Abstract: 【Abstract】 Objective To investigate mechanisms underlying the signal crosstalk of VEGF-IL-6-STAT3 between cutaneous melanoma cells and vascular endothelial cells. Methods EC-304 vascular endothelial cells were divided into 3 groups: control group cultured in conventional endothelial cell-conditioned medium, vascular endothelial growth factor （VEGF） group cultured in endothelial cell-conditioned medium containing 50 μg/L VEGF165, A375 co-culture group co-cultured with a melanoma cell line A375. After 24-, 48- and 72-hour treatment, the culture medium was collected, and enzyme-linked immunosorbent assay was performed to detect the level of interleukin-6 （IL-6）. Cultured A375 cells were divided into 4 groups: control group receiving conventional culture in Dulbecco′s modified Eagle′s medium （DMEM）, A375 + EC-304 group co-cultured with EC-304 cells, A375 + EC-304 + IL-6 group co-cultured with EC-304 cells in DMEM containing 50 μg/L IL-6 （an agonist of the signal transducer and activator of transcription-3 ［STAT3］ pathway ）, A375 + EC-304 + JSI-124 group co-cultured with EC-304 cells in DMEM containing 1 μmol/L JSI-124（a STAT3 pathway inhibitor）. After 24-, 48- and 72-hour treatment, cells were collected, and Western blot analysis, cell counting kit-8 （CCK8） assay and Transwell invasion assay were performed to determine the protein expression of STAT3 and phosphorylated （p）-STAT3, cellular proliferative activity and invasive activity, respectively. Two-way analysis of variance and t test were used for statistical analysis. Results The level of IL-6 significantly increased in the culture medium of EC-304 cells in the VEGF group and A375 co-culture group compared with the control group （FVEGF = 29.63, P < 0.001; FA375 = 11.09, P = 0.020）. Compared with the control group, the A375 + EC-304 group showed significantly enhanced protein expression of p-STAT3 in A375 cells （P < 0.001）, increased cell activity （P < 0.001）, and increased number of invasive cells (152.66 ± 16.04 vs. 86.13 ± 7.24, t = 4.43, P < 0.001）; compared with the A375 + EC-304 group, the A375 + EC-304 + IL-6 group showed significantly increased protein expression of p-STAT3 （P < 0.001）, enhanced cell activity （P < 0.001）, and increased number of invasive cells （187.34 ± 14.38, t = 2.17, P < 0.001）; compared with the A375 + EC-304 group, the A375 + EC-304 + JSI-124 group showed significantly decreased protein expression of p-STAT3 （P < 0.001）, decreased cell activity （P < 0.001）, and decreased number of invasive cells （124.92 ± 8.72, t = -1.86, P < 0.001）. Conclusion There is a signal crosstalk of VEGF-IL-6-STAT3 between cutaneous melanoma cells and vascular endothelial cells, which may play an important role in the proliferation and invasion of A375 cells.

Key words: Cutaneous malignant melanom, Vascular endothelial cell, VEGF, IL-6, STAT3, Crosstalk