Limits...
Cucurbitacin E Induces Autophagy via Downregulating mTORC1 Signaling and Upregulating AMPK Activity.

Zha QB, Zhang XY, Lin QR, Xu LH, Zhao GX, Pan H, Zhou D, Ouyang DY, Liu ZH, He XH - PLoS ONE (2015)

Bottom Line: Our results showed that CuE induced autophagy as evidenced by the formation of LC3-II and colocalization of punctate LC3 with the lysosomal marker LAMP2 in HeLa and MCF7 cells.However, CuE induced much lower levels of autophagy in ATG5-knocked down cells and failed to induce autophagy in DU145 cells lacking functional ATG5 expression, suggesting the dependence of CuE-induced autophagy on ATG5.The suppression of mTORC1 activity was further confirmed by reduced recruitment of mTOR to the lysosome, which is the activation site of mTORC1.

View Article: PubMed Central - PubMed

Affiliation: College of Life Science and Technology, Jinan University, Guangzhou, China; Department of Fetal Medicine, the First Affiliated Hospital of Jinan University, Guangzhou, China.

ABSTRACT
Cucurbitacins, the natural triterpenoids possessing many biological activities, have been reported to suppress the mTORC1/p70S6K pathway and to induce autophagy. However, the correlation between such activities is largely unknown. In this study, we addressed this issue in human cancer cells in response to cucurbitacin E (CuE) treatment. Our results showed that CuE induced autophagy as evidenced by the formation of LC3-II and colocalization of punctate LC3 with the lysosomal marker LAMP2 in HeLa and MCF7 cells. However, CuE induced much lower levels of autophagy in ATG5-knocked down cells and failed to induce autophagy in DU145 cells lacking functional ATG5 expression, suggesting the dependence of CuE-induced autophagy on ATG5. Consistent with autophagy induction, mTORC1 activity (as reflected by p70S6K and ULK1S758 phosphorylation) was inhibited by CuE treatment. The suppression of mTORC1 activity was further confirmed by reduced recruitment of mTOR to the lysosome, which is the activation site of mTORC1. In contrast, CuE rapidly activated AMPK leading to increased phosphorylation of its substrates. AMPK activation contributed to CuE-induced suppression of mTORC1/p70S6K signaling and autophagy induction, since AMPK knockdown diminished these effects. Collectively, our data suggested that CuE induced autophagy in human cancer cells at least partly via downregulation of mTORC1 signaling and upregulation of AMPK activity.

No MeSH data available.


Related in: MedlinePlus

Effects of CuE on the mTORC1 signaling pathways.The phosphorylation of downstream signaling proteins of mTORC1 in HeLa (A), MCF7 (B) and DU145 (C) cells treated with CuE (1 μM) were assayed by western blotting. Representative blots of at least three independent experiments are presented and the relative densitometry ratios are shown under each band. All blots are cropped to show only the target bands for clarity. (D) Immunofluorescent analysis of mTOR colocalization with LAMP2. HeLa cells were cultured with CuE (1 μM) and then immunostained and visualized by fluorescent microscopy. Quantitative analysis is shown in the bottom panel. Scale bars: 10 μm (2 μm in magnified images).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4430304&req=5

pone.0124355.g002: Effects of CuE on the mTORC1 signaling pathways.The phosphorylation of downstream signaling proteins of mTORC1 in HeLa (A), MCF7 (B) and DU145 (C) cells treated with CuE (1 μM) were assayed by western blotting. Representative blots of at least three independent experiments are presented and the relative densitometry ratios are shown under each band. All blots are cropped to show only the target bands for clarity. (D) Immunofluorescent analysis of mTOR colocalization with LAMP2. HeLa cells were cultured with CuE (1 μM) and then immunostained and visualized by fluorescent microscopy. Quantitative analysis is shown in the bottom panel. Scale bars: 10 μm (2 μm in magnified images).

Mentions: Previous studies have reported that cucurbitacin B could suppress mTORC1/p70S6K signaling [9]. As mTORC1 signaling plays an important role in regulating autophagy under various stresses, we assayed whether CuE suppressed mTORC1 signaling by measuring the phosphorylation of p70S6K, S6 and 4E-BP1, the established markers of mTORC1 activity. Western blotting revealed that in HeLa, MCF7, and DU145 cells, CuE rapidly downregulated the levels of phosphorylated p70S6KT389 (Fig 2A–2C), indicative of decreased mTORC1 activity. Consistent with this, the phosphorylation levels of S6, a downstream substrate of p70S6K, was also decreased (Fig 2A–2C). In contrast, the phosphorylation of another mTORC1 substrate 4E-BP1T37/46 was not decreased but instead increased over time (Fig 2A–2C); 4E-BP1 expression was also increased during CuE treatment, but the underlying mechanism needs further investigation. As a direct substrate of mTORC1 and the autophagy-initiating kinase, the phosphorylation of ULK1S758 in all tested cells was suppressed in a time-dependent manner upon CuE treatment (Fig 2A–2C). To further confirm CuE-induced downregulation of mTORC1 activity, we detected the colocalization of mTOR with the lysosomal marker LAMP2, as the lysosomal surface is the site for mTORC1 activation by a complex process via the interaction with Rag guanosine triphosphatases and/or Rheb in response to many cues, including amino acids [36–38]. Under basal conditions, mTOR showed a high colocalization with LAMP2 puncta; but upon CuE treatment, mTOR had reduced colocalization with LAMP2 (Fig 2D), reflecting decreased mTORC1 activity [37]. Together, these results indicated that CuE suppressed both mTORC1/p70S6K and mTORC1/ULK1 signaling branches but not mTORC1/4E-BP1 signaling. Given the important role of mTORC1 in regulating autophagy, our data suggested that suppression of mTORC1 activity by CuE might contribute to its autophagy-inducing effect.


Cucurbitacin E Induces Autophagy via Downregulating mTORC1 Signaling and Upregulating AMPK Activity.

Zha QB, Zhang XY, Lin QR, Xu LH, Zhao GX, Pan H, Zhou D, Ouyang DY, Liu ZH, He XH - PLoS ONE (2015)

Effects of CuE on the mTORC1 signaling pathways.The phosphorylation of downstream signaling proteins of mTORC1 in HeLa (A), MCF7 (B) and DU145 (C) cells treated with CuE (1 μM) were assayed by western blotting. Representative blots of at least three independent experiments are presented and the relative densitometry ratios are shown under each band. All blots are cropped to show only the target bands for clarity. (D) Immunofluorescent analysis of mTOR colocalization with LAMP2. HeLa cells were cultured with CuE (1 μM) and then immunostained and visualized by fluorescent microscopy. Quantitative analysis is shown in the bottom panel. Scale bars: 10 μm (2 μm in magnified images).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4430304&req=5

pone.0124355.g002: Effects of CuE on the mTORC1 signaling pathways.The phosphorylation of downstream signaling proteins of mTORC1 in HeLa (A), MCF7 (B) and DU145 (C) cells treated with CuE (1 μM) were assayed by western blotting. Representative blots of at least three independent experiments are presented and the relative densitometry ratios are shown under each band. All blots are cropped to show only the target bands for clarity. (D) Immunofluorescent analysis of mTOR colocalization with LAMP2. HeLa cells were cultured with CuE (1 μM) and then immunostained and visualized by fluorescent microscopy. Quantitative analysis is shown in the bottom panel. Scale bars: 10 μm (2 μm in magnified images).
Mentions: Previous studies have reported that cucurbitacin B could suppress mTORC1/p70S6K signaling [9]. As mTORC1 signaling plays an important role in regulating autophagy under various stresses, we assayed whether CuE suppressed mTORC1 signaling by measuring the phosphorylation of p70S6K, S6 and 4E-BP1, the established markers of mTORC1 activity. Western blotting revealed that in HeLa, MCF7, and DU145 cells, CuE rapidly downregulated the levels of phosphorylated p70S6KT389 (Fig 2A–2C), indicative of decreased mTORC1 activity. Consistent with this, the phosphorylation levels of S6, a downstream substrate of p70S6K, was also decreased (Fig 2A–2C). In contrast, the phosphorylation of another mTORC1 substrate 4E-BP1T37/46 was not decreased but instead increased over time (Fig 2A–2C); 4E-BP1 expression was also increased during CuE treatment, but the underlying mechanism needs further investigation. As a direct substrate of mTORC1 and the autophagy-initiating kinase, the phosphorylation of ULK1S758 in all tested cells was suppressed in a time-dependent manner upon CuE treatment (Fig 2A–2C). To further confirm CuE-induced downregulation of mTORC1 activity, we detected the colocalization of mTOR with the lysosomal marker LAMP2, as the lysosomal surface is the site for mTORC1 activation by a complex process via the interaction with Rag guanosine triphosphatases and/or Rheb in response to many cues, including amino acids [36–38]. Under basal conditions, mTOR showed a high colocalization with LAMP2 puncta; but upon CuE treatment, mTOR had reduced colocalization with LAMP2 (Fig 2D), reflecting decreased mTORC1 activity [37]. Together, these results indicated that CuE suppressed both mTORC1/p70S6K and mTORC1/ULK1 signaling branches but not mTORC1/4E-BP1 signaling. Given the important role of mTORC1 in regulating autophagy, our data suggested that suppression of mTORC1 activity by CuE might contribute to its autophagy-inducing effect.

Bottom Line: Our results showed that CuE induced autophagy as evidenced by the formation of LC3-II and colocalization of punctate LC3 with the lysosomal marker LAMP2 in HeLa and MCF7 cells.However, CuE induced much lower levels of autophagy in ATG5-knocked down cells and failed to induce autophagy in DU145 cells lacking functional ATG5 expression, suggesting the dependence of CuE-induced autophagy on ATG5.The suppression of mTORC1 activity was further confirmed by reduced recruitment of mTOR to the lysosome, which is the activation site of mTORC1.

View Article: PubMed Central - PubMed

Affiliation: College of Life Science and Technology, Jinan University, Guangzhou, China; Department of Fetal Medicine, the First Affiliated Hospital of Jinan University, Guangzhou, China.

ABSTRACT
Cucurbitacins, the natural triterpenoids possessing many biological activities, have been reported to suppress the mTORC1/p70S6K pathway and to induce autophagy. However, the correlation between such activities is largely unknown. In this study, we addressed this issue in human cancer cells in response to cucurbitacin E (CuE) treatment. Our results showed that CuE induced autophagy as evidenced by the formation of LC3-II and colocalization of punctate LC3 with the lysosomal marker LAMP2 in HeLa and MCF7 cells. However, CuE induced much lower levels of autophagy in ATG5-knocked down cells and failed to induce autophagy in DU145 cells lacking functional ATG5 expression, suggesting the dependence of CuE-induced autophagy on ATG5. Consistent with autophagy induction, mTORC1 activity (as reflected by p70S6K and ULK1S758 phosphorylation) was inhibited by CuE treatment. The suppression of mTORC1 activity was further confirmed by reduced recruitment of mTOR to the lysosome, which is the activation site of mTORC1. In contrast, CuE rapidly activated AMPK leading to increased phosphorylation of its substrates. AMPK activation contributed to CuE-induced suppression of mTORC1/p70S6K signaling and autophagy induction, since AMPK knockdown diminished these effects. Collectively, our data suggested that CuE induced autophagy in human cancer cells at least partly via downregulation of mTORC1 signaling and upregulation of AMPK activity.

No MeSH data available.


Related in: MedlinePlus