Interleukin-12 Inhibits The Hepatocellular Carcinoma Growth By Inducing Macrophage Polarization To The M1-like Phenotype Through Downregulation Of Stat-3

Objective: To investigate whether overexpression of L-12 in monocytes could induce the phenotype directional differentiation into tumoricidal M1 macrophages, and inhibit HCC growth in tumor microenvironment. Explore the Stat-3 pathway underlying the macrophages polarization.Method: Fresh human peripheral blood mononuclear cells were isolated by Ficoll-Paque density solution, and then pAd5F35-IL-12 recombinant adenovirus infected it. After these monocytes were cultivated for 48 h, SMMC-7721 or Hep3 B cells were added in the upper insert of the transwell apparatus. The experimental cells were divided into the SMMC-7721 or Hep3 B and SMMC-7721/GFP or Hep3B/GFP control groups, as well as the SMMC-7721/IL-12 or Hep3B/IL-12 groups. The expression of human IL-12 gene and protein was detected, and thedifferentiated phenotype markers of monocytes and the growth of HCC were teated in vitro and in vivo. The green fluorescence of monocyte/IL-12 cells was observed under fluorescence microscopy, the expression of IL-12p35 and IL-12p40 mRNA was detected by RT-PCR, the protein levels of IL-12 in the supernatant of coculture system were assayed by ELISA.The characterization of cell populations was measured with the MFI of the surface receptors CD14, CD197(M1 maker) and CD206(M2 maker) by FCM, then cytokine/chemokine( IL-10, IL-12, TGF-β, VEGF-A and MMP-9)were measured by Q-PCR and ELISA. In order to explore the mechanism underlying the macrophages polarization, we detected the Stat-3 pathway and its downstream transcription factor c-myc by Western blot. The proliferation ability of HCC cell lines was assessed by the CCK8 and colony-forming assays, the cell cycle analysis of HCC cell lines was tested by FCM, and Transwell invasion assay was used to assess the invasion ability of HCC cells in vitro. To evaluate the effect of IL-12 overexpressed monocytes on the proliferation of HCC cells in vivo,NOD-SCID mice xenograft model was established.Results: Compared with the controls, the monocytes which were infected by pAd5F35-IL-12 were able to emit green fluorescence, ELISA result showed that the protein levels of IL-12 were increased in the supernatant. Compared with control groups, the MFI of CD14+CD197+cells in the Hep3B/IL-12 group and SMMC-7721/IL-12 group increased,the MFI of CD14+CD206+ cells in the Hep3B/IL-12 and SMMC-7721/IL-12 group decreased. The transcriptions of CD206, IL-10,VEGF-A, TGF-β and MMP-9(M2 associated markers) were all decreased in the Hep3B/IL-12 and SMMC-7721/IL-12 groups compared with the controls. Furthermore, the cytokine/chemokine protein levels in the supernatants of co-cultured system were measured with ELISA which showned that IL-12 was significantly increased, and IL-10, VEGF-A,TGF-β and MMP-9 were all significantly decreased in the Hep3B/IL-12 and SMMC-7721/IL-12 group. The optical density of Stat-3 and c-myc were notably decreased. The proliferation ability of those HCC cell lines was assessed by the CCK8 and colony-forming assays, and the results definitely indicated that the growth ability of both HCC cell lines was profoundly inhibited. In other side, the cell cycle analysis also revealed that the HCC cells were decreased S phase and increased G0/G1 phase population. The results of Transwell invasion assay indicated that overexpressing IL-12 monocytes significantly weakened the invasiveness of co-cultured HCC cells. Consistent with the findings in vitro, the neoplasm induced by Hep3B/IL-12 and SMMC-7721/IL-12 grew remarkably slower than those of the controls. PCNA staining showed that the IL-12 overexpressed monocytes inhibited the proliferation of HCC cells,and the results of MVD suggested that tumor angiogenesis were obviouslydecreased in the Hep3B/IL-12 and SMMC-7721/IL-12 groups.Conclusions: The recombinant adenovirus Ad-IL-12 could effectively infect the human peripheral blood monocytes and express the IL-12 P70 protein successfully. The phenotype of monocyte/IL-12 would be polarized to M1 macrophages in the coculture system, which might be related with the downregulation of Stat-3 and c-myc. These results have proven that IL-12 overexpressed monocytes could directionally differentiate to M1-like macrophages and result in the inhibition of HCC growth in vivo and vitro.