The Effect Of Dihydrotanshinone I On The Hypoxia-Inducible Factor-1α And Its Signal Transduction Pathways In Tumor Cells

The hypoxia-inducible factor-1 (HIF-1) has been known to be correlated to the adaptation and proliferation of tumor cells and HIF-1 plays an important role in tumor progression, angiogenesis and metastasis, therefore HIF-1 has become an important target in the development of anticancer drugs. Dihydrotanshinone I (DHTS) is a phenanthrenequinone compound from Salvia miltiorrhiza Bunge, which has been used in oriental medicine for its antitumor and antibacterial activities. However, the mechanisms by which DHTS inhibits tumor growth are not fully understood. In the present study, we firstly demonstrated the potential molecular mechanisms underlying the anticancer effects of DHTS.We here demonstrated the effect of DHTS on hypoxia-inducible factor-1 (HIF-1) activation which was detected by luciferase reporter assay. DHTS dose-dependently decreased the hypoxia-induced accumulation and activation of HIF-1 a protein in human cervical cancer cell line HeLa which was detected by western blot and we next performed an immunofluorescence assay to evaluate the effect of DHTS on HIF-1 a expression in HeLa cells. Further analysis revealed that DHTS decreased newly-synthesized HIF-1 a protein, without enhancing the degradation of HIF-1 a protein or affecting the expression level of HIF-1 a mRNA by western blot. Moreover, phosphorylation levels of mammalian target of rapamycin (mTOR), extracellular signal-regulated kinase-1/2 (ERK1/2), ribosomal protein S6 kinase (p70S6K), eIF4E binding protein-1 (4E-BP1), and eukaryotic initiation factor 4E (eIF4E) were dose-dependently suppressed by DHTS, but no significant effect on total protein levels was observed. Furthermore, DHTS decreases expression of HIF-1 a target genes, including vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT-1), and erythropoietin (EPO) which were detected by western blot, reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometric analysis indicated that DHTS inhibits the proliferation of HeLa cells via blocking cell cycle progression in the G1 phase. The inhibition of HIF-1αa translation by of DHTS is naturally associated with a strong suppression of angiogenic factor VEGF in both in vitro cell culture and tumor tissue section of zenograft model. In vivo studies further confirmed the inhibitory effect of DHTS on the expression of HIF-la and VEGF proteins, leading to a decrease in tumor vascularization and growth of the xenografts.These results show that DHTS inhibited HIF-la protein synthesis by downregulating the PI3K/mTOR/p70S6K and ERK signaling pathways. These conclusions suggest that DHTS is an effective inhibitor of HIF-1 and provide new perspectives into the mechanism of its anticancer activity and hope DHTS could help for anticancer drug development.