Proteomics Investigation Of The Proliferation Inhibitory Of Levodopa Against Esophageal Squamous Cell Carcinoma

BackgroundEsophageal cancer(EC)is one of the most common cancers and the sixth leading cause of cancer-related deaths in the world.About 70% of EC occur in China.EC consists of two histological types: Esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC).In China,EC is the fourth leading cause of cancer-related deaths,with about 250,000 people dying each year from EC,most of them(> 90%)being ESCC.ESCC is difficult to be detected in the early stage due to the complicated etiology.The recurrence rate is high after treatment and the five-year survival rate is less than 20%.These threat people's lives and health seriously.Therefore,the research on chemopreventive drugs for ESCC is particularly important.In recent years,due to the highly safety and wide clinical application of FDA(Food and drug administration)-approved drugs,researching new uses of these drugs has become a hot topic.Previous researches have shown that in addition to the hypoglycemic effects,metformin can repress the progression of prostate cancer,ovarian cancer and pancreatic cancer.Therefore,it is feasible to find drugs that can inhibit cancer progress from FDA-approved drugs,and can greatly save time andeconomic costs.We screened levodopa from a number of FDA-approved drugs and found that it can inhibit the proliferation and colony formation ability of ESCC.Levodopa is the most effective drug in the treatment of Parkinson's disease.It is a precursor of dopamine,and can enter the brain through the blood-brain barrier and act as dopamine under the action of dopamine decarboxylase.Proteome refers to “a complete set of proteins expressed by a genome”,that is,all proteins expressed by a cell or even a living organism.Proteomics essentially refers to the study of protein characteristics on a large scale.Proteomics analysis can not only provide a material basis for the laws of life activities,but also provide theoretical basis and solutions for the clarification and resolution of various disease mechanisms.In this study,to investigate the inhibitory mechanism,we performed mass spectrum-based proteomics analysis of KYSE150 cells after levodopa treatment.Methods1.Cytotoxicity assay: KYSE150 and KYSE450 cells were treated with different concentration of levodopa(0,200,500,800,1000 and 1500 ?M)for 24 or 48 h,the number of cells were measured by DAPI staining,and the survival rate was calculated.The concentration at which the survival rate was 50% was selected as the maximum concentration of the cell proliferation assay.2.Cell proliferation assay: KYSE150 and KYSE450 cells were treated with different dose of levodopa(0,50,100,200,400 and 600 ?M)for 0,24,48,72 and 96 h,the number of cells were measured by DAPI staining.The curves of different concentrations at various time points were performed to evaluate the effect of levodopa on the proliferation of ESCC cells.3.Soft agar assay: KYSE150 and KYSE450 cells treated with different concentration of levodopa(0,25,50,100,200 and 400 ?M)for 2 weeks were performed to evaluate the clone formation ability.4.Proteomics analysis: KYSE150 cells were performed mass spectra-based proteomics analysis after levodopa(0,600 ?M)treatment for 24 h.5.Western blotting and immunofluorescence assay: These assays were performed to verify the proteomics data.6.Mitochondrial membrane potential detection assay: JC-1 and TMRE were used to measure the mitochondrial membrane potential after levodopa(0,600 ?M)treatment,respectively.7.Transmission electron microscopy(TEM)detection: After levodopa(0,600 ?M)treatment for 24 h,TEM was used to detect changes in mitochondrial morphology of KYSE150 and SHEE cells.8.Cell viability assay: XTT cell viability Kit was used to evaluate the effect of levodopa on cellular mitochondrial activity.Results1.The results of cytotoxicity assay showed that the drug concentration of 50% of survival rate was 600 ?M.2.The results of cell proliferation and soft agar assays revealed that the proliferation and colony formation ability were dramatically repressed with the increasing dose of levodopa.3.Proteomics analysis revealed that pathways including oxidative phosphorylation,non-alcoholic fatty liver disease(NAFLD)and Parkinson disease and some proteins such as SDHD,NDUFS4 and MT-CO3 were down-regulated after levodopa(0,600 ?M)treatment.4.The results of western blotting and immunofluorescence revealed that after levodopa treatment,the level of SDHD,NDUFS4 and MT-CO3 were indeed decreased,which affirm the proteomics data.5.The results of JC-1 and TMRE assays showed that the mitochondrial membrane potential of KYSE150 cells decreased after levodopa treatment.6.Transmission electron microscopy(TEM)detection showed that after levodopa treatment,the mitochondria of KYSE150 cells were swollen and the number of mitochondrial cristae was reduced,while the mitochondrial morphology of SHEE cells was not obviously changed.7.The results of XTT assay showed that as the concentration of levodopa increased,the absorbance at 450 nm decreased,which indicated a decrease in cellular mitochondrial viability.ConclusionLevodopa inhibits the growth of ESCC through regulating pathways including oxidative phosphorylation,non-alcoholic fatty liver disease(NAFLD)and Parkinson disease,down-regulating the levels of SDHD,NDUFS4 and MT-CO3,and inhibits oxidative phosphorylation.