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A novel pyruvate kinase M2 activator compound that suppresses lung cancer cell viability under hypoxia.

Kim DJ, Park YS, Kim ND, Min SH, You YM, Jung Y, Koo H, Noh H, Kim JA, Park KC, Yeom YI - Mol. Cells (2015)

Bottom Line: We demonstrate that PA-12 stimulates the pyruvate kinase activity of recombinant PKM2 in vitro, with a half-maximal activity concentration of 4.92 μM, and effectively suppresses both anchorage-dependent and -independent growth of lung cancer cells in non-essential amino acid-depleted medium.In addition, PA-12 blocked the nuclear translocalization of PKM2 in lung cancer cells, resulting in the inhibition of hypoxia response element (HRE)-mediated reporter activity as well as hypoxia-inducible factor 1 (HIF-1) target gene expression, eventually leading to the suppression of cell viability under hypoxia.Taken together, our data suggest that PA-12 is a novel and potent PKM2 activator that has therapeutic implications for lung cancer.

View Article: PubMed Central - PubMed

Affiliation: Medical Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 305-806, Korea.

ABSTRACT
Pyruvate kinase M2 isoform (PKM2), a rate-limiting enzyme in the final step of glycolysis, is known to be associated with the metabolic rewiring of cancer cells, and considered an important cancer therapeutic target. Herein, we report a novel PKM2 activator, PA-12, which was identified via the molecular docking-based virtual screening. We demonstrate that PA-12 stimulates the pyruvate kinase activity of recombinant PKM2 in vitro, with a half-maximal activity concentration of 4.92 μM, and effectively suppresses both anchorage-dependent and -independent growth of lung cancer cells in non-essential amino acid-depleted medium. In addition, PA-12 blocked the nuclear translocalization of PKM2 in lung cancer cells, resulting in the inhibition of hypoxia response element (HRE)-mediated reporter activity as well as hypoxia-inducible factor 1 (HIF-1) target gene expression, eventually leading to the suppression of cell viability under hypoxia. We also verified that the effects of PA-12 were dependent on PKM2 expression in cancer cells, demonstrating the specificity of PA-12 for PKM2 protein. Taken together, our data suggest that PA-12 is a novel and potent PKM2 activator that has therapeutic implications for lung cancer.

No MeSH data available.


Related in: MedlinePlus

Effects of PA-12 on lung cancer cell viability under hypoxia. (A) PA-12 suppresses lung cancer cell viability under hypoxia. (B) Effect of PA-12 on intracellular localization of PKM2 under hypoxia. For A-B, A549 lung cancer cells were pretreated with PA-12 or PKIII for 2 h and then cultured for a further 48 h (A) or 12 h (B) under hypoxia. The cytoplasmic or nuclear protein was fractionated from PA-12 or PKIII-pretreated or untreated cells, and analyzed by Western blotting. Expression of lamin B1 (nuclear protein) and α-tubulin (cytoplasmic protein) was used to verify sample loading. (C) PA-12 suppresses hypoxia-induced hypoxia response element (HRE) activity. HRE activity was analyzed by a luciferase assay. (D) PA-12 inhibits the expression of HIF target genes. A549 cells were pretreated with PA-12 or PKIII for 2 h and then cultured for 12 h under hypoxia. Expression of HIF target genes was analyzed by RT-PCR with β-actin as the loading control. For B and D, the numbers underneath the graphic western blotting and RT-PCR data are quantitative estimations of the band intensity made using Image J program (NIH, USA). Similar results were obtained from three independent experiments, and representative data are shown.
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f3-molce-38-4-373: Effects of PA-12 on lung cancer cell viability under hypoxia. (A) PA-12 suppresses lung cancer cell viability under hypoxia. (B) Effect of PA-12 on intracellular localization of PKM2 under hypoxia. For A-B, A549 lung cancer cells were pretreated with PA-12 or PKIII for 2 h and then cultured for a further 48 h (A) or 12 h (B) under hypoxia. The cytoplasmic or nuclear protein was fractionated from PA-12 or PKIII-pretreated or untreated cells, and analyzed by Western blotting. Expression of lamin B1 (nuclear protein) and α-tubulin (cytoplasmic protein) was used to verify sample loading. (C) PA-12 suppresses hypoxia-induced hypoxia response element (HRE) activity. HRE activity was analyzed by a luciferase assay. (D) PA-12 inhibits the expression of HIF target genes. A549 cells were pretreated with PA-12 or PKIII for 2 h and then cultured for 12 h under hypoxia. Expression of HIF target genes was analyzed by RT-PCR with β-actin as the loading control. For B and D, the numbers underneath the graphic western blotting and RT-PCR data are quantitative estimations of the band intensity made using Image J program (NIH, USA). Similar results were obtained from three independent experiments, and representative data are shown.

Mentions: A previous study reported that PKM2 activators suppressed the growth of xenografted tumors and prolonged cancer cell doubling time under hypoxia (Anastasiou et al., 2012). Therefore, we investigated whether PA-12 could suppress cancer cell viability under hypoxia. Cells were pretreated with PA-12 or PKIII for 2 h under normoxia and then incubated further for 48 h under hypoxia. The cell viability rate under hypoxia, measured by counting cell numbers, was significantly decreased by PA-12 in a dose-dependent manner (Fig. 3A). It was reported that dimeric PKM2 enhanced HIF-1α activity in the nucleus to facilitate the reprogramming of glucose metabolism and promote tumorigenesis (Luo et al., 2011), while PKM2 activators changed the dimeric form of PKM2 to a tetrameric form (Anastasiou et al., 2012). We, therefore, first investigated the effects of PA-12 on the nuclear localization of PKM2 under hypoxia. Cells were pretreated with PA-12 or PKIII for 2 h, cultured further for 12 h under hypoxia, and then the nuclear fraction was isolated for the immunodetection of PKM2. The nuclear PKM2 level was slightly increased under hypoxia compared to normoxia but PA-12 strongly suppressed the nuclear translocation of PKM2 (Fig. 3B). We then investigated whether PA-12 can also regulate HIF activity in A549 cells under hypoxia. At 24 h post-transfection with an HRE reporter, the cells were treated with PA-12 or PKIII for 2 h and then cultured further for 12 h under hypoxia. HRE reporter activity was strongly suppressed by PA-12 in a dose-dependent manner (Fig. 3C). In accordance, PA-12 significantly inhibited the expression of HIF target genes, including GLUT3, GLUT1 and VEGF (Fig. 3D). These results indicate that PA-12 can abolish the PKM2-mediated transcriptional activation of HIF-1 by suppressing the nuclear translocation of PKM2.


A novel pyruvate kinase M2 activator compound that suppresses lung cancer cell viability under hypoxia.

Kim DJ, Park YS, Kim ND, Min SH, You YM, Jung Y, Koo H, Noh H, Kim JA, Park KC, Yeom YI - Mol. Cells (2015)

Effects of PA-12 on lung cancer cell viability under hypoxia. (A) PA-12 suppresses lung cancer cell viability under hypoxia. (B) Effect of PA-12 on intracellular localization of PKM2 under hypoxia. For A-B, A549 lung cancer cells were pretreated with PA-12 or PKIII for 2 h and then cultured for a further 48 h (A) or 12 h (B) under hypoxia. The cytoplasmic or nuclear protein was fractionated from PA-12 or PKIII-pretreated or untreated cells, and analyzed by Western blotting. Expression of lamin B1 (nuclear protein) and α-tubulin (cytoplasmic protein) was used to verify sample loading. (C) PA-12 suppresses hypoxia-induced hypoxia response element (HRE) activity. HRE activity was analyzed by a luciferase assay. (D) PA-12 inhibits the expression of HIF target genes. A549 cells were pretreated with PA-12 or PKIII for 2 h and then cultured for 12 h under hypoxia. Expression of HIF target genes was analyzed by RT-PCR with β-actin as the loading control. For B and D, the numbers underneath the graphic western blotting and RT-PCR data are quantitative estimations of the band intensity made using Image J program (NIH, USA). Similar results were obtained from three independent experiments, and representative data are shown.
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Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400313&req=5

f3-molce-38-4-373: Effects of PA-12 on lung cancer cell viability under hypoxia. (A) PA-12 suppresses lung cancer cell viability under hypoxia. (B) Effect of PA-12 on intracellular localization of PKM2 under hypoxia. For A-B, A549 lung cancer cells were pretreated with PA-12 or PKIII for 2 h and then cultured for a further 48 h (A) or 12 h (B) under hypoxia. The cytoplasmic or nuclear protein was fractionated from PA-12 or PKIII-pretreated or untreated cells, and analyzed by Western blotting. Expression of lamin B1 (nuclear protein) and α-tubulin (cytoplasmic protein) was used to verify sample loading. (C) PA-12 suppresses hypoxia-induced hypoxia response element (HRE) activity. HRE activity was analyzed by a luciferase assay. (D) PA-12 inhibits the expression of HIF target genes. A549 cells were pretreated with PA-12 or PKIII for 2 h and then cultured for 12 h under hypoxia. Expression of HIF target genes was analyzed by RT-PCR with β-actin as the loading control. For B and D, the numbers underneath the graphic western blotting and RT-PCR data are quantitative estimations of the band intensity made using Image J program (NIH, USA). Similar results were obtained from three independent experiments, and representative data are shown.
Mentions: A previous study reported that PKM2 activators suppressed the growth of xenografted tumors and prolonged cancer cell doubling time under hypoxia (Anastasiou et al., 2012). Therefore, we investigated whether PA-12 could suppress cancer cell viability under hypoxia. Cells were pretreated with PA-12 or PKIII for 2 h under normoxia and then incubated further for 48 h under hypoxia. The cell viability rate under hypoxia, measured by counting cell numbers, was significantly decreased by PA-12 in a dose-dependent manner (Fig. 3A). It was reported that dimeric PKM2 enhanced HIF-1α activity in the nucleus to facilitate the reprogramming of glucose metabolism and promote tumorigenesis (Luo et al., 2011), while PKM2 activators changed the dimeric form of PKM2 to a tetrameric form (Anastasiou et al., 2012). We, therefore, first investigated the effects of PA-12 on the nuclear localization of PKM2 under hypoxia. Cells were pretreated with PA-12 or PKIII for 2 h, cultured further for 12 h under hypoxia, and then the nuclear fraction was isolated for the immunodetection of PKM2. The nuclear PKM2 level was slightly increased under hypoxia compared to normoxia but PA-12 strongly suppressed the nuclear translocation of PKM2 (Fig. 3B). We then investigated whether PA-12 can also regulate HIF activity in A549 cells under hypoxia. At 24 h post-transfection with an HRE reporter, the cells were treated with PA-12 or PKIII for 2 h and then cultured further for 12 h under hypoxia. HRE reporter activity was strongly suppressed by PA-12 in a dose-dependent manner (Fig. 3C). In accordance, PA-12 significantly inhibited the expression of HIF target genes, including GLUT3, GLUT1 and VEGF (Fig. 3D). These results indicate that PA-12 can abolish the PKM2-mediated transcriptional activation of HIF-1 by suppressing the nuclear translocation of PKM2.

Bottom Line: We demonstrate that PA-12 stimulates the pyruvate kinase activity of recombinant PKM2 in vitro, with a half-maximal activity concentration of 4.92 μM, and effectively suppresses both anchorage-dependent and -independent growth of lung cancer cells in non-essential amino acid-depleted medium.In addition, PA-12 blocked the nuclear translocalization of PKM2 in lung cancer cells, resulting in the inhibition of hypoxia response element (HRE)-mediated reporter activity as well as hypoxia-inducible factor 1 (HIF-1) target gene expression, eventually leading to the suppression of cell viability under hypoxia.Taken together, our data suggest that PA-12 is a novel and potent PKM2 activator that has therapeutic implications for lung cancer.

View Article: PubMed Central - PubMed

Affiliation: Medical Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 305-806, Korea.

ABSTRACT
Pyruvate kinase M2 isoform (PKM2), a rate-limiting enzyme in the final step of glycolysis, is known to be associated with the metabolic rewiring of cancer cells, and considered an important cancer therapeutic target. Herein, we report a novel PKM2 activator, PA-12, which was identified via the molecular docking-based virtual screening. We demonstrate that PA-12 stimulates the pyruvate kinase activity of recombinant PKM2 in vitro, with a half-maximal activity concentration of 4.92 μM, and effectively suppresses both anchorage-dependent and -independent growth of lung cancer cells in non-essential amino acid-depleted medium. In addition, PA-12 blocked the nuclear translocalization of PKM2 in lung cancer cells, resulting in the inhibition of hypoxia response element (HRE)-mediated reporter activity as well as hypoxia-inducible factor 1 (HIF-1) target gene expression, eventually leading to the suppression of cell viability under hypoxia. We also verified that the effects of PA-12 were dependent on PKM2 expression in cancer cells, demonstrating the specificity of PA-12 for PKM2 protein. Taken together, our data suggest that PA-12 is a novel and potent PKM2 activator that has therapeutic implications for lung cancer.

No MeSH data available.


Related in: MedlinePlus