The effect of tumor-associated macrophage on proliferation of renal carcinoma cells and related mechanism / 中华泌尿外科杂志

ABSTRACT

Objective:To investigate the effect of tumor-associated macrophage(TAM) on proliferation of renal carcinoma cells and its related mechanism.Methods:The model of TAM was established by stimulating human monocytic leukemia cell line THP-1 with phorbol myristate acetate (PMA), bacterial endotoxin (LPS) and interferon-γ (IFN- γ). Then the TAM model was co-cultured with carcinoma cell lines ACHN and 786-O in vitro .The cytokines IL-6, TNF-α and IL-1β in TAM supernatant were detected by enzyme-linked immunosorbent assay (ELISA). MTT method was used to detect the proliferation of ACHN and 786-O cells treated with supernatant of TAM or TAM/Tocilizumab. Western blot was used to detect lactate dehydrogenase A (LDHA) expression of both renal cancer cells co-cultured with TAM or TAM/Tocilizumab. The ACHN and 786-O cells with LDHA-overexpression and LDHA-knockdown were cultured in TAM supernatant in vitro. The cell proliferation was detected by MTT and the relative proliferation rate was calculated.Results:THP-1 cells was differentiated into TAM through the treatment of 80 ng/ml PMA combined with 20 ng/ml LPS and 20 ng/ml IFN- γ.The expression rate of CD68, a cell surface marker on TAM, was (36.2 ±4.5)%. When TAM was co-cultured with ACHN cells, the results of ELISA showed that the secretion of IL-6 in the supernatant was significantly elevated compared with that in the supernatant when ACHN cells cultured alone [(138.0 ±12.4) pg/ml and (19.7±4.9) pg/ml], and the secretion of TNF- α [(122.5 ±14.2) pg/ml and (12.6 ±2.3) pg/ml] and IL-1 β [(89.2 ±6.4) pg/ml and (69.2 ±3.5) pg/ml] were also significantly increased. The secretion of IL-6 [(119.2 ±14.8) pg/ml and (17.1 ±3.3) pg/ml], TNF- α [(122.6 ±14.4) pg/ml and (45.7 ±7.2) pg/ml] and IL-1 β [(95.1 ±11.8) pg/ml and (88.2 ±12.7) pg/ml] in the supernatant were also significantly elevated when 786-O cells co-cultured with TAM compared with 786-O cells cultured alone. After treated with the supernatant of TAM for 72 hours, the relative proliferation rates of ACHN and 786-O cells [(128.6 ±21.4)% and (124.2 ±19.7)%] were significantly higher than that of the control group (100.0%). At the same time, the expression of LDHA in ACHN and 786-O cells increased significantly. After 72 hours of treatment with the supernatant of TAM combined with tocilizumab, the relative proliferation rates of ACHN and 786-O cells [(76.5±13.7)% and (74.8±12.5)%] were significantly lower than that of the control group(100.0%), and the expression of LDHA was also significantly decreased at the same time. The relative proliferation rates of ACHN and 786-O cells in LDHA overexpression group [(121.5 ±17.2)% and (122.7±21.6)%]were significantly higher than that in blank-vector-transfection group[(93.3±10.7)% and (89.8±11.2)%], while the relative proliferation rates in LDHA-knockdown group [(61.4±11.2)% and (58.0 ±10.6)% ]were significantly lower than that in blank-vector-transfection group.Conclusions:By secreting IL-6, TAM can up-regulate the expression of LDHA and promote the proliferation of renal cancer cells.