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Metformin Induces Apoptosis and Downregulates Pyruvate Kinase M2 in Breast Cancer Cells Only When Grown in Nutrient-Poor Conditions.

Silvestri A, Palumbo F, Rasi I, Posca D, Pavlidou T, Paoluzi S, Castagnoli L, Cesareni G - PLoS ONE (2015)

Bottom Line: Our results showed that metformin can induce apoptosis in breast cancer cells when cultured at physiological glucose concentrations and that the pro-apoptotic effect was completely abolished when cells were grown in high glucose/high amino acid medium.Induction of apoptosis was found to be dependent on AMPK activation but, at least partially, independent of TORC1 inactivation.Not only glucose levels but also amino acid concentration can influence the observed metformin inhibitory effect on the mTOR pathway as well as its pro-apoptotic effect.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Genetics, Department of Biology, Tor Vergata University, Rome, Italy.

ABSTRACT

Introduction: Metformin is proposed as adjuvant therapy in cancer treatment because of its ability to limit cancer incidence by negatively modulating the PI3K/AKT/mTOR pathway. In vitro, in addition to inhibiting cancer cell proliferation, metformin can also induce apoptosis. The molecular mechanism underlying this second effect is still poorly characterized and published data are often contrasting. We investigated how nutrient availability can modulate metformin-induced apoptosis in three breast cancer cell lines.

Material and methods: MCF7, SKBR3 and MDA-MB-231 cells were plated in MEM medium supplemented with increasing glucose concentrations or in DMEM medium and treated with 10 mM metformin. Cell viability was monitored by Trypan Blue assay and treatment effects on Akt/mTOR pathway and on apoptosis were analysed by Western Blot. Moreover, we determined the level of expression of pyruvate kinase M2 (PKM2), a well-known glycolytic enzyme expressed in cancer cells.

Results: Our results showed that metformin can induce apoptosis in breast cancer cells when cultured at physiological glucose concentrations and that the pro-apoptotic effect was completely abolished when cells were grown in high glucose/high amino acid medium. Induction of apoptosis was found to be dependent on AMPK activation but, at least partially, independent of TORC1 inactivation. Finally, we showed that, in nutrient-poor conditions, metformin was able to modulate the intracellular glycolytic equilibrium by downregulating PKM2 expression and that this mechanism was mediated by AMPK activation.

Conclusion: We demonstrated that metformin induces breast cancer cell apoptosis and PKM2 downregulation only in nutrient-poor conditions. Not only glucose levels but also amino acid concentration can influence the observed metformin inhibitory effect on the mTOR pathway as well as its pro-apoptotic effect. These data demonstrate that the reduction of nutrient supply in tumors can increase metformin efficacy and that modulation of PKM2 expression/activity could be a promising strategy to boost metformin anti-cancer effect.

No MeSH data available.


Related in: MedlinePlus

AMPK activation by AICAR is sufficient to induce apoptosis and downregulation of PKM2.A) MCF7 cells were plated at 8X105 cells/well in 6-well plates in MEM medium and treated with 10 mM metformin, 3 mM AICAR, 100 nM rapamycin or PBS as control, for 48 hours. After treatment cells were lysed and protein extracts were analysed by Western Blot with antibodies directed against mTOR (p) Ser2448, RPS6 (p) Ser240/244 and PARP (Cl). GAPDH was used as loading control. B) MCF7 cells were plated at 8X105 cells/well in 6-well plates in MEM medium and treated with 10 mM metformin, 3 mM AICAR, 100 nM Rapamycin or PBS as control for 48 hours. After treatment cells were lysed and protein extracts were analysed by Western Blot with anti-mTOR (p) Ser2448 and anti-PKM2 antibodies. GAPDH was used as loading control.
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pone.0136250.g006: AMPK activation by AICAR is sufficient to induce apoptosis and downregulation of PKM2.A) MCF7 cells were plated at 8X105 cells/well in 6-well plates in MEM medium and treated with 10 mM metformin, 3 mM AICAR, 100 nM rapamycin or PBS as control, for 48 hours. After treatment cells were lysed and protein extracts were analysed by Western Blot with antibodies directed against mTOR (p) Ser2448, RPS6 (p) Ser240/244 and PARP (Cl). GAPDH was used as loading control. B) MCF7 cells were plated at 8X105 cells/well in 6-well plates in MEM medium and treated with 10 mM metformin, 3 mM AICAR, 100 nM Rapamycin or PBS as control for 48 hours. After treatment cells were lysed and protein extracts were analysed by Western Blot with anti-mTOR (p) Ser2448 and anti-PKM2 antibodies. GAPDH was used as loading control.

Mentions: The most invoked mechanism underlying the cell-autonomous effect of metformin on cancer cells implies the ability to inhibit the mTOR pathway via the activation of AMPK. We asked whether this mechanism is involved not only in the metformin-induced reduction of cell proliferation but also in its pro-apoptotic effect. We first asked whether the direct activation of AMPK could induce apoptosis. Cells were plated in MEM medium at normoglycemic concentration and treated for 24 and 48 hours with 3 mM AICAR, an AMP analog that binds to and activates AMPK. As shown in Fig 6A, treatment with AICAR induced downregulation of mTOR phosphorylation at a level comparable to that achieved with metformin. After 48 hours treatment, PARP cleavage was higher than the control in both AICAR and metformin treated cells indicating that the induction of AMPK is sufficient to induce apoptosis in our experimental conditions (Fig 6A). Next we asked whether this effect could be recapitulated by mTOR inhibition. To this end cells were treated with 100 nM rapamycin for 24 and 48 hours. Although the treatment induced a significant downregulation of the mTOR pathway, as measured by phosphorylation of Ser2448 in mTOR and phosphorylation of the downstream target RPS6, it did not cause apoptosis (Fig 6A). All together these results indicate that sustained induction of AMPK is sufficient to induce apoptosis and that signalling routes, other than the ones inhibited by rapamycin, need to be perturbed in order to cause cell death.


Metformin Induces Apoptosis and Downregulates Pyruvate Kinase M2 in Breast Cancer Cells Only When Grown in Nutrient-Poor Conditions.

Silvestri A, Palumbo F, Rasi I, Posca D, Pavlidou T, Paoluzi S, Castagnoli L, Cesareni G - PLoS ONE (2015)

AMPK activation by AICAR is sufficient to induce apoptosis and downregulation of PKM2.A) MCF7 cells were plated at 8X105 cells/well in 6-well plates in MEM medium and treated with 10 mM metformin, 3 mM AICAR, 100 nM rapamycin or PBS as control, for 48 hours. After treatment cells were lysed and protein extracts were analysed by Western Blot with antibodies directed against mTOR (p) Ser2448, RPS6 (p) Ser240/244 and PARP (Cl). GAPDH was used as loading control. B) MCF7 cells were plated at 8X105 cells/well in 6-well plates in MEM medium and treated with 10 mM metformin, 3 mM AICAR, 100 nM Rapamycin or PBS as control for 48 hours. After treatment cells were lysed and protein extracts were analysed by Western Blot with anti-mTOR (p) Ser2448 and anti-PKM2 antibodies. GAPDH was used as loading control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4546379&req=5

pone.0136250.g006: AMPK activation by AICAR is sufficient to induce apoptosis and downregulation of PKM2.A) MCF7 cells were plated at 8X105 cells/well in 6-well plates in MEM medium and treated with 10 mM metformin, 3 mM AICAR, 100 nM rapamycin or PBS as control, for 48 hours. After treatment cells were lysed and protein extracts were analysed by Western Blot with antibodies directed against mTOR (p) Ser2448, RPS6 (p) Ser240/244 and PARP (Cl). GAPDH was used as loading control. B) MCF7 cells were plated at 8X105 cells/well in 6-well plates in MEM medium and treated with 10 mM metformin, 3 mM AICAR, 100 nM Rapamycin or PBS as control for 48 hours. After treatment cells were lysed and protein extracts were analysed by Western Blot with anti-mTOR (p) Ser2448 and anti-PKM2 antibodies. GAPDH was used as loading control.
Mentions: The most invoked mechanism underlying the cell-autonomous effect of metformin on cancer cells implies the ability to inhibit the mTOR pathway via the activation of AMPK. We asked whether this mechanism is involved not only in the metformin-induced reduction of cell proliferation but also in its pro-apoptotic effect. We first asked whether the direct activation of AMPK could induce apoptosis. Cells were plated in MEM medium at normoglycemic concentration and treated for 24 and 48 hours with 3 mM AICAR, an AMP analog that binds to and activates AMPK. As shown in Fig 6A, treatment with AICAR induced downregulation of mTOR phosphorylation at a level comparable to that achieved with metformin. After 48 hours treatment, PARP cleavage was higher than the control in both AICAR and metformin treated cells indicating that the induction of AMPK is sufficient to induce apoptosis in our experimental conditions (Fig 6A). Next we asked whether this effect could be recapitulated by mTOR inhibition. To this end cells were treated with 100 nM rapamycin for 24 and 48 hours. Although the treatment induced a significant downregulation of the mTOR pathway, as measured by phosphorylation of Ser2448 in mTOR and phosphorylation of the downstream target RPS6, it did not cause apoptosis (Fig 6A). All together these results indicate that sustained induction of AMPK is sufficient to induce apoptosis and that signalling routes, other than the ones inhibited by rapamycin, need to be perturbed in order to cause cell death.

Bottom Line: Our results showed that metformin can induce apoptosis in breast cancer cells when cultured at physiological glucose concentrations and that the pro-apoptotic effect was completely abolished when cells were grown in high glucose/high amino acid medium.Induction of apoptosis was found to be dependent on AMPK activation but, at least partially, independent of TORC1 inactivation.Not only glucose levels but also amino acid concentration can influence the observed metformin inhibitory effect on the mTOR pathway as well as its pro-apoptotic effect.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Genetics, Department of Biology, Tor Vergata University, Rome, Italy.

ABSTRACT

Introduction: Metformin is proposed as adjuvant therapy in cancer treatment because of its ability to limit cancer incidence by negatively modulating the PI3K/AKT/mTOR pathway. In vitro, in addition to inhibiting cancer cell proliferation, metformin can also induce apoptosis. The molecular mechanism underlying this second effect is still poorly characterized and published data are often contrasting. We investigated how nutrient availability can modulate metformin-induced apoptosis in three breast cancer cell lines.

Material and methods: MCF7, SKBR3 and MDA-MB-231 cells were plated in MEM medium supplemented with increasing glucose concentrations or in DMEM medium and treated with 10 mM metformin. Cell viability was monitored by Trypan Blue assay and treatment effects on Akt/mTOR pathway and on apoptosis were analysed by Western Blot. Moreover, we determined the level of expression of pyruvate kinase M2 (PKM2), a well-known glycolytic enzyme expressed in cancer cells.

Results: Our results showed that metformin can induce apoptosis in breast cancer cells when cultured at physiological glucose concentrations and that the pro-apoptotic effect was completely abolished when cells were grown in high glucose/high amino acid medium. Induction of apoptosis was found to be dependent on AMPK activation but, at least partially, independent of TORC1 inactivation. Finally, we showed that, in nutrient-poor conditions, metformin was able to modulate the intracellular glycolytic equilibrium by downregulating PKM2 expression and that this mechanism was mediated by AMPK activation.

Conclusion: We demonstrated that metformin induces breast cancer cell apoptosis and PKM2 downregulation only in nutrient-poor conditions. Not only glucose levels but also amino acid concentration can influence the observed metformin inhibitory effect on the mTOR pathway as well as its pro-apoptotic effect. These data demonstrate that the reduction of nutrient supply in tumors can increase metformin efficacy and that modulation of PKM2 expression/activity could be a promising strategy to boost metformin anti-cancer effect.

No MeSH data available.


Related in: MedlinePlus