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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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Related in: MedlinePlus

Role of PI3K/Akt/mTOR in the accumulation of in U266 cells. (a) U266 cells were exposed to normoxia or hypoxia in the presence or absence of different concentrations of GA (0 and 0.2 μM) for 4 h. Then, the protein expression involved PI3K/Akt/mTOR pathway as analyzed by western blots. β-actin was used as a loading control. (b) For quantity of (a), images were analyzed using Image J. Bars were the mean ± SD (n = 3). The comparisons were made relative to hypoxia alone group, and the different levels of significance was indicated as **P < 0.01. (c) U266 cells were exposed to normoxia or hypoxia in the presence or absence of LY294002 (20 μM) or rapamycin (20 nM) for 4 h. Then, the protein expression involved PI3K/Akt/mTOR pathway was analyzed by western blots. β-actin was used as a loading control. (d) For quantity of (c), images were analyzed using Image J. Bars are the mean ± SD (n = 3). The comparisons were made relative to hypoxia alone group, and the different levels of significance was indicated as **P < 0.01. (e) U266 cells were exposed to normoxia or hypoxia in the presence or absence of LY294002 (20 μM) or rapamycin (20 nM) for 4 h. vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) mRNA were detected by real-time polymerase chain reaction (PCR) and analyzed by the ΔΔCt method. Bars are shown as mean ± SD (n = 3). The comparisons were made relative to hypoxia alone group, and the different levels of significance was indicated as **P < 0.01.
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fig04: Role of PI3K/Akt/mTOR in the accumulation of in U266 cells. (a) U266 cells were exposed to normoxia or hypoxia in the presence or absence of different concentrations of GA (0 and 0.2 μM) for 4 h. Then, the protein expression involved PI3K/Akt/mTOR pathway as analyzed by western blots. β-actin was used as a loading control. (b) For quantity of (a), images were analyzed using Image J. Bars were the mean ± SD (n = 3). The comparisons were made relative to hypoxia alone group, and the different levels of significance was indicated as **P < 0.01. (c) U266 cells were exposed to normoxia or hypoxia in the presence or absence of LY294002 (20 μM) or rapamycin (20 nM) for 4 h. Then, the protein expression involved PI3K/Akt/mTOR pathway was analyzed by western blots. β-actin was used as a loading control. (d) For quantity of (c), images were analyzed using Image J. Bars are the mean ± SD (n = 3). The comparisons were made relative to hypoxia alone group, and the different levels of significance was indicated as **P < 0.01. (e) U266 cells were exposed to normoxia or hypoxia in the presence or absence of LY294002 (20 μM) or rapamycin (20 nM) for 4 h. vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) mRNA were detected by real-time polymerase chain reaction (PCR) and analyzed by the ΔΔCt method. Bars are shown as mean ± SD (n = 3). The comparisons were made relative to hypoxia alone group, and the different levels of significance was indicated as **P < 0.01.

Mentions: The PI3K/Akt/mTOR pathway is a crucial regulator of cell proliferation and angiogenesis. It has been reported that PI3K/Akt signaling pathway may be involved in hypoxia-induced HIF-1α protein accumulation and its downstream target gene expression.(14,15) To explore whether GA can inhibit hypoxia-mediated activation of PI3K/Akt/mTOR, U266 cells were treated with 0.2 μM GA for 4 h under hypoxia. Our data showed hypoxia augmented phosphorylation of Akt and mTOR in U266 cells, which were all attenuated by GA (Fig. 4a,b). Interestingly, GA did not affect the total protein levels of these kinases, suggesting that the action of GA was specific to the protein activation.


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)

Role of PI3K/Akt/mTOR in the accumulation of in U266 cells. (a) U266 cells were exposed to normoxia or hypoxia in the presence or absence of different concentrations of GA (0 and 0.2 μM) for 4 h. Then, the protein expression involved PI3K/Akt/mTOR pathway as analyzed by western blots. β-actin was used as a loading control. (b) For quantity of (a), images were analyzed using Image J. Bars were the mean ± SD (n = 3). The comparisons were made relative to hypoxia alone group, and the different levels of significance was indicated as **P < 0.01. (c) U266 cells were exposed to normoxia or hypoxia in the presence or absence of LY294002 (20 μM) or rapamycin (20 nM) for 4 h. Then, the protein expression involved PI3K/Akt/mTOR pathway was analyzed by western blots. β-actin was used as a loading control. (d) For quantity of (c), images were analyzed using Image J. Bars are the mean ± SD (n = 3). The comparisons were made relative to hypoxia alone group, and the different levels of significance was indicated as **P < 0.01. (e) U266 cells were exposed to normoxia or hypoxia in the presence or absence of LY294002 (20 μM) or rapamycin (20 nM) for 4 h. vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) mRNA were detected by real-time polymerase chain reaction (PCR) and analyzed by the ΔΔCt method. Bars are shown as mean ± SD (n = 3). The comparisons were made relative to hypoxia alone group, and the different levels of significance was indicated as **P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig04: Role of PI3K/Akt/mTOR in the accumulation of in U266 cells. (a) U266 cells were exposed to normoxia or hypoxia in the presence or absence of different concentrations of GA (0 and 0.2 μM) for 4 h. Then, the protein expression involved PI3K/Akt/mTOR pathway as analyzed by western blots. β-actin was used as a loading control. (b) For quantity of (a), images were analyzed using Image J. Bars were the mean ± SD (n = 3). The comparisons were made relative to hypoxia alone group, and the different levels of significance was indicated as **P < 0.01. (c) U266 cells were exposed to normoxia or hypoxia in the presence or absence of LY294002 (20 μM) or rapamycin (20 nM) for 4 h. Then, the protein expression involved PI3K/Akt/mTOR pathway was analyzed by western blots. β-actin was used as a loading control. (d) For quantity of (c), images were analyzed using Image J. Bars are the mean ± SD (n = 3). The comparisons were made relative to hypoxia alone group, and the different levels of significance was indicated as **P < 0.01. (e) U266 cells were exposed to normoxia or hypoxia in the presence or absence of LY294002 (20 μM) or rapamycin (20 nM) for 4 h. vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) mRNA were detected by real-time polymerase chain reaction (PCR) and analyzed by the ΔΔCt method. Bars are shown as mean ± SD (n = 3). The comparisons were made relative to hypoxia alone group, and the different levels of significance was indicated as **P < 0.01.
Mentions: The PI3K/Akt/mTOR pathway is a crucial regulator of cell proliferation and angiogenesis. It has been reported that PI3K/Akt signaling pathway may be involved in hypoxia-induced HIF-1α protein accumulation and its downstream target gene expression.(14,15) To explore whether GA can inhibit hypoxia-mediated activation of PI3K/Akt/mTOR, U266 cells were treated with 0.2 μM GA for 4 h under hypoxia. Our data showed hypoxia augmented phosphorylation of Akt and mTOR in U266 cells, which were all attenuated by GA (Fig. 4a,b). Interestingly, GA did not affect the total protein levels of these kinases, suggesting that the action of GA was specific to the protein activation.

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