Resveratrol inhibits cancer cell metabolism by down regulating pyruvate kinase M2 via inhibition of mammalian target of rapamycin

Abstract

Metabolism of cancer cells with pyruvate kinase M2 (PKM2) at its centre stage has assumed a prime significance in cancer research in recent times. Cancer cell metabolism, characterized by enhanced glucose uptake, production of lactate and anabolism is considered an ideal target for therapeutic interventions. Expression of PKM2 switches metabolism in favor of cancer cells, therefore, the present study was designed to investigate the hitherto unknown effect of resveratrol, a phytoalexin, on PKM2 expression and resultant implications on cancer metabolism. We observed that resveratrol down-regulated PKM2 expression by inhibiting mTOR signaling and suppressed cancer metabolism, adjudged by decreased glucose uptake, lactate production (aerobic glycolysis) and reduced anabolism (macromolecule synthesis) in various cancer cell lines. A contingent decrease in intracellular levels of ribose-5-phosphate (R5P), a critical intermediate of pentose phosphate pathway, accounted for a reduced anabolism. Consequently, the state of suppressed cancer metabolism resulted in decreased cellular proliferation. Interestingly, shRNA-mediated silencing of PKM2 inhibited glucose uptake and lactate production, providing evidence for the critical role of PKM2 and its mediation in the observed effects of resveratrol on cancer metabolism. Further, an over-expression of PKM2 abolished the observed effects of resveratrol, signifying the role of PKM2 downregulation as a critical function of resveratrol. The study reports a novel PKM2-mediated effect of resveratrol on cancer metabolism and provides a new dimension to its therapeutic potential.

Figure 1. Resveratrol decreases PKM2 expression.

Resveratrol treatment decreases PKM2 mRNA in (A), HeLa; (B), HepG2 and (C), MCF-7; by approximately two-folds. β-actin was taken as endogenous control and normalized to PKM2 mRNA. Relative quantification analysis was done using SDS 1.1 RQ software. Western blot showing decreased PKM2 protein in all the cell lines studied on treatment with 50 µM resveratrol (D). C- Mock treated control and R- resveratrol treated. All experiments were repeated 3 times and data is expressed as mean ± SE. *p<0.05.

Figure 2. Western blots showing the effect of resveratrol on mTOR signaling and rapamycin (mTOR inhibitor) on PKM2 expression.

Resveratrol (50 µM) inhibited mTOR signaling in all the three cell lines studied as evident from decreased phosphorylation of p-p70S6K upon resveratrol treatment (A); C- Mock treated control and R- resveratrol treated. mTOR inhibition by 20 nM rapamycin reduced PKM2 expression in all experimental cell lines (B); C- Mock treated control and Rapa- 20 nM Rapamycin.

Figure 3. Resveratrol inhibits aerobic glycolysis (glucose uptake and lactate production characteristic of cancer metabolism).

A significant decrease in glucose uptake (A) and lactate production (B), was observed in HeLa and HepG2 (*p<0.05), following 48 hours resveratrol treatment. However, in MCF-7 aerobic glycolysis did not show significant signs of inhibition (p>0.05). All experiments were repeated 3 times and data is expressed as mean ± SE.

Figure 4. PKM2 knockdown reduced aerobic glycolysis.

PKM2 was knocked down (nearly 60% – using lentiviral pGIPZ vector containing PKM2 shRNA) as analyzed by: real time PCR (A), decreased glucose uptake (B), lactate production (C), and cellular proliferation (D) following knock down. All experiments were repeated 3 times and data is expressed as mean ± SE. *p<0.05.

Figure 5. Resveratrol decreased intracellular levels of R5P.

R5P is a pentose phosphate pathway intermediate, an important marker for anabolism (nucleotide biosynthesis). Approximately 20–25% decrease in intracellular levels of R5P in HeLa and HepG2 was observed upon 50 µM resveratrol treatment. However, there was an insignificant decrease of around 5% in MCF-7 cells (see text). Results indicated inhibited anabolism following resveratrol treatment. All experiments were repeated 3 times. Values are expressed as mean ± SE. *p<0.05.

Figure 6. Cellular proliferation retarded as a result of suppressed cancer metabolism.

To examine how inhibition of cancer metabolism affects proliferation of cell lines, we trypsinized and counted cells at 0, 24, 48, 72 and 96 hours. There was a decrease in proliferation rate in all the three cell lines studied with prominent decrease in HeLa (A), HepG2 (B) and least prominent in MCF -7 (C). All experiments were repeated 3 times and data is expressed as mean ± SE. *p<0.05.

Figure 7. PKM2 over-expression abolished the effects of resveratrol.

Over-expression of PKM2 using myc tagged pCDNA vector (see text) was confirmed using anti myc and anti PKM2 antibodies (A). Increased cellular proliferation in PKM2 transfected cells, compared to mock transfected cells, in continuous presence of inhibitory concentrations of resveratrol (B). Glucose uptake (C) and lactate production (D) was substantially enhanced in PKM2 transfected cells indicating that PKM2 over-expression abrogates effects of resveratrol on cancer metabolism and cellular proliferation. These results further validate PKM2 as critical target of resveratrol. All experiments were repeated 3 times and data expressed as mean ± SE. *p<0.05. PKM2 – indicates mock transfected cells whereas PKM2+indicates PKM2 transfected cells.