Apoptosis Effects Of ODC And AdoMetDC Biantisense Virus On Esophageal Cancer Cell Eca109 Cancer Cells

Polyamine is multi-polyion of aliphatic in all living beings or organisms, including Put,Spd and Spm,have critical physiological functions in cell growth and differentiation.Polyamines could regulate the genetic expression through changing the constructions of DNA or regulate the signal transmission of the cells.Decarboxylase(ODC) and S-adenosylmethionine decarboxylase(AdoMetDC) are the key enzymes in the biosynthesis of polyamines.High polyamine levels and elevated polyamine synthesis activity were found in many tumors.And the overexpression of ODC or AdoMetDC was also reported to cause malignant transformation. Therefore,inhibition of ODC and/or AdoMetDC activity might induce a depletion of intracellular polyamines,providing an effective anticancer treatment strategy,we prepare to construct an adenoviral vector harboring an additional antisense AdoMetDC sequence and ODC sequence to suppress Ecol09 cells.Our study showed that adenovirus-mediated antisense ODC and AdoMetDC expression inhibits tumor cell growth through blocking the polyamine synthesis pathway.It suggested that the recombinant adenovirus Ad-ODC-AdoMetDCas might be a new anticancer reagent in the treatment of esophageal cancers.【Methods】①The construction of ODC and AdoMetDC Biantisense Virus.②Adenovirus-mediated gene transduction efficiency was assessed with counting GFP-positive cells using FACS. ③The MTT method were used to evaluate the effects of recombinant adenovirus(Ad-ODC-AdoMetDCas) at different MOI on cell proliferation.And make a growth curve of the cells.④Western Blot was used to analysis the protein of the ODC and AdoMetDC expression in Ecol09 cells respectively.The ECAI09 cells were treated with phosphate-buffered saline(PBS),Ad-GFP,Ad-ODCas,or Ad-ODC-AdoMetDCas for 72 h,and total cell lysates were prepared in extraction buffer.Protein concentrations were quantified using the bicinchoninic acid protein assay.BCA method test the concentration of the protein.⑤HPLC system was used to measure polyamine content in ECAI09 cells infected Ad-ODC-AdoMetDCas.The Ecol09 cells were harvested by scraping and permeabilized with 5%trichloroacetic acid.The polyamines in the supernatant were separated and quantified on an ion-paired, reversed-phase HPLC system.⑥TUNEL was used to analyze cell apoptosis.The tumor cells were treated with Ad-GFP,Ad-ODCas,and hd-ODC-AdoMetDCas at an MOI of 50 or with PBS as a control.TUNEL assay kit was supplied by SantaiBiological Company and used to detect apoptotic cells.⑦The change of morphology of apoptotic cells was observed by electron microscope.【Results】①Ecol09 tumor cells were infected with AdGFP at MOIs of 5,10,20,50 and 100 for 48 h.We demonstrated that 75%of Ecol09 cells were positive for GFP at an MOI of 50,this MOI was used for further study.②Ad-ODC-AdoMetDCas could inhibit Ecol09 cell growth and invasive ability.Ad-ODC-AdoMetDCas vector-mediated gene transfer inhibited tumor cell growth through the blockade of polyamine synthesis pathway.③The Western Blot method shows that Ad-ODC-AdoMetDCas induced a greater reduction of both ODC and AdoMetDC protein in Eco109 cells compared with Ad-GFP-infected or uninfected cells.④The HPLC system shows that the both Ad-ODCas and Ad-ODCAdoMetDCas decreased the polyamine content of Ecol09 cells(p＜0.05),correlating with the downregulation of polyamine biosynthesis.⑤TUNEL proved that the rate of apoptosis in cells infected by Ad-ODC-AdoMetDCas was significantly higher than in cells infected by Ad-GFP or no virus-treated cells.⑥Characterized morphology was observed by electron microscope(chromatin condensation,nuclear disintegration,formation of apoptotic bodies).【Conclusions】Ad-ODC-AdoMetDCas has significant inhibitory effects on esophageal cancer cell proliferation,leads to cell apoptosis and bears therapeutic potential for the treatment of esophageal cancer.