing increased significant comet tailing and apoptosis after treatd with ATO and CDDP in ICC cell lines.Conclusion:(1)ATO treatment made ICC cell more sensitive to CDDP,and inhibited the proliferation of RBE and HUCCT1 cells.(2)ATO combined with CDDP treatment inhibited BCAT1 protein expression and increased the expression of H2AX.(3)BCAT1 silencing can increase cell DNA damage induced by ATO in combination with CDDP chemotherapy,suggesting that BCAT1 may be involved in the cell DNA damage of ICC cells induced by ATO combined with CDDP. |
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