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Induction of apoptosis and autophagy via sirtuin1- and PI3K/Akt/mTOR-mediated pathways by plumbagin in human prostate cancer cells.

Zhou ZW, Li XX, He ZX, Pan ST, Yang Y, Zhang X, Chow K, Yang T, Qiu JX, Zhou Q, Tan J, Wang D, Zhou SF - Drug Des Devel Ther (2015)

Bottom Line: Our study has shown that PLB had potent pro-apoptotic and pro-autophagic effects on PC-3 and DU145 cells.In addition, PLB downregulated pre-B cell colony-enhancing factor/visfatin, and the inhibition of pre-B cell colony-enhancing factor/visfatin significantly enhanced basal and PLB-induced apoptosis and autophagy in both cell lines.Moreover, reduction of intracellular reactive oxygen species (ROS) level attenuated the apoptosis- and autophagy-inducing effects of PLB on both PC-3 and DU145 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA ; Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guizhou Medical University, Guiyang, Guizhou, People's Republic of China.

ABSTRACT
Plumbagin (PLB) has been shown to have anticancer activities in animal models, but the role of PLB in prostate cancer treatment is unclear. This study aimed to investigate the effects of PLB on apoptosis and autophagy and the underlying mechanisms in human prostate cancer cell lines PC-3 and DU145. Our study has shown that PLB had potent pro-apoptotic and pro-autophagic effects on PC-3 and DU145 cells. PLB induced mitochondria-mediated apoptosis and autophagy in concentration- and time-dependent manners in both PC-3 and DU145 cells. PLB induced inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase (MAPK) pathways and activation of 5'-AMP-dependent kinase (AMPK) as indicated by their altered phosphorylation, contributing to the pro-autophagic activity of PLB. Modulation of autophagy altered basal and PLB-induced apoptosis in both cell lines. Furthermore, PLB downregulated sirtuin 1 (Sirt1), and inhibition of Sirt1 enhanced autophagy, whereas the induction of Sirt1 abolished PLB-induced autophagy in PC-3 and DU145 cells. In addition, PLB downregulated pre-B cell colony-enhancing factor/visfatin, and the inhibition of pre-B cell colony-enhancing factor/visfatin significantly enhanced basal and PLB-induced apoptosis and autophagy in both cell lines. Moreover, reduction of intracellular reactive oxygen species (ROS) level attenuated the apoptosis- and autophagy-inducing effects of PLB on both PC-3 and DU145 cells. These findings indicate that PLB promotes apoptosis and autophagy in prostate cancer cells via Sirt1- and PI3K/Akt/mTOR-mediated pathways with contribution from AMPK-, p38 MAPK-, visfatin-, and ROS-associated pathways.

No MeSH data available.


Related in: MedlinePlus

Effect of PLB on the expression level of molecule targets in autophagy signaling pathway.Notes: The phosphorylation levels of PI3K, AMPK, p38 MAPK, and Akt, and the total levels of mTOR, PTEN, beclin 1, LC3-I, and LC3-II in PC-3 and DU145 cells were determined by Western blotting assay. β-actin was used as the internal control. (A) Representative blots showing the phosphorylation levels of PI3K, AMPK, p38 MAPK, and Akt, and the total levels of mTOR, PTEN, beclin 1, LC3-I, and LC3-II in PC-3 and DU145 cells treated with PLB at 0.1 μM, 1 μM, and 5 μM for 24 hours. (B) Bar graphs showing the phosphorylation levels of PI3K, AMPK, p38 MAPK, and Akt, and the total levels of mTOR, PTEN, beclin 1, LC3-I, and LC3-II in PC-3 and DU145 cells. Data represent the mean ± SD of three independent experiments. *P<0.05; **P<0.01; and ***P<0.001 by one-way ANOVA.Abbreviations: AMPK, 5′-AMP-dependent kinase; ANOVA, analysis of variance; hr, hour; LC3, microtubule-associated protein 1A/1B-light chain 3; mTOR, mammalian target of rapamycin; p38 MAPK, p38 mitogen-activated protein kinase; PI3K, phosphatidylinositide 3-kinase; PLB, plumbagin; PTEN, phosphatase and tensin homolog; SD, standard deviation.
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f5-dddt-9-1511: Effect of PLB on the expression level of molecule targets in autophagy signaling pathway.Notes: The phosphorylation levels of PI3K, AMPK, p38 MAPK, and Akt, and the total levels of mTOR, PTEN, beclin 1, LC3-I, and LC3-II in PC-3 and DU145 cells were determined by Western blotting assay. β-actin was used as the internal control. (A) Representative blots showing the phosphorylation levels of PI3K, AMPK, p38 MAPK, and Akt, and the total levels of mTOR, PTEN, beclin 1, LC3-I, and LC3-II in PC-3 and DU145 cells treated with PLB at 0.1 μM, 1 μM, and 5 μM for 24 hours. (B) Bar graphs showing the phosphorylation levels of PI3K, AMPK, p38 MAPK, and Akt, and the total levels of mTOR, PTEN, beclin 1, LC3-I, and LC3-II in PC-3 and DU145 cells. Data represent the mean ± SD of three independent experiments. *P<0.05; **P<0.01; and ***P<0.001 by one-way ANOVA.Abbreviations: AMPK, 5′-AMP-dependent kinase; ANOVA, analysis of variance; hr, hour; LC3, microtubule-associated protein 1A/1B-light chain 3; mTOR, mammalian target of rapamycin; p38 MAPK, p38 mitogen-activated protein kinase; PI3K, phosphatidylinositide 3-kinase; PLB, plumbagin; PTEN, phosphatase and tensin homolog; SD, standard deviation.

Mentions: Next, we investigated the mechanisms for the autophagy-inducing effect of PLB in PC-3 and DU145 cells. First we examined the phosphorylation levels of PI3K at Tyr199, AMPK at Thr172, and p38 MAPK at Thr180/Tyr182 that are upstream signaling molecules of Akt/mTOR pathway and play an important role in the regulation of cell proliferation and death.10,11 PI3K catalyzes the formation of phosphatidylinositol-3,4,5-triphosphate via phosphorylation of phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate.30 Growth factors and hormones trigger this phosphorylation event, which in turn coordinates cell growth, cell cycle, cell migration, and cell survival. In this study, PLB significantly inhibited the phosphorylation of PI3K at Tyr199 in both cell lines in a concentration-dependent manner compared to the control cells (Figure 5A and B). Exposure of PC-3 cells to 5 μM PLB for 24 hours decreased the phosphorylation level of PI3K at Tyr199 by 38.2% (P<0.05; Figure 5A and B). Treatment of DU145 cells with 5 μM PLB for 24 hours reduced the phosphorylation level of PI3K at Tyr199 by 16.0% (P<0.01; Figure 5A and B). However, incubation of both cell lines with PLB did not significantly affect the expression of total PI3K. The ratio of p-PI3K over total PI3K was concentration-dependently decreased by PLB in both cell lines compared to the control cells. In PC-3 cells, the p-PI3K/PI3K ratio decreased from 2.3 at basal level to 2.0, 1.7, and 1.5, when PC-3 cells were treated with PLB at 0.1 μM, 1 μM, and 5 μM, respectively (P<0.05 or 0.01; Figure 5A and B). In DU145 cells, treatment with 5 μM PLB decreased the ratio of p-PI3K/PI3K by 27.1% compared to the control cells (2.0 vs 2.7) (P<0.05; Figure 5A and B).


Induction of apoptosis and autophagy via sirtuin1- and PI3K/Akt/mTOR-mediated pathways by plumbagin in human prostate cancer cells.

Zhou ZW, Li XX, He ZX, Pan ST, Yang Y, Zhang X, Chow K, Yang T, Qiu JX, Zhou Q, Tan J, Wang D, Zhou SF - Drug Des Devel Ther (2015)

Effect of PLB on the expression level of molecule targets in autophagy signaling pathway.Notes: The phosphorylation levels of PI3K, AMPK, p38 MAPK, and Akt, and the total levels of mTOR, PTEN, beclin 1, LC3-I, and LC3-II in PC-3 and DU145 cells were determined by Western blotting assay. β-actin was used as the internal control. (A) Representative blots showing the phosphorylation levels of PI3K, AMPK, p38 MAPK, and Akt, and the total levels of mTOR, PTEN, beclin 1, LC3-I, and LC3-II in PC-3 and DU145 cells treated with PLB at 0.1 μM, 1 μM, and 5 μM for 24 hours. (B) Bar graphs showing the phosphorylation levels of PI3K, AMPK, p38 MAPK, and Akt, and the total levels of mTOR, PTEN, beclin 1, LC3-I, and LC3-II in PC-3 and DU145 cells. Data represent the mean ± SD of three independent experiments. *P<0.05; **P<0.01; and ***P<0.001 by one-way ANOVA.Abbreviations: AMPK, 5′-AMP-dependent kinase; ANOVA, analysis of variance; hr, hour; LC3, microtubule-associated protein 1A/1B-light chain 3; mTOR, mammalian target of rapamycin; p38 MAPK, p38 mitogen-activated protein kinase; PI3K, phosphatidylinositide 3-kinase; PLB, plumbagin; PTEN, phosphatase and tensin homolog; SD, standard deviation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4366042&req=5

f5-dddt-9-1511: Effect of PLB on the expression level of molecule targets in autophagy signaling pathway.Notes: The phosphorylation levels of PI3K, AMPK, p38 MAPK, and Akt, and the total levels of mTOR, PTEN, beclin 1, LC3-I, and LC3-II in PC-3 and DU145 cells were determined by Western blotting assay. β-actin was used as the internal control. (A) Representative blots showing the phosphorylation levels of PI3K, AMPK, p38 MAPK, and Akt, and the total levels of mTOR, PTEN, beclin 1, LC3-I, and LC3-II in PC-3 and DU145 cells treated with PLB at 0.1 μM, 1 μM, and 5 μM for 24 hours. (B) Bar graphs showing the phosphorylation levels of PI3K, AMPK, p38 MAPK, and Akt, and the total levels of mTOR, PTEN, beclin 1, LC3-I, and LC3-II in PC-3 and DU145 cells. Data represent the mean ± SD of three independent experiments. *P<0.05; **P<0.01; and ***P<0.001 by one-way ANOVA.Abbreviations: AMPK, 5′-AMP-dependent kinase; ANOVA, analysis of variance; hr, hour; LC3, microtubule-associated protein 1A/1B-light chain 3; mTOR, mammalian target of rapamycin; p38 MAPK, p38 mitogen-activated protein kinase; PI3K, phosphatidylinositide 3-kinase; PLB, plumbagin; PTEN, phosphatase and tensin homolog; SD, standard deviation.
Mentions: Next, we investigated the mechanisms for the autophagy-inducing effect of PLB in PC-3 and DU145 cells. First we examined the phosphorylation levels of PI3K at Tyr199, AMPK at Thr172, and p38 MAPK at Thr180/Tyr182 that are upstream signaling molecules of Akt/mTOR pathway and play an important role in the regulation of cell proliferation and death.10,11 PI3K catalyzes the formation of phosphatidylinositol-3,4,5-triphosphate via phosphorylation of phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate.30 Growth factors and hormones trigger this phosphorylation event, which in turn coordinates cell growth, cell cycle, cell migration, and cell survival. In this study, PLB significantly inhibited the phosphorylation of PI3K at Tyr199 in both cell lines in a concentration-dependent manner compared to the control cells (Figure 5A and B). Exposure of PC-3 cells to 5 μM PLB for 24 hours decreased the phosphorylation level of PI3K at Tyr199 by 38.2% (P<0.05; Figure 5A and B). Treatment of DU145 cells with 5 μM PLB for 24 hours reduced the phosphorylation level of PI3K at Tyr199 by 16.0% (P<0.01; Figure 5A and B). However, incubation of both cell lines with PLB did not significantly affect the expression of total PI3K. The ratio of p-PI3K over total PI3K was concentration-dependently decreased by PLB in both cell lines compared to the control cells. In PC-3 cells, the p-PI3K/PI3K ratio decreased from 2.3 at basal level to 2.0, 1.7, and 1.5, when PC-3 cells were treated with PLB at 0.1 μM, 1 μM, and 5 μM, respectively (P<0.05 or 0.01; Figure 5A and B). In DU145 cells, treatment with 5 μM PLB decreased the ratio of p-PI3K/PI3K by 27.1% compared to the control cells (2.0 vs 2.7) (P<0.05; Figure 5A and B).

Bottom Line: Our study has shown that PLB had potent pro-apoptotic and pro-autophagic effects on PC-3 and DU145 cells.In addition, PLB downregulated pre-B cell colony-enhancing factor/visfatin, and the inhibition of pre-B cell colony-enhancing factor/visfatin significantly enhanced basal and PLB-induced apoptosis and autophagy in both cell lines.Moreover, reduction of intracellular reactive oxygen species (ROS) level attenuated the apoptosis- and autophagy-inducing effects of PLB on both PC-3 and DU145 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA ; Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guizhou Medical University, Guiyang, Guizhou, People's Republic of China.

ABSTRACT
Plumbagin (PLB) has been shown to have anticancer activities in animal models, but the role of PLB in prostate cancer treatment is unclear. This study aimed to investigate the effects of PLB on apoptosis and autophagy and the underlying mechanisms in human prostate cancer cell lines PC-3 and DU145. Our study has shown that PLB had potent pro-apoptotic and pro-autophagic effects on PC-3 and DU145 cells. PLB induced mitochondria-mediated apoptosis and autophagy in concentration- and time-dependent manners in both PC-3 and DU145 cells. PLB induced inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase (MAPK) pathways and activation of 5'-AMP-dependent kinase (AMPK) as indicated by their altered phosphorylation, contributing to the pro-autophagic activity of PLB. Modulation of autophagy altered basal and PLB-induced apoptosis in both cell lines. Furthermore, PLB downregulated sirtuin 1 (Sirt1), and inhibition of Sirt1 enhanced autophagy, whereas the induction of Sirt1 abolished PLB-induced autophagy in PC-3 and DU145 cells. In addition, PLB downregulated pre-B cell colony-enhancing factor/visfatin, and the inhibition of pre-B cell colony-enhancing factor/visfatin significantly enhanced basal and PLB-induced apoptosis and autophagy in both cell lines. Moreover, reduction of intracellular reactive oxygen species (ROS) level attenuated the apoptosis- and autophagy-inducing effects of PLB on both PC-3 and DU145 cells. These findings indicate that PLB promotes apoptosis and autophagy in prostate cancer cells via Sirt1- and PI3K/Akt/mTOR-mediated pathways with contribution from AMPK-, p38 MAPK-, visfatin-, and ROS-associated pathways.

No MeSH data available.


Related in: MedlinePlus