V-ATPase Subunit Modulates Biobehavior Of Gastric Cancer Cells Through Polarizing Macrophage

[Objective]:To investigate the role that a2 NTD played in modulating macrophage polarization and its impact on the biological behavior of gastric cancer cell line SGC-7901.[Methods]:The peripheral blood mononuclear cells were isolated from healthy donor through Ficoll density gradient centrifugation and induced into macrophages, which were then divided into four groups:M-CSF group(rh M-CSF 100ng/m L and RPMI 1640),a2 NTD group(rha2NTD 500ng/m L and RPMI 1640),a2NTD+M-CSF group(rha2NTD 500ng/m L, rh M-CSF 100ng/m L and RPMI 1640) and control group(RPMI 1640 only). When it arrived forty-eight hours, double color immunofluorescence cytochemistry was used to evaluate the expression of macrophage membrane antigen; Enzyme linked immunosorbent assay(ELISA) was adopted to estimate the volume of cytokines IL-10 and IL-12 secreted in different subgroups. Transwell system was adopted to co-culture gastric cancer cells SGC-7901 with the a2 NTD group(rha2NTD 500ng/m L and RPMI 1640), control group(RPMI 1640 only) and blank group(no cell cultured). After all, the impact of macrophages on gastric cancer cell SGC-7901 proliferation was evaluated by CCK-8 assay; Apoptosis rate was assessed through Hoechst dyeing kit; Invasiveness of gastric cancer cells SGC-7901 was detected by transwell invasive test.[Results]:The expression of CD68 was detected on macrophage membrane in all four subgroups(+); The mean optical density(MD) was 0.092±0.005 in control group, 0.095±0.006 in M-CSF group, 0.094±0.005 in a2 NTD group and 0.094±0.005 in a2NTD+M-CSF group. The differences were not statistically significant(P>0.05). Meanwhile, the expression of CD206 which mainly exits on M2 macrophage membrane was hard to detect in control group(-) with MD value of 0.025±0.004, normal in M-CSF group with MD value of 0.191±0.012 and a2 NTD group(+) with MD value of 0.197±0.136, and significantly elevated in a2NTD+M-CSF group(+++) with MD value of 0.285±0.011. There was an significant difference between each group except for M-CSF group in compare with a2 NTD group(all P<0.01). The level of IL-10 was(85.65±13.64)ng/L in M-CSF group and(87.77±14.25)ng/L in a2 NTD group, both of which were higher than control group [(71.67±7.56)ng/L, P<0.01]. The level of IL-12 was(9.91±1.50)ng/L in M-CSF group and(10.15±1.80)ng/L in a2 NTD group, both of which were lower than control group [(16.87±1.10)ng/L, P<0.01]. In a2NTD+M-CSF group, the level of IL-10 was(116.98±14.27)ng/L which was higher than all other groups(all P<0.01) and the level of IL-12 was(5.31±0.88)ng/L which was lower than all other groups(all P<0.01). Macrophages from all three groups showed the ability to promote proliferation of gastric cancer cell SGC-7901, and those in a2 NTD group manifested the most powerful effect in compared with those in blank group and control group(all P<0.001). Apoptosis rate in all three groups was significant different when compared with each other(all P<0.01), and that in a2 NTD group was lower than blank group and control group(all P<0.01).The number of permeating cells were significant different when compared with each other(all P<0.01), and that of a2 NTD group(295.79±11.27) was higher than that of blank group(175.87±4.16) and control group [(224.93±7.97),(P<0.01)].[Conclusion]:(1) a2 NTD shows an effect on inducing macrophage into M2 phenotype.(2) The polarized M2 type macrophage significantly enhance proliferation and invasion ability of gastric cancer cell.