Inhibitory Effects Of ODC And AdoMetDC Biantisense Virus On Lung Neoplasms Cell A-549

【Objective】Polyamines including spermidine,spermine,and their diamine precursor, putrescine,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.In mammalian cells,the intracellular polyamine biosynthetic pathway is primarily regulated by the action of two rate-limiting enzymes.Ornithine decarboxylase(ODC)is the first key enzyme required for polyamine synthesis,decarboxylating ornithine to produce putrescine. The second,rate-limiting enzyme is S-adenosylmethionine decarboxylase (AdoMetDC).It generates the aminopropyl donor,decarboxylated Sadenosylmethionine(dcSAM),by decarboxylating adenosylmethionine.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.Difluoromethylornithine(DFMO)irreversibly inactivates ODC activity and has been used in clinical chemoprevention trials for epithelial cancers.AdoMetDC inhibitors,such as methylglyoxalbisguanylhydrazone, have also been shown to inhibit tumor growth.AdoMetDC inhibitors,such as methylglyoxalbis(MGBG),have also been shown to inhibit tumor growth.SAM486A is a new AdoMetDC inhibitor that has been shown to possess anti-proliferative activity in both tissue culture cells and preclinical animal studies.But not only the DFMO but also the MGBC and SAM486A are all instability and toxiferous.So we prepare to construct an adenoviral vector harboring an additional antisense AdoMetDC sequence and ODC sequence to suppress A-549 cells.Our study showed thatadenovirus-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 lung cancers.【Methods】①The construction of ODC and AdoMetDC Biantisense Virus.②Adenovirus-mediated gene transduction efficiency was assessed with counting GFP-positive cells using FACS.A-549 cells(3×105 cells/well)seeded in 6-well plates were infected.With Ad-GFP at different multiplicities of infection(MOIs)of 5,10,20, 50 and 100.GFP expression was analyzed at 48 h after the infection using a flow cytometer.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 A-549 cells respectively.The A-549 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 A-549 cells infected Ad-ODC-AdoMetDCas.The A-549 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 Ad-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. 【Results】①A-549 tumor cells were infected with AdGFP at MOIs of 5,10,20,50 and 100 for 48 h.We demonstrated that 75%of A-549 cells were positive for GFP at an MOI of 50,this MOI was used for further study.②Ad-ODC-AdoMetDCas could inhibit A-549 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 than 60%(p＜0.05)reduction of both ODC and AdoMetDC protein in A-549 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 A-549 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.【Conclusions】Ad-ODC-AdoMetDCas has significant inhibitory effects on lung neoplasms cell proliferation and invasion and bears therapeutic potential for the treatment of lung neoplasms.