Effect Of Allicin Combined With Cisplatin On Apoptosis And Expression Of Death Receptor Related Proteins In MEC-1 Cells

Objective:To study the effect of allicin combined with cisplatin on apoptosis and expression of death receptor related proteins in mucodermoid carcinoma cells,and to provide experimental basis for the application of allicin combined with cisplatin in clinical chemotherapy of mucoepidermoid carcinoma.Methods:The concentration gradient of drug action was determined by pre-experiment with MEC-1 cell line as the research object.Allicin concentration gradients were0,30.0,60.0,90.0,120.0,150.0,180.0mg/L and DDP gradients were 0,2.5,5.0,7.5,10.0,12.5,15.0 mg/L.（1）The proliferation inhibition rate of MEC-1 cells treated with different concentrations of Allicin and DDP for 24 h,48 h and 72 h was detected by CCK-8,and the 24 h IC₅₀ of MEC-1 treated with Allicin and DDP was calculated by SPSS21.0.The 1/4IC₅₀,1/2IC₅₀ and IC₅₀ values of Allicin and DDP and the combination of them were selected to treat MEC-1 cells for 24 h.The inhibition rate of cell proliferation was calculated and Q value method was used to judge the combined effect of the two drugs.Allicin group,DDP group and combined group were selected for follow-up experiment.（2）MEC-1 cells were treated with 85.5 mg/L Allicin,5.5mg/LDDP and 85.5 mg/L Allicin+5.5 mg/LDDP,and the control group was treated with PBS phosphate buffer of 0.1%DMSO.24 hours later,the growth morphology of cells in each group was observed by inverted microscope.（3）DAPI fluorescence staining was used to detect the morphological changes of nuclei in per group after MEC-1 for 24 h.（4）flow cytometry was used to explore the difference of apoptosis rate in each group after treatment with MEC-1 for 24 hours.（5）RT-q PCR was used to detect the expression of Fas,TRAIL and Caspase-8m RNA in each drug group after MEC-1for 24 hours.（6）Western blot was used to explore the expression of Fas,TRAIL and Caspase-8 proteins in each drug group after MEC-1 for 24 hours.Results:（1）at the same time point,,the proliferation inhibition rate of MEC-1 cells increased when the concentration of Allicin and DDP increased,and after MEC-1 was treated with Allicin and DDP at the same concentration,the inhibition rate of cell proliferation increased with the extension of time（P<0.05）.The values of Allicin IC₅₀and cisplatin IC₅₀ were 171.0 mg/L and 11.0 mg/L respectively.The cell proliferation inhibition rate of each group was computed 24 hours after therapy with Allicin concentration 42.75 mg/L,85.5 mg/L,171.0 mg/L group and DDP concentration 2.75mg/L,5.5 mg/L,11.0 mg/L group,and combined group（42.75 mg/L+2.75 mg/L）,（85.5mg/L+5.5 mg/L）and（171.0 mg/L+11.0 mg/L）,respectively.Only in the group of Allicin combined with DDP（85.5 mg/L+5.5 mg/L）,the Q value calculated by Kim Jong-un method was 1.25>1.15,and the combination of the two drugs had synergistic effect.Therefore,the experiment was divided into control group,Allicin group（85.5mg/L）,DDP group（5.5 mg/L）and combined group（85.5 mg/L+5.5 mg/L）.（2）under the inverted microscope,the growth morphology of MEC-1 in the control group was better and the speed was faster.The growth rate of MEC-1 in the Allicin group,the DDP group and the combination group was significantly slower,and the exfoliated cells and cell adhesion in the combined group were significantly higher than those in the single drug group.（3）DAPI staining showed that there was no obvious apoptosis in MEC-1 in control group,but the nuclear morphology of apoptosis could be observed in Allicin group,DDP group and combination group,and the number of apoptotic bodies in combination group was higher than that in Allicin and DDP group.（4）flow cytometry showed that apoptosis of MEC-1 cells could be induced in Allicin group,DDP group and combination group,and the apoptosis rate in combination group was upper than that in single drug group.（5）The results of RT-q PCR showed that matched with the control group,both Allicin and DDP alone or in combination could up-regulate the relative expression of Fas,TRAIL and caspase-8m RNA in MEC-1 cells,and the combination of drugs had the most meaningfully effect on the regulation of target genes.（6）Western blot detection showed that the expression of Fas,TRAIL,caspase-8 protein in Allicin group,DDP group and combination group,which was clearly upper than that in the control group,and the protein expression in the combination group was higher than that in the single drug group（P<0.05）.Conclusions:（1）Both Allicin and DDP could inhibit the proliferation of MEC-1 cells in a time-concentration dependent manner,and the combination of Allicin and DDP had a synergistic effect.Compared with the single drug group,Allicin combined with DDP could increase the apoptosis rate of MEC-1 cells.（2）Allicin cooperates with DDP to induce apoptosis in MEC-1 cells,which may be related to the up-regulation of Fas,TRAIL,caspase-8,a key protein in death receptor pathway.