SDA-33 Inhibits Human Colon Cancer Cell Proliferation And Angiogenesis By Targeting HIF-1α

Objective:Tanshinone is a fat-soluble phenanthroquinone compound extracted from the root of Salvia miltiorrhiza Bunge,which has the effects of prostration and antibacterial.Among a series of tanshinone derivatives designed and synthesized,we experimentally found that SDA-33 has some antitumor activity,however its molecular mechanism is not clear.In this study,we compared tanshinone Ⅱ A to explore the anti-tumor molecular mechanism of action of the tanshinone derivative SDA-33 and determine its effectiveness.Methods:1.Luciferase reporter assay was used to detect the effect of SDA-33 on the transcriptional activity of hypoxia-inducible factor-1α(HIF-1α)in tumor cells;Cell survival was measured by MTT assay.2.Western blot and immunofluorescence staining were used to detect the effect of SDA-33 on HIF-1α protein level in different cancer cell lines.3.Western blot assay was performed to examine the effect of SDA-33 on HIF-1αprotein synthesis and degradation in human colon cancer HCT116 cells.4.Western blot assay was used to further examine the effect of SDA-33 on mTOR/p70S6K/4E-BP1 and MAPK signaling pathways.5.Reverse transcription-polymerase chain reaction(RT-PCR)and Western blot experiments were performed to determine the effects of SDA-33 on vascular endothelial growth factor(VEGF)and erythropoietin(EPO)protein and mRNA levels,which are related target genes regulated by HIF-1α.6.EdU assay and cell colony formation assay were used to determine the effect of SDA-33 on HCT116 cell proliferation;Cell migration and invasion assay were used to detect the effect of SDA-33 on cell angiogenesis.7.The effect of SDA-33 on apoptosis induced by tumor necrosis factor-a(TNF-α)in HCT116 cells was examined by flow cytometry.Result:1.The tanshinone derivative-SDA-33 reduced the transcriptional activity and protein level of HIF-1α in a dose-dependent manner,with a more significant inhibitory effect compared with tanshinone Ⅱ A,and no significant toxic effect on tumor cells.2.SDA-33 inhibited the protein synthesis of HIF-1α in HCT116 cells without affecting its protein degradation.3.SDA-33 inhibits HIF-1α protein synthesis by down-regulating mTOR/p70S6K/4E-BP1 and MAPK signal transduction pathways.4.SDA-33 inhibits the protein and mRNA levels of VEGF and EPO in a dose-dependent manner.5.SDA-33 inhibits tumor angiogenesis by inhibiting cell migration and invasion through down-regulation of HIF-1α.6.SDA-33 inhibits tumor cell proliferation by down-regulating HIF-1α,and promotes apoptosis.Conclusion:The tanshinone derivative-SDA-33 inhibited the expression of HIF-1αthrough mTOR/p70S6K/4E-BP1 and MAPK signaling pathways,and then inhibited the proliferation and angiogenesis of human colon cancer cells,and the effect was more obvious compared with tanshinone Ⅱ A.In this study,we investigated the anti-tumor molecular mechanism of the tanshinone derivative-SDA-33 and provided a new scientific basis for the development of targeted therapeutics.