Effect Of 12-Deoxyphorbol-13-Palmitate On Expressions Of VEGF And HIF-1α In MCF-7 Cells And Its Related Mechanism

ObjectivesThe present research aimed at investigating the effect of 12-Deoxyphorbol 13-palmitate (DP) on the expressions of vascular endothelial growth factor (VEGF) and hypoxia induced factor lα (HIF-1α) in breast cancer MCF-7 cells. Simultaneously, we further explored the involvement of phosphatidylino-sitol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) in DP’s regulation.Methods1. MTT method was used to detect the effect of DP on MCF-7 cell viability.2. ELISA assay was applied to measure the influence of DP and PI3K inhibitor wortmannin in the expression of VEGF.3. Real time PCR was used to explore the mRNA levels of VEGF and HIF-1α.4. IF was adopted to investigate the protein level and the location of HIF-1α in MCF-7 cells.5. Western blot was applied to detect the effect of DP on phosphorylation of PI3K, Akt and mTOR, and the effect of co-treatment with wortmannin on protein levels of TSC1 and TSC2.Results1. The cells were divided into control group, hypoxic group (150 μM CoCl2),10 μM DP group (150 μM CoCl2+10 μM DP),20 μM DP group (150 μM CoCl2+20 μM DP),40μM DP group (150 μM CoCl2+40 μM DP). We found that the cell viability in hypoxic control group exhibited no difference compared with control group, but the treatments of DP depressed the cell viability in a concentration- and time-depended manner (P<0.05).2. The effect of DP on VEGF expression was investigated by ELISA and RT-PCR methods. Under hypoxic condition, VEGF expression was higher than that in normoxic condition (P<0.05). However, this enhancement of VEGF was blocked significantly by the addition of DP (10, 20,40 μM) (P<0.05).3. We found the hypoxic condition could enhance HIF-1α expression obviously (P<0.05), But the treatments of DP eliminated this increase in a concentration-depended way (P<0.05). IF assay result demostrated that in the presence of DP, the HIF-1α fluorescence intensity became weaker. Simultaneously the accumulation of HIF-1α in and around the nucleus in hypoxic control was obviously decreased after the addition of DP (20 μM). Interestingly, the mRNA level of HIF-1α remained unchanged by RT-PCR (P>0.05).4. Western blot was used to measure the effect of DP on PI3K/Akt/mTOR signaling pathway. Under hypoxic condition, treatments of DP down-regulated the expression of p-PI3K (P<0.05), which was correlative to its effectors p-Akt and p-mTOR, but then up-regulated the TSC1 and TSC2 expressions (P<0.05). Whereas the increasements of TSC1 and TSC2 and downregulation of VEGF by DP was blocked by PI3K inhibitor wortmannin.Conclusions1. DP inhibited the viability of MCF-7 cells in a time- and dose-dependented manner.2. DP depressed the hypoxia-induced expression of VEGF3. DP downregulated the expression of HIF-1α without affecting its transcriptional activity, and re-distributed the location of HIF-1α.4. PI3K/Akt/mTOR signaling pathway was involved in the effect of DP on VEGF and HIF-1α expressions.