Effect Of3-deazaneplanocin A On Proliferation, Apoptosis And Migration Of Esophageal Squamous Cell Carcinoma

3-deazaneplanocin A (DZNep) is a kind of histone methylation inhibitors, and ithas been applied to anti-tumor, anti-parasitic, anti-viral, anti-arthritic and immunesuppressive activity and other pharmacological effects. It has been reported thatDZNep can inhibit proliferation of tumor cells by reactivate silenced tumorsuppressor genes, which provides a new target for the treatment of tumors. In addition,DZNep has been shown to play an important role in treatment of liver cancer, breastcancer, medulloblastoma, acute myeloid leukemia, glioblastoma multiforme tumors,non-small cell lung cancer, cardiovascular disease, and AIDS, etc. However, reportson the role of DZNep in esophageal squamous cell carcinoma (ESCC) is rarely, so theeffects of DZNep on ESCC cells will be investigated in our study.Esophageal squamous cell carcinoma is one of the most common malignantdigestive tumor, and the morbidity and mortality of ESCC has ranked fourth in thenation. Normally when the patient has been diagnosed as metaphase or terminalcancer, its success rate for treatment is very low and it’s easy to relapse and itsprognosis is poor. The traditional methods in treatment of esophageal squamouscarcinoma include surgery, chemotherapy and radiotherapy, but the cure rate andprognosis of the patient is not optimistic. Due to the change of tumor-related genes isan accumulation of multiple stages, different stages of esophageal squamous cellcarcinoma are involved in activation of multiple cancer genes and inactivation oftumor suppressor genes. Therefore, to find the molecular targeting drug for the treatment of esophageal squamous cell carcinoma, combination with other treatmentsfor comprehensive treatment of esophageal squamous cell carcinoma is imminent.S-adenosylhomocysteine hydrolase (SAHH) is an important rate-limitingenzyme in methylation pathway, and it catalyzes hydrolysis of S-adenosyl-homocysteine (SAH) to homocysteine (Hcy) and adenosine (Ado), the hydrolysisreaction is the only way of S-adenosyl homocysteine metabolism, and the reaction isreversible. Adenosine is catalyzed by a series of enzyme and generates cAMP, whichcan be involved in cellular signal transduction as an intracellular second messenger.At the same time, homocysteine generates S-adenosylmethionine (SAM) through aseries of catalytic reaction. SAM provides methyl for methyltransfer reactions in vivoand it generates SAH, thus the methylation cycle is completed. Obviously, SAHH isan important regulator of methyl transfer reactions in vivo, and it regulates variousreceptor molecules, such as proteins, nucleic acids and some other signal transductionmolecules in methylation reaction. EZH2is histone methyl-transferase of PRC2family, mainly mediates histone demethylation in tumor cells, and expresses in manytumor cells. EZH2induces gene silencing by catalyzing H3K27me3modifications,and it also plays a role in transcriptional regulation and adjusting the chromatin state.Existing evidences prove that DZNep has antitumor activity, partly by reducing theexpression of EZH2and its activity resulting in the inhibition of PRC2.In this study, we treated esophageal squamous cell carcinoma cell line EC9706Eca109cells with a certain concentration of DZNep and detected the changes in cellcycle, proliferation, migration, apoptosis and adhesion ability of esophagealsquamous cancer. Thus the study was divided into two parts, the first part was toexplore and optimize the conditions in which DZNep treated esophageal squamouscell carcinoma, and the second part was to detect the changes of esophagealsquamous cell in cell cycle, proliferation, migration, apoptosis and adhesion ability.Methods1Cell culture The ESCC cell line EC9706cells and Eca109cells were saved by ourlaboratory. All of cells in our work were cultured in RPMI1640mediumsupplemented with10%standard fetal bovine serum (FBS) at37℃, under saturatedhumidity,5%CO2incubator.2Condition optimization of DZNep treatment of esophagealsquamous cell carcinomaIn this part, we extracted total cellular protein of Eca109cells at different timeafter treating Eca109cells with different concentrations of DZNep, and thendenatured them by boiling, and detected the expression of SAHH and EZH2proteinsand SAHH mRNA levels through Western Blot and RT-PCR methods to determinethe optimal conditions that DZNep treatment of esophageal squamous cancer cells.3Detection of the effect of DZNep on cell cycle, proliferation,migration, apoptosis and adherence factor of ESCC cellsESCC cells were treated with DZNep at optimal conditions, and then wedetected the impact on cell cycle and apoptosis of EC9706and Eca109cells by flowcytometry, tested its effect on cell proliferation through the CCK-8assay and EdUmethod, measured its effect on cell migration by Transwell assay, and detected thechanges of cell adherence factor by Western Blot.Results1Condition optimization of DZNep treatment of esophagealsquamous cell carcinoma and expression of related proteinsEca109cells were treated with different concentrations of DZNep for48and72h and then the expression of SAHH protein levels were detected by Western Blot.The results showed that the expression of SAHH in Eca109cells was decreasedobviously after treatment with5μmol/L DZNep for48h, and death cells began to appear. So5μmol/L DZNep was chosen as the optimal concentration and48h as theoptimal time. However, the results suggested that SAHH expression was lower thanthe control group after treatment with DZNep, but with no significant difference(P>0.05). SAHH expression at protein and mRNA levels in EC9706and Eca109cellswere detected after treated with DZNep by Western Blot and RT-PCR, and the resultsshowed that SAHH expression at protein and mRNA levels did not changesignificantly (P>0.05). The change of the expression of EZH2was detected byWestern Blot, and the results suggested that the expression of EZH2in treatmentgroup was significantly lower than the control group (P<0.05).2Detection of the effect of DZNep on cell cycle, proliferation,migration, apoptosis and adherence factor of ESCC cellsFlow cytometry was used to detect changes of cell cycle in EC9706and Eca109cells, and we found that the cell cycle of EC9706cells had no significant changecompared with control group (P>0.05) after treating with DZNep, while the cell cycleof Eca109cells changed significantly (P<0.05). The results of EdU and CCK-8assayshowed that proliferation rates of Eca109and EC9706cells treated with DZNep wassignificantly lower than the control group (P<0.05). The results of Transwell assayshowed that the number of migrating cells of EC9706and Eca109cells treated withDZNep was significantly less than the control group (P<0.05). Meanwhile, cellapoptosis was detected by flow cytometry, and the results showed that the cellapoptosis rates of the two cell lines in treated group was significantly higher than thecontrol group (P<0.05). Finally, the expression of E-cadherin was detected throughWestern Blot, suggesting that EC9706and Eca109cells treatment group wassignificantly higher than the control group (P<0.05).ConclusionThe optimal conditions treating the Eca109cells with DZNep were successfullydetected and it was5μmol/L for48h. We found that DZNep can inhibit theexpression of SAHH and EZH2, and the latter effect seemed more obvious. In addition, DZNep can inhibit the proliferation and migration ability of EC9706andEca109cells, promote apoptosis, and up-regulate the levels of adherence factor.