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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: 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.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: 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.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