Effects And Related Mechanisms Of 5-fluorouracil Combined With 5-chloro-2,4-dihydroxypyridine On Malignant Phenotype Of Colorectal Cancer Cells

Background and ObjectiveColorectal cancer is one of the most commonly diagnosed cancers worldwide and the 5-year survival rate is still less than 60% in China.Invasion and metastasis of colorectal cancer are major factors that affect the survival and prognosis of patients.Janus kinase 2/Signal transducer and activator of transcription3 signaling(JAK2/STAT3)is well known for crucial role in transducing a variety of extracellular and intracellular signals to regulate gene expression and coordinate cell proliferation,survival,metastasis,angiogenesis and immunosuppression.5-FU is widely used to treat a range of cancers,including colorectal,liver,and breast cancers.However,it has toxicity to normal cells and cancer cells can develop resistance to it,which are two major barriers to successful cancer treatment.Tegafur,gimeracil and oteracil porassium capsules(S-1)is the first-line therapy for advanced gastric cancer in Asia and is used with increased frequency in Western counties.S-1 has been widely approved for gastric cancer treatment in many countries in Asia and Europe and for several other cancers in Japan.It is composed of tegafur(FT),5-chloro-2,4-dihydroxypyridine(CDHP),and oteracil potassium(OXO)in a molar ratio of 1:0.4:1.In this study,5-FU and CDHP at a ratio of 1:0.4 was used as S-1 because they play the main role in the S-1 activity.The purpose of the research is to investigate the effects of 5-FU treatment with CDHP in colorectal cancer cells and the roles of JAK2/STAT3 signaling pathway during the therapy.Methods1.Various concentrations of 5-FU,CDHP,the combination of S-1(5-FU with CDHP in a ratio of 1:0.4),AG490 were used to deal with the colorectal cancer Lo Vo cells and HT29 cells,cell viability was measured by CCK-8 assay.2.CDHP,5-FU and S-1(5-FU + CDHP)were used to treat both cells for 24 h respectively.Through the CCK-8 assay,plate colony formation assay,cell scratch test and transwell invasion assay,the ability of proliferation,clone formation,migration and invasion were detedted.3.CDHP,5-FU and S-1(5-FU + CDHP)were used to treat both cells for 24 h respectively.q PCR and Western Blot technology were used to detect the JAK2/STAT3 signaling pathway related genes and proteins in colorectal cancer cells.4.S-1(5-FU + CDHP),AG490,S-1(5-FU + CDHP)+ AG490 were used to treat both cells for 24 h respectively.Through the CCK-8 assay,plate colony formation assay,transwell invasion assay and flow cytometry,the ability of proliferation,clone formation,invasion and apotosis rate were detedted.5.S-1(5-FU + CDHP),AG490,S-1(5-FU + CDHP)+ AG490 were used to treat both cells for 24 h respectively.q PCR and Western Blot technology were used to detect the JAK2/STAT3 signaling pathway related genes and proteins in colon cancer cells.Results1.5-FU treatment with or without CDHP inhibited the proliferation of Lo Vo and HT29 cells both in a dose and time dependent manner.Results for IC50 of 5-FU at 24 h,48 h and 72 h were 178.63 ?M,79.87?M,51.82?M(Lo Vo)and 240.72?M,120.29?M,76.44?M(HT29).CDHP alone did not affect the proliferation of either cell lines and the addition of CDHP enhanced the growth inhibitory effect of 5-FU.Results for IC50 of AG490 at 24 h were 103.88?M(Lo Vo)and 124.3?M(HT29).2.Through the CCK-8 assay,cell colony formation assay,cell scratch test and transwell assay,both cells had shown CDHP alone did not have the effects(P>0.05).5-FU+CDHP showed with less migration cell number compared to 5-FU group(p < 0.05),as well as the cell scratch test,and the abilities of cell proliferation and colony formation were all decreased(P<0.05).3.q PCR and Western Blot showed that compared to 5-FU group,S-1(5-FU+CDHP)group had lower Bcl-XL and Survivin gene and protein expression.Western Blot also had showed that S-1(5-FU+CDHP)group showed less expression of p-JAK2 and p-STAT3 compared to 5-FU group.4.Through the CCK-8 assay,cell colony formation assay,transwell assay and Flow cytometry,using S-1(5-FU + CDHP),AG490,S-1(5-FU + CDHP)+ AG490 treatment of cells for 24 h,Compared with S-1 group or AG490 group,the proliferation of S-1(5-FU+CDHP)+AG490 group significantly decreased,as well as the ability of cell proliferation,colony formation and invasion.what's more,the apoptosis of S-1(5-FU+CDHP)+AG490 group were increased significantly in both cell lines.5.The results of q PCR and Western Blot showed that the gene and protein expression of Bcl-XL and Survivin and p-JAK2,p-STAT3 were higher in S-1(5-FU+CDHP)+AG490 group than in S-1 group or AG490 group.Conclusions1.The combination of 5-fluorouracil and 5-chloro-2,4-dihydroxypyridine has a synergistic effect on the inhibition of proliferation,migration and inwasion in colorectal cancer cells.2.The combination of 5-fluorouracil and 5-chloro-2,4-dihydroxypyridine may regulate the malignant biological behavior of cells by inhibiting the phosphorylation of JAK2,STAT3,and the expression of Bcl-XL and Survivin proteins.3.Targeted the inhibition of JAK2/STAT3 signaling pathway enhances the inhibitory effect of 5-fluorouracil and 5-chloro-2,4-dihydroxypyridine in colorectal cancer cells.