5-Fu Preferably Induces Apoptosis In Braf V600E Colorectal Cancer Cells Via Down-Regulation Of Bcl-xL

The overwhelming majority?>95%?of BRAF mutations in mCRC occur in codon600,involving a T1799A transversion in exon 15,which results in the substitution of a valine amino acid for a glutamic acid?V600E mutation?.Indeed,overall survival?OS?was only 21 months in BRAFV600E-mutant patients,whereas it was 39.4 months in patients with non-V600E BRAF mutations.In this study,We selected a variety of different types of drugs and natural active components to test whether there was variation of sensitivity of paired isogenic colorectal cancer cell lines RKO with either BRAF WT?+/-?or mutant?600E/-?.Previous research has shown only low concentration of 5-FU can induce apoptosis the BRAF mutant colon cells while the BRAF wild type colon cancer was uniformity.5-FU as main backbone of the first-line treatment of patients with colon cancer shows a mighty efficacy with consociation of oxaliplatin and irinotecan in the clinic trial.However,it is known little about the efficacy of 5-FU alone in colorectal cancer patients with BRAFV600E.In this study,we aim to investigate the sensitivity of paired isogenic colorectal cancer cell lines RKO with either BRAF WT?+/-?or mutant?600E/-?to 5-FU in vitro and in vivo to further elucidate its underlying functioning patterns.Objectives:The aim of study was to ascertain that 5-FU induces marked apoptosis in BRAF mutant colorectal cancer cells.Then,determine the effect of Bcl-xL on the sensitivity of 5-FU in the cells with BRAFV600E.It describes mechanism of 5-FU on BRAF mutant CRC cells as potential supports for treatment of CRC patients with BRAF V600E.Methods and results:1.BRAF V600E mutant colorectal cancer cells are more sensitive to Fluorouracil?5-FU?.One pair of isogenic colorectal cancer cell lines RKO with either WT?+/-?or mutant?600E/-?BRAF was treated with 5-FU or different types of drugs and natural active components to determine inhibition of cell proliferation and calculate IC₅₀ by MTT colorimetric assay.And then,explore that how BRAF mutation regulate the process of 5-FU-induced apoptosis tested by Flow Cytometry and Hoechst 33258staining.These results indicate that,in contrast to resistance to many therapeutic drugs,BRAF mutant colon cancer cells are sensitive to 5-FU.The outcomes demonstrate 5-FU-induced apoptosis is preferably regulated by cell apoptosis pathway.Collectively,BRAF V600E mutant colorectal cancer cells are more sensitive to 5-FU.2.5-FU-induced mitochondrial apoptosis is preferably regulated by down-regulating Bcl-xL in BRAF V600E mutant colorectal cancer cells.Paired RKO cell lines was treated with 5-FU to explore the expression of caspase-3/-8/-9 of mitochondrial apoptosis pathway by Western Blot.We observed that BRAF V600E mutant cells that were exhibited markedly increased levels of cleavage Caspase-3/-9 pretreated with 5-FU for 24h but slight increasing in Caspase-8 cleavage.Collectively,these outcomes demonstrate 5-FU-induced apoptosis is preferably regulated by Caspase-9/-3 mediated mitochondrial apoptosis pathway.Following,Paired RKO cell lines was treated with 5-FU to explore mRNA expression and expression of Bcl-2 family member by fluorescence quantitative RT-PCR analysis and Western blot.And then detect whether suppression of Bcl-xL can enhance the apoptosis of BRAF WT cells with or without 5-FU treatment by siRNA technology,Flow Cytometry,qRT-PCR analysis and Western blot.Surprisingly,all the three pro-apoptotic Bcl-2 members were reduced after 5-FU treatment in both of BRAF WT and mutant cells,with almost equal reduction level in each cell line.Among the four members of anti-apoptotic Bcl-2 family,three of them increased after 5-FU treatment in both of BRAF WT and mutant cells.Only Bcl-xL was reduced after 5-FU treatment in BRAF mutant cells.Bcl-xL knockdown restored apoptosis in the BRAF WT cells which is equal to the BRAF mutant cell.These results suggest that,5-FU down-regulates Bcl-xL in BRAF mutant colorectal cancer cells,which may be associated with enhanced 5-FU-induced apoptosis.3.BRAF mutant tumors are sensitive to 5-FU in vivo with reduced Bcl-xLTo determine the preferent sensitivity of BRAF mutant colorectal cancer cells to5-FU in vivo,BRAF WT and mutant cells were implanted subcutaneously into opposite flanks of the same Nu/Nu mice to establish xenograft tumors.Then detect mRNA expression and expression of Bcl-xL in the Xenograft by fluorescence quantitative RT-PCR analysis and Western blot.5-FU significantly suspended the proliferation of BRAF mutant tumors cell in vivo but BRAF WT tumors.Following,immunoblotting result indicated marked attenuation in the protein expression of Bcl-xL in treated-BRAF Mutant tumors.The mRNA expression of Bcl-xL was also reduced in treated-BRAF Mutant tumors,which consistent to that in vitro.These results demonstrate that BRAF mutant tumors are sensitive to 5-FU in vivo with reduced Bcl-xL what strongly demonstrated the important role of bcl-xL in the sensitivity of BRAF mutant CRC cells treated with 5-FU.Conclusion:Our results reveal 5-FU preferably induces marked apoptosis in BRAF mutant colorectal cancer cells,and we firstly focuses on the mechanism of 5-FU in the BRAF WT or Mutant cells.We identify attenuation of expression of Bcl-xL mediated activation of caspase-3/9 by BRAF V600E to be an important mechanism of apoptosis that contributes to a great therapeutic efficacy in BRAF Mut cells.Not only that,5-FU significantly retarded the growth of BRAF Mut tumors in the xenograft-beating mice that was associated with enhanced tumor cell apoptosis,verifying a promising therapeutic strategy of mutant BRAF CRCs.