Silvestri Alessandra, Palumbo Francesco, Rasi Ignazio, Posca Daniela, Pavlidou Theodora, Paoluzi Serena, Castagnoli Luisa, Cesareni Giovanni
Laboratory of Molecular Genetics, Department of Biology, Tor Vergata University, Rome, Italy.
Laboratory of Molecular Genetics, Department of Biology, Tor Vergata University, Rome, Italy; IRCCS, Fondazione Santa Lucia, Rome, Italy.
PLoS One. 2015 Aug 20;10(8):e0136250. doi: 10.1371/journal.pone.0136250. eCollection 2015.
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.
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.
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.
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.
二甲双胍被提议作为癌症治疗的辅助疗法，因为它能够通过负向调节PI3K/AKT/mTOR信号通路来降低癌症发病率。在体外，除了抑制癌细胞增殖外，二甲双胍还能诱导细胞凋亡。这种第二种效应的分子机制仍未得到充分表征，且已发表的数据往往相互矛盾。我们研究了营养物质的可利用性如何调节二甲双胍在三种乳腺癌细胞系中诱导的细胞凋亡。
将MCF7、SKBR3和MDA-MB-231细胞接种于添加了逐渐增加的葡萄糖浓度的MEM培养基或DMEM培养基中，并用10 mM二甲双胍处理。通过台盼蓝试验监测细胞活力，并通过蛋白质免疫印迹法分析处理对Akt/mTOR信号通路和细胞凋亡的影响。此外，我们测定了丙酮酸激酶M2（PKM2）的表达水平，PKM2是一种在癌细胞中表达的著名糖酵解酶。
我们的结果表明，当在生理葡萄糖浓度下培养时，二甲双胍可诱导乳腺癌细胞凋亡，而当细胞在高葡萄糖/高氨基酸培养基中生长时，促凋亡作用完全消失。发现细胞凋亡的诱导依赖于AMPK激活，但至少部分独立于TORC1失活。最后，我们表明，在营养匮乏的条件下，二甲双胍能够通过下调PKM2表达来调节细胞内糖酵解平衡，且该机制由AMPK激活介导。
我们证明二甲双胍仅在营养匮乏的条件下诱导乳腺癌细胞凋亡和PKM2下调。不仅葡萄糖水平，氨基酸浓度也会影响观察到的二甲双胍对mTOR信号通路的抑制作用及其促凋亡作用。这些数据表明，减少肿瘤中的营养供应可提高二甲双胍的疗效，调节PKM2的表达/活性可能是增强二甲双胍抗癌作用的一种有前景的策略。
