The Mechanism Study Of Mefloquine Inhibits Esophageal Squamous Cell Carcinoma Proliferation

Background and objectiveEsophageal cancer(EC)is a malignant tumor occurring in the upper digestive tract,and its incidence rate and mortality rate remain high in the world.The latest global cancer statistics data showed that the incidence and mortality rate of EC ranks seventh and sixth respectively in the world.EC is divided into esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC),and ESCC accounts for more than 90%of EC and is highly prevalent in Asian countries.In recent years,medical technology has developed rapidly,but the prognosis of patients with ESCC has not improved significantly,and the 5-year survival rate is still at 15-25%.At present,Except for surgical resection,radiotherapy and chemotherapy as the main methods for tumor treatment and prevention of postoperative recurrence,while the side effects are obvious and long-term use of chemotherapeutic drugs is easy to produce drug resistance.Therefore,the prevention and treatment of esophageal cancer are still problems which need to be solved in the world.The occurrence and development of tumor is a complex process,in addition to the abnormal regulation of oncogenes and tumor suppressor genes involved in the occurrence and development of tumor,the abnormal metabolism of cells is also an important reason for the occurrence and development of tumor.The metabolic abnormalities of tumor cells include Warburg effect,vigorous metabolism of fatty acid and lipid membrane,negative nitrogen balance of protein and amino acid metabolism,etc.Among these,Warburg effect is a significant feature of glucose metabolism in malignant tumor cells,and tumor cells obtain energy by glycolysis(Pyruvate produced by glycolysis enters mitochondria,and then under the action of pyruvate dehydrogenase,acetyl coenzyme A is produced,which enters the tricarboxylic acid cycle and produces ATP),and they require a large amount of glucose to produce ATP to maintain their rapid growth.Mitochondria,as the place of energy production in cells,participates in the process of glycolysis to produce energy,and mitochondria also regulates a variety of cellular functions,including the regulation of energy,redox state,production of reactive oxygen species(ROS),control of intracellular calcium level,etc.These changes of cell function will affect biosynthesis pathway,cell signaling transduction pathway,regulation of transcription factors and so on.In recent years,more and more evidence showed that mitochondria were closely related to the occurrence and development of tumor.And mitochondrial gene mutations can affect mitochondrial metabolism,such as succinate dehydrogenase(SDH)mutation,fumarate hydratase(FH)mutation and others,which can lead to tumor.Autophagy,which is a process of lysosomal degradation and circulation of proteins and organelles.On the one hand,autophagy can inhibit the occurrence of tumor by inhibiting oxidative stress,chronic tissue damage and carcinogenic signaling pathways;on the other hand,autphagy maintains mitochondrial function and energy production through mediating recycling of cell contents,and it’s conducive to better survival of tumor cells.Therefore,the regulation of mitochondrial autophagy provides a new strategy for tumor treatment.In recent years,research data show that some non-anticancer drugs approved by the US Food and Drug Administration(FDA)have shown a certain degree of anti-tumor effect,such as metformin,statins and others.These non-anti-cancer drugs are highly safe,and their application for cancer treatment can save a lot of cost and time compared with the long period from the devolpment of new drugs to the market.Hence,the new use of old drugs provides a new strategy for the prevention and treatment of ESCC.In our previous study,we screened from a lot of FDAapproved non-anti-cancer drugs by testing the inhibitory effect on clone formation of ESCC cells,and screened out mefloquine(MQ),which was a clinical drug used to treat chloroquine resistant or multidrug resistant falciparum malaria.Some research data showed that MQ exhibited antitumor effects in liver cancer and colon cancer,etc,but its role and mechanism in esophageal cancer are not clear.Methods(1)To explore the anti-tumor effect of MQ in ESCC cells in vitroFirstly,immortalized normal esophageal epithelial cells(SHEE cells)were used to detect the toxic effect of MQ on SHEE cells;then,cell proliferation and clone formation assay were used to detect the inhibitory effect of MQ on the proliferation and clone formation ability of ESCC.(2)To explore the underlying mechsnisms of MQ-induced anti-tumor effect with proteomics assayKYSE150 cells of ESCC were treated with MQ(10μM)for 24 hours,and then collected the cells for analysis.The differentially expressed proteins between the MQ-treated group and the control group were screened by mass spectrometry and database search;next,based on this data,subcellular localization analysis,gene ontology term enrichment,KEGG pathway enrichment analysis and Venn analysis were performed to find the most influenced organelles,signaling pathways and molecules after MQ treatment.(3)To test the changes of mitochondrial function with several related kitsReactive oxygen species(ROS)detection kit was used to detect ROS production in ESCC cell lines KYSE150 and KYSE450 after treatment with MQ;Western blot was used to detect the protein level of oxidative phosphorylation complex(OXPHOS complex)in KYSE150 and KYSE450 cells after treatment with MQ;nicotinamide adenine dinucleotide(NAD)assay kit was applied to test the value of NAD+/NADH in KYSE150 and KYSE450 cells treated with MQ;adenosine triphosphate(ATP)assay kit was performed to detect ATP production in KYSE150 and KYSE450 cells treated with MQ;N-acridine orange dye was used to test the mitochondrial integrity in KYSE150 and KYSE450 cells after MQ treatment;transmission electron microscope was used to observe the morphological changes of mitochondria after MQ treatment,and the number of autophagic mitochondria in each field was counted;immunofluorescence(IF)was performed to observe the signaling of autophagic marker LC3,and the co-localization of mitochondrial marker MTCO1 and autophagic marker LC3 after MQ treatment;Western blot was used to test the protein level of LC3 in KYSE150 cells after MQ treatment;at the same time,the apoptosis assay was applied to analyze the apoptosis in KYSE150 and KYSE450 cells after MQ treatment.(4)To explore the role of SDHC in ESCCFirstly,TCGA database was applied to analyze the expression of the 4 proteins obtained from proteomics analysis in esophageal cancer tissues and normal tissues,including succinate dehydrogenase complex subunit C(SDHC),succinate dehydrogenase complex subunit D(SDHD),mitochondrially encoded cytochrome c oxidase Ⅲ(MTC03)and NADH:ubiquinone oxidoreductase subunit V3(NDUFV3);next,Western blot was applied to test the protein level of SDHC in KYSE150 and SHEE cells after MQ treatment;next,immunohistochemistry(IHC)was used to detect the expression of SDHC in tissue array;at last,knocking down SDHC in KYSE150 and KYSE450 cell lines with shRNA,and cell proliferation and clone formation assays were applied to detect the role of SDHC in cell proliferation and clone formation.(5)To explore the anti-tumor effect of MQ in ESCC in vivoFirstly,Western blot was performed to detect the protein level of SDHC in established PDX model tissues,and chose 2 cases of PDX models with SDHC high expression and 2 cases of PDX models with SDHC low expression;the 4 PDX models were divided into different groups after stable passage,and mice were given with solvent control(sterile water),low dose of MQ(50 mg/kg)and high dose of MQ(200 mg/kg)by gavage;then,IHC was performed to detect the SDHC expression in different groups.Results(1)MQ suppressed cell proliferation and clone formation of ESCCThe cytoxicity assay result showe that MQ didn’t exihibit obvious toxic effect on SHEE cells;cell proliferation and clone formation results showed that MQ obviously inhibited cell proliferation and clone formation of KYSE150 and KYSE450 cells in a time and dose-dependent manner.(2)Mitochondira may play a role in MQ-induced anti-tumor effectMass spectrometry and bioinformatics analysis results showed that a total of 6167 proteins were identified,of which 5151 proteins had quantitative information,among these-protiens,with 1.5 times as the change threshold,t-test p-value<0.5 as the standard,a total of 147 differential proteins were got,which including 86 up-regulated proteins and 61 down-regulated proteins;meanwhile,the quality control test of mass spectrometry results showed that the sample preparation met the requirements;the heatmap showed the expression of differential proteins between the MQ treatment group and the control group,and the down-regulated differentially expressed proteins were selected for the next study;WoLF PSORT software prediction results showed that the differentially expressed proteins were mainly distributed in palsma membrane and mitochondria;GO annotation analysis results showed that the top 3 cell components contain organelle membrane,mitochondrial membrane and mitochondria;KEGG pathway enrichment analysis results showed that the most obviously down-regulated signaling pathways contain Alzheimer’s disease,nonalcoholic fatty liver disease,Parkinson’s disease and oxidative phosphorylation and Huntington’s disease,they were all related to mitochondrial function;the results of Venn diagram analysis showed that there were 4 common mitochondrial proteins involved in the enriched 5 signaling pathways,including SDHC,SDHD,MTC03 and NDUFV3.(3)MQ affected the function of mitochondria in ESCC cellsROS detection kit results showed that the production of ROS increased significantly in KYSE150 and KYSE450 cells after MQ treatment;Western blot results showed that the protein levels of OXPHOS complexes decreased in KYSE150 and KYSE450 cells after MQ treatment;NAD+/NADH detection kit results showed that the value of NAD+/NADH in KYSE150 and KYSE450 cells decreased significantly after MQ treatment;ATP detection kit results showed that the production of ATP in KYSE150 and KYSE450 cells decreased significantly after MQ treatment;NAO staining results showed that the NAO staining in KYSE150 and KYSE450 cells significantly decreased after MQ treatment;transmission electron microscopy results showed that there’re many double membrane vesicles in KYSE150 cells after MQ treatment,and the average number of mitochondria with autophagic changes in each field was significantly increased;at the same time,IF results showed that the fluorescence signal of autophagic marker LC3 increased after MQ treatment,and the signal of LC3 and the mitochondrial marker MTCO1 showed colocalization in KYSE150 and KYSE450 cells;finally,Western blot results showed that the protein expression level of LC3-Ⅱ significantly increased after MQ treatment;apoptosis assay results showed that MQ treatment didn’t induce cell apoptosis.(4)SDHC played a cancer promoting role in ESCC progressionTCGA database analysis showed that compared with normal tissues,SDHC was obviously highly expressed in esophageal adenocarcinoma(EAC)and esophageal squamous cell carcinoma(ESCC)tissues,while SDHD and NDUFV3 didn’t show obvious difference between ESCC and normal tissues,the expression of MTC03 in esophageal cancer has not been reported;Western blot results showed that the protein levels of SDHC in KYSE150 cells treated with MQ(10 μM)for 24 h decreased significantly;meanwhile,Western blot results showed that the protein levels of SDHC in SHEE cells treated with MQ(10 μM)for 24 h didn’t exhibit obvious change;IHC results showed that the expression of SDHC in cancer tissues was significantly higher than that in normal tissues in ESCC tissue array;cell proliferation and clone formation assay results showed that the cell proliferation and clone formation ability were obviously suppressed in KYSE150 and KYSE450 cells after knocking down SDHC.(5)MQ suppressed ESCC tumor growth in vivoWestern blot results showed that in tissues of cases EG59 and EG60,SDHC was high expressed,while in cases EG2,EG20,EG37 and EG84,SDHC was low expressed,and the protein levels of SDHD、MTC03 and NDUFV3 didn’t show obvious change;in vivo study results showed that MQ obviously inhibited tumor growth of EG59 and EG60,while didn’t exhibit antitumor effect in EG20 and EG84;IHC results showed that the protein level of SDHC obviously decreased in MQ group in tissues of EG59 and EG60,while the protein level of SDHC didn’t show obvious change in tissues of EG20 and EG84.Conclusions(1)MQ inhibited the proliferation of ESCC in vitro and in vivo.(2)MQ affected the function of mitochondria and induced autophagy of mitochondria without inducing apoptosis.(3)SDHC played an important role in MQ-induced anti-tumor effect,suggesting that SDHC has the potential as a new target for tumor treatment.