The Effect Of Shikonin On The Hypoxia-inducible Factor-1? And Its Signal Transduction Pathways In Tumor Cells

Objective:Shikonin has the effect of inhibiting HIF-la through high-throughput screening.Shikonin is a naphthoquinone derived from the roots of the herb Lithospermum erythrorhizon.These findings suggest that shikonin could be considered for use as a potential drug in human colon cancer therapy.Methods:We demonstrated the effect of shikonin on hypoxia-inducible factor-1(HIF-1)activation which was detected by luciferase reporter assay.MTT assay was used to detect the survival rate of HCT116 and SW620 cells.Immunofluorescence assay was used to detect HIF-la nuclear expression.The expression of HIF-la mRNA was detected by RT-PCR.Further analysis revealed that shikonin decreased newly-synthesized HIF-1? protein,without enhancing the degradation of HIF-1?protein by western blot.The effect of shikonin on intracellular ROS expression was detected by reactive oxygen species test.We next investigated the effects of shikonin on the Akt/mTOR/p70S6K/4E-BP1/eIF4E pathway by western blot.Flow cytometry was used to detect the effect of shikonin on cell cycle of HCT116.The proliferation of colon cancer cells was detected by colony formation assay and EdU assay.The effect of shikonin in vivo was examined by zenograft model experiments in nude mice.Result:Luciferase reporter gene assay show that shikonin inhibits hypoxia-inducible factor-1(HIF-1)activation,and the best effect is the highest dose(10 ?M).MTT assay show that shikonin have no significant cytotoxicity at 10 uM.Immunofluorescence assay further indicated that shikonin inhibited nuclear accumulation of HIF-la.Western blot showed that shikonin inhibits the protein synthesis of HIF-la but not its degradation and the mRNA levels.Shikonin inhibits HIF-1? protein synthesis by blocking Akt/mTOR/p70S6K/4E-BP1/eIF4E signaling.We performed cell cycle analysis by flow cytometry and showed that shikonin induced G1/S phase arrest in HCT116 cells and Influencing cell growth.Similarly,Western blotting experiments have demonstrated that drugs can inhibit the expression of cyclin in a dose-dependent manner and promote the expression of tumor suppressor genes.Colony formation assay and EdU assay showed that shikonin could inhibit the proliferation of colon cancer cells.Shikonin inhibits the growth of HCT116 cells by reducing HIF-la protein expression in tumor tissue in a xenograft.Conclusion:Shikonin is considered as a potent HIF-1? inhibitor,inhibited HIF-1?protein synthesis by down-regulating the Akt/mTOR/p70S6K/4E-BP1/eIF4E signaling pathways.These findings suggest that shikonin could be considered for use as a potential drug in human colon cancer therapy.