The Effect Of Inhibitor Of Growth 4 On Colorectal Cancer Cell Growth And Invasion And Its Underlying Mechanism

Objective: To investigate the effect of inhibitor of growth 4(ING4) on colorectal cancer cell growthand invasion and its underlying mechanismMethods:We quantified ING4 m RNA in high(Lo Vo and SW620) and low(LS174T and SW480)metastatic cell lines, and normal human colorectalmucous epithelial FHC cellsby q RT-PCRanalysis. The humanized ING4 coding sequence(CDS) fragment was amplified by PCR using p Ad Track-CMV/ING4 as a template and subsequently subcloned into p Lenti6.3/IRES/GFP lentiviral plasmid between Nhe I and Sgs I restriction enzyme sites to construct p Lenti6.3/ING4/IRES/GFP. Then the p Lenti6.3/ING4/IRES/GFP or blank control p Lenti6.3/IRES/GFP was cotransfected into 293 T cells with helper packaging plasmids including p LP1, p LP2 and VSVG by Lipofectamine2000. The lentivirus-expressing ING4(LV-ING4) or blank lentivirus(LV) was consequently generated and titre was determined. After infection of the Lo Vo cellswith 10 MOI LV-ING4 or LV followed by selection with Blasticidin S(BSD), we obtained the Lo Vo-ING4 and Lo Vo-Mocktransfectants. To further detect thelentiviral-mediated GFP and ING4 transgeneexpression, the Lo Vo-ING4, Lo Vo-Mock and Lo Vocontrol cells were analyzed by fluorescence microscopy, RT-PCR and Westernblot, respectively. The effect oflentiviral-mediated ING4 expression on human Lo Vo CRC cell growth, cell cycle and invasionin vitrowas assessed by MTT assay, cell cycle assay and Transwell chamber invasion assay. Theeffect of ING4 on CRC cell growth in vivo was monitored by measurement of tumor volume and tumor weight using Lo Vo CRC subcutaneously(s.c.) transplanted tumor in athymic BALB/c nude mouse model. The expression of cell cycle-related proteins such as P21, Cyclin E and CDK2, and epithelial mesenchymal transition(EMT)-associated proteins such as E-Cadherin, N-Cadherin, Vimentin, Snail1, Snail2, ZEB1 and Twist was analyzed by Western blot. The CD31 expression and microvessel density(MVD) in s.c.transplanted tumor was evaluated by immunohistochemistry analysis. The level of IL-6, IL-8 and VEGF of Lo Vo cell supernatant was measured by ELISA assay.Results:1) The ING4 expression in CRC cell is downregulated; 2) Lentivirus-mediated ING4 overexpression suppresses Lo Vo CRC cell growth in vitro and in vivo in athymic nude mouse; 3) ING4 induces cell cycle G1 phase arrest in Lo Vo CRC cells possibly via upregulation of P21 and downregulation of Cyclin E; 4)ING4 inhibits tumor angiogenesis via downregulation of IL-6, IL-8 and VEGF proangiogenic factors;5) ING4 suppresses Lo Vo CRC invasion via downregulation of Snail1 EMT-inducing transcription factor(EMT-TF), N-Cadherin and Vimentin mesenchymal markers as well as upregulation of E-Cadherin epithelial marker.Conclusions: ING4 can suppress Lo Vo CRC cell growth possibly via induction of G1 phasearrest through regulating G1 phase checkpoint molecules, and inhibition of tumor angiogenesis through reducing proangiogenic factors. ING4 is also capable of inhibiting Lo Vo CRC cell invasion and metastasis via downregulation of EMT-TF Snail1 and reversal of EMT.