Effect Of Gambogic Acid On Expression Of Hypoxia-induced Hypoxia-Inducible Factor-1?/Vascular Endothelial Growth Factor In Multiple Myeloma Cells

ObjectiveThe hypoxic environment is an important factor causing tumor angiogenesis which is strongly correlated to tumor metastasis,angiogenesis and tumor resistance therapy by activating the key transcription factor,hypoxia-inducible factor-la(HIF-1?).HIF-1? is a constant hallmark in multiple myeloma(MM)induced angiogenesis which plays an important role in the pathogenesis and progression of MM.Gambogic Acid(GA)is the major active ingredient of gamboge,which has been shown to possess antitumor effect by in vitro and in vivo study.However,the underlying molecular mechanism that whether GA inhibits MM angiogenesis remains not well understood.In this study,we investigated the effects and mechanism of GA on hypoxia induced expression of HIF-1?,and its downstream target gene vascular endothelial growth factor(VEGF)in human MM cells.MethodsThe effects of Gambogic Acid,Wogonin and Curcumin on proliferation of U266 and 8226 cells were evaluated by CCK-8 assay.After the drugs of non-cytotoxic concentration treated the hypoxia induced MM cells,secreted VEGF was assayed by ELISA and VEGF mRNA was detected by quantitative real time PCR.The effective drug was selected for the next study.The effect of drug on HIF-1? and VEGF protein in hypoxia induced MM cells was detected by Western Blot.An immunofluorescence staining in vitro that the intracellular expression of HIF-1? was assayed to confirm the Western Blot results.The activity of PI3K/Akt/mTOR pathway in hypoxia and the effect of PI3K and mTOR inhibitor on HIF-1 ?/VEGF were determined by Western Blot,as well as the effect of selected drug on PI3K/Akt/mTOR pathway in hypoxic MM cells.In vivo,the effect of selected drug on growth of tumor in MM xenografts was observed and immunohistochemistry staining of CD31,HIF-1? and VEGF was performed.ResultsRPMI-8226 and U266 MM cells were cultured in hypoxia,the VEGF mRNA expressions were up-regulated 1.50� 0.10 and 4.77�0.55 folds for 2 hours,2.88 �0.25 and 9.19�0.64 folds for 4 hours,2.74�0.23 and 4.32�0.38 folds for 8 hours,1.36 �0.12 and 2.57 � 0.25 folds for 16 hours,compared with cells cultured in normoxia,respectively.Different concentration of Gambogic Acid,Wogonin and Curcumin treated MM cells,and the max non-cytotoxic concentrations were 0.2 ?M,20 ?M and 4 ?M,respectively.Gambogic Acid of non-cytotoxic concentration(0 ?M,0.05 ? M,0.1 ? M and 0.2 ?M)treated hypoxia induced U266 cell for 4 hours,VEGF mRNA expressions were up-regulated 9.19 �0.64,7.33 �0.51,5.90�0.40 and 4.76�0.44 folds compared with normoxic control,respectively.And for 8226 cells,VEGF mRNA expressions were 2.88 �0.25,2.67 �0.15,2.34 �0.18 and 1.99 �0.21 folds,respectively.There was no effect of Gambogic Acid on VEGF mRNA expression in normoxia groups.And there were no effects of Wogonin and Curcumin on VEGF mRNA expression in either normoxia or hypoxia MM cells.The secreted VEGF in normoxic culture for 8 hours was 15.47� 1.25 and 22.47�2.05 pg/(ml*105 cells),respectively.After 0.05,0.1 and 0.2 u M Gambogic Acid treated MM cells in normoxia for 8 hours,the secreted VEGF were unchanged except for U266 cell treated with 0.2?M Gambogic Acid.However,in hypoxia for 8 hours,the secreted VEGF of U266 and 8226 cells were 31.30� 1.57 and 45.10�3.25 pg/(m*105?cells).The secreted VEGF of U266 cells were 26.28 � 1.46 and 20.74�1.39 pg/(ml*105cells)after 0.1 and 0.2 ?M Gambogic Acid treatment.And in 8226 cells,it was 33.07�3.41 pg/(ml*105cells)after 0.2 ?M Gambogic Acid treatment.The HIF-1? and VEGF proteins of U266 were up-regulated in hypoxia for 4 hours compared with normoxic group.After treatment of U266 cells with 0.05,0.1 and 0.2?M Gambogic Acid,the expression of HIF-1? and VEGF markedly decreased which was confirmed by an immunofluorescence assay in vitro that the intracellular expression of HIF-la was relatively sparser and weaker compared to those before GA treatment.Hypoxia induced increase in the level of HIF-1? subunit protein and activated the phosphatidylinositol 3-kinase(PI3K)/Akt/mammalian target protein of rapamycin(mTOR)pathway.Moreover,the treatment with PI3K and mTOR inhibitors markedly decreased HIF-1? and VEGF expressions under hypoxic condition.Mechanistic studies exhibited that GA inhibited the production of HIF-la by reducing phosphorylation of Akt and mTOR in U266 cells.Furthermore,in vivo study revealed that intravenous injection of GA once every other day for two weeks could suppress tumor growth in a dosage-dependent manner.Moreover,administration of GA did not affect the body weight of mice.Immunohistochemistry was performed and the results showed that the presence of endothelial-specific antibody CD31-stained capillaries in xenografts was dose-dependently reduced by GA treatment and the tumor sections treated with GA showed significant lower levels of VEGF and HIF-1? than that of control tumor tissue.ConclusionVEGF mRNA and protein can be up-regulated in U266 and RPMI-826 MM cells after hypoxia.GA can suppress hypoxia-activated VEGF mRNA and protein expression.HIF-1? protein can be up-regulated in U266 cells after hypoxia,and GA suppress hypoxia-induced HIF-1? expression,while the level of HIF-1? mRNA have no obviously change,suggesting that GA can control HIF-1? expression via post transcriptional regulation.Hypoxia treatment significantly increase the expression of phosphorylated PI3K/Akt/mTOR pathway in U266 cells,and the inhibitors of PI3K/Akt/mTOR pathway can decrease HIF-1?/VEGF expression.GA can suppress HIF-1?/VEGF expression by the inhibition of PI3K/Akt/mTOR signaling pathway.GA can suppress tumor volumes by antiangiogenesis activity in vivo.Therefore,GA may be a new potent therapeutic agent against human MM cells.