Research On The Inhibitory Effect And Mechanism Of Cisplatin And Salidroside On Human Gastric Cancer Cell Lines HGC-27 And MGC-803

Objective: To investigate the inhibitory effect of DDP combined with salidroside on gastric cancer cells and the molecular mechanism,thus,providing new ways of thinking and method for clinical treatment of gastric cancer.Methods:Human gastric cancer cell lines HGC-27 and MGC-803 were selected as experimental subjects.Blank control group,DDP group(10 ?M),salidroside group(8000 ?M),combination group with DDP(10 ?M)and salidroside(8000 ?M)were set for experimental study.1.Study on the inhibitory effect of DDP and salidroside on gastric cancer cells: MTT method was used to detect the effect of DDP,salidroside and their combination on the cell viability of HGC-27 and MGC-803 cell lines;The morphological change was observed by light microscope and fluorescence microscope;Detection of the effect of the combined administration on proliferation of gastric cancer cells was conducted by cell clone formation assay;2.Study of the signaling pathway for which DDP combined with salidroside inhibited gastric cancer cells: JC-1 staining was used to detect mitochondrial membrane potential;The mRNA levels of Bcl-2 and Bax related to mitochondrial apoptosis pathway were detected by q-PCR;3.Study on the transcriptome of DDP combined with Salidroside in inhibiting gastric cancer cells: blank control HGC-27 cells and cells treat with the combination of DDP and salidroside were used for the transcriptome analysis,and GO and KEGG enrichment analysis were conducted to analyze the changed genes;q-PCR was used to verify the related genes.RESULT:1.MTT results showed that DDP,salidroside and the combination treatment with DDP and salidroside significantly inhibited the proliferation of human gastric cancer cells HGC-27 and MGC-803 in vitro.Particularly,the combination treatment obviously inhibited cell viability in comparison with DDP treatment and salidroside treatment alone(P<0.05).The morphological changes such as chromatin concentration,nuclear fragmentation and cell edge irregularity were observed under light microscope,the most significant change of cell morphology was observed in the combination group.Under fluorescence microscope,the green fluorescence of DDP combined with salidroside was weaker,orange fluorescence was more obvious,and most of the cells were pyknotic,it is suggested that combined administration can induce apoptosis of gastric cancer cells.The results of the colony formation experimental showed that the colony formation of HGC-27 and MGC-803 cells was significantly reduced by the combination treatment with DDP and salidroside compared to the control group,indicating that the combination treatment with DDP and salidroside could significantly inhibit the proliferation of gastric cancer cells.2.The results of JC-1 staining showed that compared with the control group,DDP group and salidroside group,the combination group with DDP and salidroside showed weaker red fluorescence and stronger green fluorescence,combined administration can significantly reduce the mitochondrial membrane potential of gastric cancer cells and induce mitochondrial dysfunction;The results of q-PCR analysis showed that DDP combined with salidroside up-regulated the mRNA level of Bax,meanwhile,down-regulated the mRNA level of Bcl-2,resulting in the decrease of the ratio of Bcl-2/Bax,and the subsequent activation of mitochondrial apoptosis pathway.3.Through transcriptome sequencing analysis,2694 differential genes were detected,including 1227 up-regulated genes and 1467 down-regulated genes;The analysis of GO function enrichment showed that the main biological functions of the significantly different genes were nucleic acid binding transcription factor process,protein binding process,cell morphological change,cell killing and so on;The analysis of KEGG showed that the number of differentially annotated genes under PI3K/AKT signaling pathway was the largest,suggesting that PI3K/AKT signaling pathway may be the key pathway for combined administration of drugs to promote apoptosis of gastric cancer cells.The results of Q-PCR showed that the combination of DDP and salidroside could significantly inhibit the expression of PI3 K and Akt mRNA in gastric cancer cells(P< 0.01).It is suggested that DDP combined with salidroside inducing apoptosis of gastric cancer cells is related to its inhibition of PI3 K / Akt signaling pathway.CONCLUSIONS:1.DDP combined with salidroside can inhibit the proliferation of gastric cancer cells in vitro,it can also cause morphological changes and decrease of cell colony,and it could also induce the apoptosis of gastric cancer cells.2.DDP combined with salidroside can significantly reduce the mitochondrial membrane potential of gastric cancer cells,at the same time,Bax mRNA was up-regulated and Bcl-2 mRNA was down regulated,and induce the apoptosis of gastric cancer cells by activating the endogenous mitochondrial apoptosis pathway.3.DDP combined with salidroside can inhibit the exogenous PI3K/AKT signaling pathway to induce gastric cancer cell apoptosis.