Hif-1α Independent Down-Regulation of VEGF Expression in HL60 Cells after In Vitro Exposure to ATRA.

Background: Although the biologic effects of ATRA so far are well characterized, the molecular mechanisms regulating these processes are largely unknown. The expression of VEGF has been widely demonstrated in various human malignant tumors including haematological malignancies. Some results demonstrated that high expression of VEGF can promote the proliferation and colony formation and inhibit the apoptosis of leukemic cells, which promotes the progression of hematopoietic tumors not only by stimulating vascular endothelial growth. But the molecular mechanism of modulation of VEGF secretion and expression by ATRA in leukemia cells, for example HL60 cells, has not been elucidated clearly. The study was aimed to investigate the anti-leukemic mechanism of ATRA in respect of vascular endothelial growth factor (VEGF) production and which pathway contributed to the molecular mechanism of VEGF expression modulation.

Methods: HL60 cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, glutamine, and antibiotics in a humidified 5% CO2 in air atmosphere at 37°C. The MTT assay was performed to detect the proliferation of HL60 cells. Analysis of cell cycle phase distribution was analyzed using a FACScan (Becton Dickinson, Mountain View, CA) and the MacCycle software (CELLQUEST). The differentiation of HL60 cells after exposure to ATRA was assessed by NBT reduction assay and percent of CD11b positive cells. Total MMP2/MMP-9 enzyme-linked immunosorbent assay of secretion. Semi-quantitative RT-PCR and ELISA assay were used to detect the expression of VEGF mRNA and secretion of VEGF protein in HL-60 cell lines treated by ATRA respectively. ChIP assay was taken to detect the binding of c-Myc on target gene VEGF.

Results: The results showed that the proliferation of HL60 cells was obviously inhibited with the time and density dependent. The differentiation was indicated by the up-regulation of NBT positive and CD11b positive cells. The expression of VEGF mRNA and secretion of VEGF protein were found in HL-60 cells, which could be down-regulated by exposure to ATRA for 48 and 72 hours. Level of Metalloproteinase MMP9 but not MMP2 in the culture supernatant of HL60 cells was correlated with VEGF secretion, which was demonstrated by the special inhibition of MMP inhibitor batimastat (BB-94) or ATRA treatment. It indicated that MMP9 played a role in the release of biologically active VEGF. ATRA exerts its anti-leukemia effects by reduction of the expression of VEGF mRNA and VEGF protein secretion. The VEGF mRNA or c-Myc mRNA expression was down-regulated, otherwise, HIF-1α mRNA was up-regulated in HL-60 cells after exposure to ATRA. The antisense c-Myc could block the VEGF mRNA expression, but there is not obvious variation of c-Myc binding on promoter of target gene VEGF. The results indicated that the down-regulation of VEGF by ATRA was not Hif-1α dependent, otherwise it was related to the indirect pathway of c-Myc.

Conclusions: c-Myc but not HIF-1α indirect pathway contributes to the down-regulation of VEGF expression in HL60 cells induced by ATRA.