Effects Of TAK-242 Combined With Oxaliplatin On Esophageal Squamous Cell Carcinoma Cell Line Eca-109

ObjectiveIn this study,the effects of TLR4 signaling pathway on human esophageal squamous cell carcinoma cell line Eca-109 were investigated through cell culture,cell immigration technics,Immunocytochemistry,Real-time PCR,and Western blotting.In addition,the synergetic roles of TLR4 inhibitor,TAK-242,and chemotherapy drugs,oxaliplatin(OXA)on Eca-109 Cells were explored.The results would provide the clues of researches in development of esophageal cancer targeted drug therapy and guideline of clinical combination therapy.Methods1.MTT assay for Cell viability Cell density of 1×105/mL of human esophageal squamous carcinoma Cell,Eca-109,Cells suspension of 200?L in one hole of 96 holes plate measured by MTT activity.Cells were divided into blank group;vehicle control group;OXA group,the final concentration were 6.25,12.5,25,50,100?M respectively;TAK-242 group,the final concentration 12.5,25,50,100,200 nM respectively;combination group: 25?M OXA + 100 nM TAK-242 group.The Cells were washed with PBS,and adding 200?L MTT(5mg /m L),after incubation for 4h,adding DMSO,shake in the shaker 10 min,the absorbance value of each micropore in490 nm was measured by enzyme marker,the optimal concentration of statistical analysis in combination of the inhibition rate of each group selection.Repeat the experiment for 3 times.2.Cell Combined medication culture Cells were divided into vehicle group,100 nM TAK-242 group,25?M OXA group,100 nM TAK-242 combined with 25?M OXA group;the 6,12 and 24 hole plate,a petri dish with 10 cm,Transwell Cell culture Cells were extracted from RNA,protein,preparation of Cell climbing patch et al,to complete the corresponding experiment.3.Transwell for Cell migration Cells treated with 12 h after dosing,first with PBS suspending cleaning two times,1640 medium containing 0.1% BSA heavysuspension,cell count and using a density of 5×105/mL,with Transwell room 200 L,so that each hole about 10 thousand cells;upper and lower chamber is formed concentration gradient to drive cell perforation,after incubation for 48 hours;making the Transwell chamber dry,after 0.1% crystal violet staining 1h,carefully wipe the upper chamber membrane no perforation of Cells;under the inverted microscope,5visual fields were randomly taken,and the number of cells counted and the average number of cells showed the ability of cell migration.Repeat the experiment for 3times.4.Immunocytochemistry The preparation of Cell sheets;4% paraformaldehyde fixation 10min;0.5% TritonX-100 perforation 20min;3% H2O2 closed 10min;0.5%BSA prepared the first antibody(MyD88,TRIF,NF?B-p65)and incubated at 4?overnight;the second day incubated at room temperature for 1h;adding the second antibody contained HRP 37? 30 min incubation;DAB staining;dehydrated;transparenting;observation under inverted microscope,optical density analysis of Image-Pro Plus software.Repeat the experiment for 3 times.5.RT-qPCR Trizol method was used to extract the total RNA of Cells in each group,and the quality of RNA extraction was determined according to the ratio of A260/A280 and the ratio of A260/A230.RNA reverse transcription cDNA was synthesized mainly comprises the following steps: total RNA amount of 1g,other reagent dosage refer to cDNA Reverse Transcription Kit specification,ribozyme free water up to the vlume 20?L;mixing system;short centrifugation;RT-PCR steps:25? 10 min,37? 120 min,85? 5min,4?forever;cDNA store at-20?.qPCR reaction system(single sample): Forward Primer(10uM)0.8 ?L,Reverse Primer(10uM)0.8?L,SYBR Green Real time PCR Master Mix 10.0?L,cDNA 2.0?L,deionized water 6.4?L,the total vlume of 20?L;qPCR reaction program: 95? 1 min;95? 15 s,60? 15 s,72? 45 s collecting dates,40 cycles;the melting curve of 65?-95?.The result of the experiment was to observe the amplification curve and the melting curve.According to the Ct value,the relative expression of mRNA was calculated by using 2-?Ctmethod,and each sample was made of 3 replicates.6.Western-blotting The Cells of each group were washed and collected,adding RIPA lysis buffer with 1% inhibitor cocktail,cracking on ice for 30 minutes,and the supernatant was collected at EP,4? centrifugation of 12000 rpm for15 min.The standard curve was drawn according to the kit of BCA protein concentration,and the absorbance value at 562 nm was measured.PolyAcrylamide gel electrophoresis(6%,10%,12% separation gel,5% concentrated gum),each of the samples is 40 g and 90 V with constant pressure in concentration gel,20 min,separation gel of 120 V,1h,200 mA with constant current wet to the protein transferred to PVDF membrane,5% skim milk closed.4? overnight,fully washing after adding the first antibody(GAPDH,TLR4,MyD88,TRIF,NF?B-p65),fully washing after adding the corresponding species the second antibody,ECL color,the optical density analysis software Image J.Each experiment was repeated three times to calculate the average optical density value,and the specific experimental time was determined according to the size of the target protein.7.statistical analysis Data are presented as mean±SD.SPSS Statistics software(17.0)was used to analyze the data.Statistical analysis of the data was performed by Student's t-test.P < 0.05 were considered to be statistical significance,P > 0.05 were considered to be statistical no-significance.Results1.MTT experimental results The survival rate of Eca-109 cells was decreased with the drug in a concentration gradient dependent manner,OXA concentration more than 25?M can significantly inhibit the growth of Eca-109Cells(P<0.001),TAK-242 concentration more than 100 nM can significantly inhibit the growth of Eca-109 Cells(P<0.001),25?M OXA and 100 nM TAK-242 combination on the growth of Eca-109 significantly inhibited(P < 0.001).2.Experimental results of cell migration Compared with the blank group,the ability of Eca-109 Cells in Dosing group to pass through the membrane was decreased(P < 0.01).Compared with the single drug group,The ability of Eca-109 Cells in 25?M OXA combined with 100 nM TAK-242 group to pass through the membrane was significantly lower(P < 0.01).3.Immunocytochemistry results The positive results of immunocytochemical staining of human esophageal squamous carcinoma Cell line Eca-109 were stained with brown or brown granules in cytoplasm or nucleus.With Image-pro-Plus 5.1 weremeasured in four groups of MyD88,TRIF,and NF?B-p65 optical density,results showed that the average optical density of TAK-242 group and combination group were significant lesser than the vehicle group(P < 0.05).4.The mRNA results of Cellular expression The expression of mRNA Cells in the drug group compared with the vehicle group,medication of TAK-242 can down regulate the MyD88,TRIF,NF?B-p65 mRNA level of the Eca-109 Cells(P<0.05),and the combination group is lower than the other groups(P < 0.05).5.Western-blotting results Protein expression were detected four groups of GAPDH,TLR4,MyD88,TRIF,NF?B-p65.The results showed that,compared with the vehicle group,medication of TAK-242 can down-regulation TLR4,MyD88,TRIF,NF?B-p65 protein expression(P < 0.05).ConclusionTAK-242 can enhance the proliferation of OXA in human esophageal squamous cancer cell inhibition and promotes the migration ability effectively is through down-regulation mRNA of MyD88,TRIF and NF?B-p65 and inhibitor TLR4 and its downstream protein MyD88,TRIF,the expression of NF?B-p65.Indicated that the effect of TLR4 signal pathway in cancer is very important,TAK-242 combined with OXA can inhibit tumor growth inhibition of cancer metastasis has great clinical application value.