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


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.


CONCLUSIONS
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.
Laboratory of Molecular Genetics, Department of Biology, Tor Vergata University, Rome, Italy.
Alessandra
A
Silvestri
Laboratory of Molecular Genetics, Department of Biology, Tor Vergata University, Rome, Italy.
Francesco
F
Palumbo
Laboratory of Molecular Genetics, Department of Biology, Tor Vergata University, Rome, Italy.
Ignazio
I
Rasi
Laboratory of Molecular Genetics, Department of Biology, Tor Vergata University, Rome, Italy.
Daniela
D
Posca
Laboratory of Molecular Genetics, Department of Biology, Tor Vergata University, Rome, Italy.
Theodora
T
Pavlidou
Laboratory of Molecular Genetics, Department of Biology, Tor Vergata University, Rome, Italy.
Serena
S
Paoluzi
Laboratory of Molecular Genetics, Department of Biology, Tor Vergata University, Rome, Italy.
Luisa
L
Castagnoli
Laboratory of Molecular Genetics, Department of Biology, Tor Vergata University, Rome, Italy; IRCCS, Fondazione Santa Lucia, Rome, Italy.
Giovanni
G
Cesareni
D000277:Adjuvants, Pharmaceutic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms / Q000201:enzymology / Q000378:metabolism*; D045744:Cell Line, Tumor / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D003470:Culture Media; D015536:Down-Regulation / Q000187:drug effects; D005260:Female; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D061986:MCF-7 Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D008687:Metformin / Q000494:pharmacology*; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors*; D060888:Real-Time Polymerase Chain Reaction
D000277:Adjuvants, Pharmaceutic; D003470:Culture Media; D007004:Hypoglycemic Agents; D008687:Metformin; D011770:Pyruvate Kinase