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Dose-Dependent AMPK-Dependent and Independent Mechanisms of Berberine and Metformin Inhibition of mTORC1, ERK, DNA Synthesis and Proliferation in Pancreatic Cancer Cells.

Ming M, Sinnett-Smith J, Wang J, Soares HP, Young SH, Eibl G, Rozengurt E - PLoS ONE (2014)

Bottom Line: Berberine treatment also reduced (by 70%) the growth of MiaPaCa-2 cell growth when implanted into the flanks of nu/nu mice.Similar results were obtained with metformin used at doses that induced either modest or pronounced reductions in intracellular ATP levels, which were virtually identical to the decreases in ATP levels obtained in response to berberine.We propose that berberine and metformin inhibit mitogenic signaling in PDAC cells through dose-dependent AMPK-dependent and independent pathways.

View Article: PubMed Central - PubMed

Affiliation: Division of Digestive Diseases, Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
Natural products represent a rich reservoir of potential small chemical molecules exhibiting anti-proliferative and chemopreventive properties. Here, we show that treatment of pancreatic ductal adenocarcinoma (PDAC) cells (PANC-1, MiaPaCa-2) with the isoquinoline alkaloid berberine (0.3-6 µM) inhibited DNA synthesis and proliferation of these cells and delay the progression of their cell cycle in G1. Berberine treatment also reduced (by 70%) the growth of MiaPaCa-2 cell growth when implanted into the flanks of nu/nu mice. Mechanistic studies revealed that berberine decreased mitochondrial membrane potential and intracellular ATP levels and induced potent AMPK activation, as shown by phosphorylation of AMPK α subunit at Thr-172 and acetyl-CoA carboxylase (ACC) at Ser79. Furthermore, berberine dose-dependently inhibited mTORC1 (phosphorylation of S6K at Thr389 and S6 at Ser240/244) and ERK activation in PDAC cells stimulated by insulin and neurotensin or fetal bovine serum. Knockdown of α1 and α2 catalytic subunit expression of AMPK reversed the inhibitory effect produced by treatment with low concentrations of berberine on mTORC1, ERK and DNA synthesis in PDAC cells. However, at higher concentrations, berberine inhibited mitogenic signaling (mTORC1 and ERK) and DNA synthesis through an AMPK-independent mechanism. Similar results were obtained with metformin used at doses that induced either modest or pronounced reductions in intracellular ATP levels, which were virtually identical to the decreases in ATP levels obtained in response to berberine. We propose that berberine and metformin inhibit mitogenic signaling in PDAC cells through dose-dependent AMPK-dependent and independent pathways.

No MeSH data available.


Related in: MedlinePlus

Berberine inhibits mTORC1 signaling and ERK activation in PDAC cells.Cultures of MiaPaCa-2 (Panels A and B) or PANC-1 cells (panels C and D) were incubated in the absence or in the presence of increasing concentrations of berberine. Then, the cells were stimulated for 1 h with 5 nM neurotensin and 10 ng/ml insulin and lysed with 2X SDS-PAGE sample buffer. The samples were analyzed by SDS-PAGE and immunoblotting with antibodies that detect the phosphorylated state of S6K at Thr389, S6 at Ser240/244, and ERK at Thr202 and Tyr204. Immunoblotting with total S6K, S6 and ERK was used to verify equal gel loading. The quantification of the immune signals was performed using Multi Gauge V3.0. The results are presented in the plots shown in panels B and D. The values represent the mean ± SEM (n = 3) of S6K, S6 and ERK phosphorylation expressed as a percentage of the maximal response obtained in 3 independent experiments.
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pone-0114573-g004: Berberine inhibits mTORC1 signaling and ERK activation in PDAC cells.Cultures of MiaPaCa-2 (Panels A and B) or PANC-1 cells (panels C and D) were incubated in the absence or in the presence of increasing concentrations of berberine. Then, the cells were stimulated for 1 h with 5 nM neurotensin and 10 ng/ml insulin and lysed with 2X SDS-PAGE sample buffer. The samples were analyzed by SDS-PAGE and immunoblotting with antibodies that detect the phosphorylated state of S6K at Thr389, S6 at Ser240/244, and ERK at Thr202 and Tyr204. Immunoblotting with total S6K, S6 and ERK was used to verify equal gel loading. The quantification of the immune signals was performed using Multi Gauge V3.0. The results are presented in the plots shown in panels B and D. The values represent the mean ± SEM (n = 3) of S6K, S6 and ERK phosphorylation expressed as a percentage of the maximal response obtained in 3 independent experiments.

Mentions: The activation of the PI3K/Akt/mTORC1 and MEK/ERK pathways plays a pivotal role in stimulating DNA synthesis, cell cycle progression and proliferation of PDAC cells and are negatively regulated by AMPK [21]. Consequently, we determined whether berberine inhibits mTORC1 and ERK activation in PDAC cells. Cultures of MiaPaCa-2 cells were treated with increasing doses of berberine and then stimulated with a combination of insulin and neurotensin to elicit positive crosstalk (Fig. 4). Lysates of these cells were analyzed by immunoblotting using antibodies that detect the phosphorylated state of S6K at Thr389, a residue directly phosphorylated by mTORC1, using Western blot analysis with antibodies that specifically detect the phosphorylated state of this residue. To corroborate that phosphorylation of S6K at Thr389 reflects its activation within PDAC cells, we examined the phosphorylation of the 40S ribosomal protein subunit S6, a downstream target of S6K [77]. As shown in Fig. 4 A, stimulation with insulin and neurotensin induced a marked increase in mTORC1 activity, as scored by phosphorylation of S6K and S6 protein (pS6K and pS6). Treatment with berberine prevented mTORC1 activation in a dose-dependent manner (Fig. 4A; quantification in Fig. 4 B).


Dose-Dependent AMPK-Dependent and Independent Mechanisms of Berberine and Metformin Inhibition of mTORC1, ERK, DNA Synthesis and Proliferation in Pancreatic Cancer Cells.

Ming M, Sinnett-Smith J, Wang J, Soares HP, Young SH, Eibl G, Rozengurt E - PLoS ONE (2014)

Berberine inhibits mTORC1 signaling and ERK activation in PDAC cells.Cultures of MiaPaCa-2 (Panels A and B) or PANC-1 cells (panels C and D) were incubated in the absence or in the presence of increasing concentrations of berberine. Then, the cells were stimulated for 1 h with 5 nM neurotensin and 10 ng/ml insulin and lysed with 2X SDS-PAGE sample buffer. The samples were analyzed by SDS-PAGE and immunoblotting with antibodies that detect the phosphorylated state of S6K at Thr389, S6 at Ser240/244, and ERK at Thr202 and Tyr204. Immunoblotting with total S6K, S6 and ERK was used to verify equal gel loading. The quantification of the immune signals was performed using Multi Gauge V3.0. The results are presented in the plots shown in panels B and D. The values represent the mean ± SEM (n = 3) of S6K, S6 and ERK phosphorylation expressed as a percentage of the maximal response obtained in 3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4262417&req=5

pone-0114573-g004: Berberine inhibits mTORC1 signaling and ERK activation in PDAC cells.Cultures of MiaPaCa-2 (Panels A and B) or PANC-1 cells (panels C and D) were incubated in the absence or in the presence of increasing concentrations of berberine. Then, the cells were stimulated for 1 h with 5 nM neurotensin and 10 ng/ml insulin and lysed with 2X SDS-PAGE sample buffer. The samples were analyzed by SDS-PAGE and immunoblotting with antibodies that detect the phosphorylated state of S6K at Thr389, S6 at Ser240/244, and ERK at Thr202 and Tyr204. Immunoblotting with total S6K, S6 and ERK was used to verify equal gel loading. The quantification of the immune signals was performed using Multi Gauge V3.0. The results are presented in the plots shown in panels B and D. The values represent the mean ± SEM (n = 3) of S6K, S6 and ERK phosphorylation expressed as a percentage of the maximal response obtained in 3 independent experiments.
Mentions: The activation of the PI3K/Akt/mTORC1 and MEK/ERK pathways plays a pivotal role in stimulating DNA synthesis, cell cycle progression and proliferation of PDAC cells and are negatively regulated by AMPK [21]. Consequently, we determined whether berberine inhibits mTORC1 and ERK activation in PDAC cells. Cultures of MiaPaCa-2 cells were treated with increasing doses of berberine and then stimulated with a combination of insulin and neurotensin to elicit positive crosstalk (Fig. 4). Lysates of these cells were analyzed by immunoblotting using antibodies that detect the phosphorylated state of S6K at Thr389, a residue directly phosphorylated by mTORC1, using Western blot analysis with antibodies that specifically detect the phosphorylated state of this residue. To corroborate that phosphorylation of S6K at Thr389 reflects its activation within PDAC cells, we examined the phosphorylation of the 40S ribosomal protein subunit S6, a downstream target of S6K [77]. As shown in Fig. 4 A, stimulation with insulin and neurotensin induced a marked increase in mTORC1 activity, as scored by phosphorylation of S6K and S6 protein (pS6K and pS6). Treatment with berberine prevented mTORC1 activation in a dose-dependent manner (Fig. 4A; quantification in Fig. 4 B).

Bottom Line: Berberine treatment also reduced (by 70%) the growth of MiaPaCa-2 cell growth when implanted into the flanks of nu/nu mice.Similar results were obtained with metformin used at doses that induced either modest or pronounced reductions in intracellular ATP levels, which were virtually identical to the decreases in ATP levels obtained in response to berberine.We propose that berberine and metformin inhibit mitogenic signaling in PDAC cells through dose-dependent AMPK-dependent and independent pathways.

View Article: PubMed Central - PubMed

Affiliation: Division of Digestive Diseases, Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
Natural products represent a rich reservoir of potential small chemical molecules exhibiting anti-proliferative and chemopreventive properties. Here, we show that treatment of pancreatic ductal adenocarcinoma (PDAC) cells (PANC-1, MiaPaCa-2) with the isoquinoline alkaloid berberine (0.3-6 µM) inhibited DNA synthesis and proliferation of these cells and delay the progression of their cell cycle in G1. Berberine treatment also reduced (by 70%) the growth of MiaPaCa-2 cell growth when implanted into the flanks of nu/nu mice. Mechanistic studies revealed that berberine decreased mitochondrial membrane potential and intracellular ATP levels and induced potent AMPK activation, as shown by phosphorylation of AMPK α subunit at Thr-172 and acetyl-CoA carboxylase (ACC) at Ser79. Furthermore, berberine dose-dependently inhibited mTORC1 (phosphorylation of S6K at Thr389 and S6 at Ser240/244) and ERK activation in PDAC cells stimulated by insulin and neurotensin or fetal bovine serum. Knockdown of α1 and α2 catalytic subunit expression of AMPK reversed the inhibitory effect produced by treatment with low concentrations of berberine on mTORC1, ERK and DNA synthesis in PDAC cells. However, at higher concentrations, berberine inhibited mitogenic signaling (mTORC1 and ERK) and DNA synthesis through an AMPK-independent mechanism. Similar results were obtained with metformin used at doses that induced either modest or pronounced reductions in intracellular ATP levels, which were virtually identical to the decreases in ATP levels obtained in response to berberine. We propose that berberine and metformin inhibit mitogenic signaling in PDAC cells through dose-dependent AMPK-dependent and independent pathways.

No MeSH data available.


Related in: MedlinePlus