The Suppressive Effect Of Adenovirus-expressing Tumor Necrosis Factor (TNF)-alpha-induced Protein8-like2(TIPE2) On Gastric Cancer Cells And Its Underlying Mechanism

Objective: To investigate the effect of tumor necrosis factor （TNF）-alpha-inducedprotein8-like2（TIPE2） on gastric cancer cells and its underlying molecular mechanism.Methods: The total RNA was purified from human peripheral blood mononuclearcells （PBMCs）. The human TIPE2cDNA fragment was then amplified by reversetranscription-polymerase chain reaction （RT-PCR）, subcloned in pMD19-T vector andsequenced. The human TIPE2coding sequence （CDS） fragment was amplified by PCRusing pMD19-T-TIPE2plasmid as template, subcloned into pAdTrack-CMV transferplasmid between SalI and XhoI sites and identified by PCR, double endonuclease digestionand sequencing, to generate pAdTrack-CMV-TIPE2recombinant transfer plasmid. ThePmeI-linearized pAdTrack-CMV-TIPE2plasmid and pAdEasy-1backbone plasmid werecotransformed into BJ5183competent bacteria for homologous recombination. Theresultant pAdEasy-1-pAdTrack-CMV-TIPE2（pAd-TIPE2） homologous recombinantplasmid purified from BJ5183bacteria was further transformed into Top10competentbacteria to amplify pAd-TIPE2plasmid. Then pAd-TIPE2plasmid was linearized withPacI and the large fragment was transfected into293human embryonic kidney cells byLipofectamine2000, leading to formation of recombinant adenovirus AdVTIPE2expressing human TIPE2. The AdVTIPE2adenovirus was abundantly amplified in293cells and purified by cesium chloride （CsCl） density-gradient ultracentrifugation. Theexpression of endogenic human TIPE2in AGS, HGC27and SGC-7901human gastriccancer cells and GES-1normal human mucous epithelial cells were detected by RT-PCR.Adenovirus-mediated human TIPE2expression in293cells and AGS gastric cancer cellswere detected by RT-PCR and Western blot, respectively. The effect ofadenovirus-mediated human TIPE2expression on growth, apoptosis, migration and invasion in AGS gastric cancer cells was examined by3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay, flowcytometric analysis of Annexin V-PE/7-AAD double staining, wound healing assay andTranswell chamber assay, respectively. The effect of adenovirus-mediated human TIPE2expression on expression of Bcl-2, Bcl-XL, Bax, Bak, Caspase-9, Caspase-3, PARP,p-ERK1/2, ERK1/2, p-AKT, AKT, p-GSK3β, GSK3β, E-cadherin, N-cadherin, Vimentin,Snail1, Snail2/Slug, Twist and Zeb1in AGS gastric cancer cells were determined byWestern blot.Results:（1） The specific684bp human TIPE2cDNA fragment containing555bpCDS was successfully cloned from human PBMCs and subcloned into pMD19-T vector,which is consistent with the sequence reported in GenBank （NM₀24575.4）.（2） ThepAdTrack-CMV-TIPE2containing555bp human TIPE2CDS recombinant transferplasmid and pAdEasy-1-pAdTrack-CMV-TIPE2（pAd-TIPE2） adenoviral homologousrecombinant plasmid was successfully constructed, and the recombinant adenovirusexpressing human TIPE2（AdVTIPE2） was obtained.（3） AdVTIPE2was capable ofeffectively directing human TIPE2gene transfer and expression in mammalian cells suchas293cells and AGS gastric cells.（4） Human TIPE2was detected in GES-1humanmucous epithelial cells but not in AGS, HGC27and SGC-7901human gastric cancer cells.（5） AdVTIPE2significantly inhibited AGS gastric cancer cell growth （relative vitality,42.7±4.5%PBS control at hour72post infection） and induced AGS apoptosis （apoptoticrate,49.2±4.5%at hour48post infection）.（6） AdVTIPE2remarkably suppressed AGSgastric cancer cell migration （relative migratory ability,65.3±5.5%PBS control） andinvasion （relative invasive ability,56.5±6.5%PBS control）.（7） AdVTIPE2exhibitedobvious effect on upregulation of Bax, cleaved Caspase-9, cleaved Caspase-3, cleavedPARP and E-cadherin as well as downregulation of Bcl-XL, p-ERK1/2, p-AKT, p-GSK3β,N-cadherin, Vimentin, Snail1, Snail2/Slug and Zeb1, but not on Bcl-2, Bak, ERK1/2, AKT,GSK3β and Twist, in AGS gastric cancer cells.Conclusions:（1） To our knowledge, we for the first time demonstrated the TIPE2was lost in human gastric cancer cells.（2） TIPE2was capable of directly inhibited gastriccancer cell growth and induced gastric cancer apoptosis very possibly via activation ofintrinsic apoptotic pathway and downregulation of PI3K/AKT and Ras/Raf/MEK/ERK1/2signaling pathway.（3） TIPE2suppressed gastric cancer metastasis very possibly through downregulation of epithelial-mesenchymal transition （EMT） transcription factors andswitch from N-cadherin to E-cadherin.