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Gambogic acid suppresses hypoxia-induced hypoxia-inducible factor-1α/vascular endothelial growth factor expression via inhibiting phosphatidylinositol 3-kinase/Akt/mammalian target protein of rapamycin pathway in multiple myeloma cells.

Wang F, Zhang W, Guo L, Bao W, Jin N, Liu R, Liu P, Wang Y, Guo Q, Chen B - Cancer Sci. (2014)

Bottom Line: We found that hypoxia induced increase in the level of HIF-1α subunit protein and activated the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target protein of rapamycin (mTOR) pathway.Mechanistic studies exhibited that GA inhibited the production of HIF-1α by reducing phosphorylation of Akt and mTOR in U266 cells.Taken together, our results identify that GA suppresses hypoxia-activated pathways that are linked to MM progression, at least partly, by the inhibition of the PI3K/Akt/mTOR signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Oncology (Key Department of Jiangsu Medicine), Medical School, Zhongda Hospital, Southeast University, Nanjing, China.

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Gambogic acid (GA) attenuates the hypoxia-induced HIF-1α activation in U266 cells. (a) Effect of GA on hypoxia-inducible factor-1α (HIF-1α) mRNA level under normoxia and hypoxia. U266 cells were treated with various concentrations of GA (0, 0.05, 0.1, and 0.2 μM) for 4 h under normoxia and hypoxia, and HIF-1α mRNA level was detected by real-time PCR and analyzed by the ΔΔCt method. Bars are shown as mean ± SD (n = 3) and represent HIF-1α/β-actin fold relative to the untreated group. (b) Effect of GA on HIF-1α protein expression under normoxia and hypoxia. U266 cells were treated with various concentrations of GA (0, 0.05, 0.1, and 0.2 μM) for 4 h under normoxia and hypoxia. HIF-1α protein expression was analyzed by western blots. (c) For quantity of (b), images were analyzed using Image J. Bars are the mean ± SD (n = 3). The comparisons were made relative to the hypoxia alone group, and the different levels of significance was indicated as **P < 0.01. (d) Immunofluorescent staining analysis of the effect of GA on intracellular HIF-1α expression in normoxic and hypoxic U266 cells. Cells were treated with various concentrations of GA (0, 0.05, 0.1, and 0.2 μM) for 4 h under normoxia and hypoxia. Green color was detected for HIF-1α, while nuclei were counterstained with blue color using DAPI (4′6′-diamidino-2-phenylindole dihydrochloride).
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fig03: Gambogic acid (GA) attenuates the hypoxia-induced HIF-1α activation in U266 cells. (a) Effect of GA on hypoxia-inducible factor-1α (HIF-1α) mRNA level under normoxia and hypoxia. U266 cells were treated with various concentrations of GA (0, 0.05, 0.1, and 0.2 μM) for 4 h under normoxia and hypoxia, and HIF-1α mRNA level was detected by real-time PCR and analyzed by the ΔΔCt method. Bars are shown as mean ± SD (n = 3) and represent HIF-1α/β-actin fold relative to the untreated group. (b) Effect of GA on HIF-1α protein expression under normoxia and hypoxia. U266 cells were treated with various concentrations of GA (0, 0.05, 0.1, and 0.2 μM) for 4 h under normoxia and hypoxia. HIF-1α protein expression was analyzed by western blots. (c) For quantity of (b), images were analyzed using Image J. Bars are the mean ± SD (n = 3). The comparisons were made relative to the hypoxia alone group, and the different levels of significance was indicated as **P < 0.01. (d) Immunofluorescent staining analysis of the effect of GA on intracellular HIF-1α expression in normoxic and hypoxic U266 cells. Cells were treated with various concentrations of GA (0, 0.05, 0.1, and 0.2 μM) for 4 h under normoxia and hypoxia. Green color was detected for HIF-1α, while nuclei were counterstained with blue color using DAPI (4′6′-diamidino-2-phenylindole dihydrochloride).

Mentions: To investigate why GA exhibited more marked effect on VEGF secretion and expression of U266 cells under hypoxia, real time-PCR, western blots and immunofluorescence were used to evaluate the expression of HIF-1α, the key regulator of hypoxia. The results showed that in both of normoxia and hypoxia, the levels of HIF-1α mRNA were unchanged with or without GA treatment on U266 cells (Fig. 3a), but compared with little expression under normoxia, hypoxic condition elicited a robust increase of HIF-1α protein level, which can be reduced by GA in a concentration-dependent manner (Fig. 3b,c). This observation was confirmed by an immunofluorescence assay in vitro that the intracellular expression of HIF-1α was relatively sparser and weaker compared to those before GA treatment (Fig. 3d). Taken together, these findings suggested that GA might downregulate HIF-1α through decreasing translation.


Gambogic acid suppresses hypoxia-induced hypoxia-inducible factor-1α/vascular endothelial growth factor expression via inhibiting phosphatidylinositol 3-kinase/Akt/mammalian target protein of rapamycin pathway in multiple myeloma cells.

Wang F, Zhang W, Guo L, Bao W, Jin N, Liu R, Liu P, Wang Y, Guo Q, Chen B - Cancer Sci. (2014)

Gambogic acid (GA) attenuates the hypoxia-induced HIF-1α activation in U266 cells. (a) Effect of GA on hypoxia-inducible factor-1α (HIF-1α) mRNA level under normoxia and hypoxia. U266 cells were treated with various concentrations of GA (0, 0.05, 0.1, and 0.2 μM) for 4 h under normoxia and hypoxia, and HIF-1α mRNA level was detected by real-time PCR and analyzed by the ΔΔCt method. Bars are shown as mean ± SD (n = 3) and represent HIF-1α/β-actin fold relative to the untreated group. (b) Effect of GA on HIF-1α protein expression under normoxia and hypoxia. U266 cells were treated with various concentrations of GA (0, 0.05, 0.1, and 0.2 μM) for 4 h under normoxia and hypoxia. HIF-1α protein expression was analyzed by western blots. (c) For quantity of (b), images were analyzed using Image J. Bars are the mean ± SD (n = 3). The comparisons were made relative to the hypoxia alone group, and the different levels of significance was indicated as **P < 0.01. (d) Immunofluorescent staining analysis of the effect of GA on intracellular HIF-1α expression in normoxic and hypoxic U266 cells. Cells were treated with various concentrations of GA (0, 0.05, 0.1, and 0.2 μM) for 4 h under normoxia and hypoxia. Green color was detected for HIF-1α, while nuclei were counterstained with blue color using DAPI (4′6′-diamidino-2-phenylindole dihydrochloride).
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Related In: Results  -  Collection

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fig03: Gambogic acid (GA) attenuates the hypoxia-induced HIF-1α activation in U266 cells. (a) Effect of GA on hypoxia-inducible factor-1α (HIF-1α) mRNA level under normoxia and hypoxia. U266 cells were treated with various concentrations of GA (0, 0.05, 0.1, and 0.2 μM) for 4 h under normoxia and hypoxia, and HIF-1α mRNA level was detected by real-time PCR and analyzed by the ΔΔCt method. Bars are shown as mean ± SD (n = 3) and represent HIF-1α/β-actin fold relative to the untreated group. (b) Effect of GA on HIF-1α protein expression under normoxia and hypoxia. U266 cells were treated with various concentrations of GA (0, 0.05, 0.1, and 0.2 μM) for 4 h under normoxia and hypoxia. HIF-1α protein expression was analyzed by western blots. (c) For quantity of (b), images were analyzed using Image J. Bars are the mean ± SD (n = 3). The comparisons were made relative to the hypoxia alone group, and the different levels of significance was indicated as **P < 0.01. (d) Immunofluorescent staining analysis of the effect of GA on intracellular HIF-1α expression in normoxic and hypoxic U266 cells. Cells were treated with various concentrations of GA (0, 0.05, 0.1, and 0.2 μM) for 4 h under normoxia and hypoxia. Green color was detected for HIF-1α, while nuclei were counterstained with blue color using DAPI (4′6′-diamidino-2-phenylindole dihydrochloride).
Mentions: To investigate why GA exhibited more marked effect on VEGF secretion and expression of U266 cells under hypoxia, real time-PCR, western blots and immunofluorescence were used to evaluate the expression of HIF-1α, the key regulator of hypoxia. The results showed that in both of normoxia and hypoxia, the levels of HIF-1α mRNA were unchanged with or without GA treatment on U266 cells (Fig. 3a), but compared with little expression under normoxia, hypoxic condition elicited a robust increase of HIF-1α protein level, which can be reduced by GA in a concentration-dependent manner (Fig. 3b,c). This observation was confirmed by an immunofluorescence assay in vitro that the intracellular expression of HIF-1α was relatively sparser and weaker compared to those before GA treatment (Fig. 3d). Taken together, these findings suggested that GA might downregulate HIF-1α through decreasing translation.

Bottom Line: We found that hypoxia induced increase in the level of HIF-1α subunit protein and activated the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target protein of rapamycin (mTOR) pathway.Mechanistic studies exhibited that GA inhibited the production of HIF-1α by reducing phosphorylation of Akt and mTOR in U266 cells.Taken together, our results identify that GA suppresses hypoxia-activated pathways that are linked to MM progression, at least partly, by the inhibition of the PI3K/Akt/mTOR signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Oncology (Key Department of Jiangsu Medicine), Medical School, Zhongda Hospital, Southeast University, Nanjing, China.

Show MeSH
Related in: MedlinePlus