Effect Of Macrophage Migration Inhibitory Factor On The Expression Of Angiogenesis Factor And PI3K Signal Transduction Pathway In Human Colon Cancer Cells

【Objectives】To investigate the effects of Macrophage migration inhibitory factor on the proliferation and expression of vascular endothelial growth factor (VEGF) and Interleukin-8 (IL-8) in colon carcinoma cell line HT-29,and to further analyze the signal transduction mechanism of MIF in regulating the growth of HT-29 cells and angiogenesis via PI3K/Akt pathway.【Methods】The cultured HT-29 cells were treated with different concentrations of recombinant human Macrophage migration inhibitory factor ( rhMIF ) (0 , 25, 50,100μg/L) and 50μg/L rhMIF combined LY294002(25μmol/L) for different time,and the cells were harvested. Using:(1) MTT assay were used to measure the proliferation of HT-29 in 0,12,24,48 hours respectively.(2) The expression of VEGF and IL-8 mRNA in HT-29 cells were examined by real-time quantitative reverse-transcription-polymerase chain reaction (RT-PCR).(3) The levels of VEGF and IL-8 protein in the supernatants were determined by enzyme-linked immunosorbent assay (ELISA).(4) Western blotting was performed to detect the expressions of p-Akt after the rhMIF treatment for 0,15,30,60 minutes,and the inhibition of PI3K/Akt by LY294002 for 30 minutes.【Results】 (1) RhMIF promoted the proliferation of HT-29 cells in a concentration- and time-dependent manner (P <0.05), and the proliferating activity was raised with the increase of concentration and prolonging of action time. LY294002 suppressed the proliferation of HT-29 cells (P < 0.01),LY294002 combined with rhMIF couldn't promote the proliferation of HT-29 cells(P>0.05).(2) The expression of VEGF and IL-8 was significantly increased at mRNA levels in the cells treated by rhMIF in a concentration- and time-dependent manner(P<0.05), but the up-regulation of VEGF and IL-8 was obviously inhibited by LY294002 pre-treatment (P<0.01).(3) The levels of VEGF and IL-8 protein in supernatants were raised gradually after rhMIF treatment with the increase of concentration or action time (P<0.05),the highest levels of VEGF and IL-8 was six times and thirty times than control group, respectively. But that in LY294002 pre-treatment groups was lower than the rhMIF-induced (P< 0.01).(4) Western-blot analysis demonstrated that:50μg/L rhMIF significantly induced the activity of Akt at 15,30,60minutes.The maximal activation of Akt took place at 30min,with increased doses of rhMIF,the activation of Akt increased gradually.PI3K inhibitor, LY294002, could significantly inhibited the activation of Akt induced by rhMIF in HT-29.【Conclusions】(1) MIF promotes the growth and proliferation of colon carcinoma cells,MIF plays a crucial role in the angiogenesis of colon cancer and the roles of MIF may be realized via increasing the expression of angiogenic factors such as VEGF and IL-8.(2) MIF can promote the proliferation and regulate the expression of VEGF and IL-8 of colon carcinoma cells via PI3K/Akt pathway.