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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

PA-12 suppresses lung cancer cell growth. (A) In-vitro pyruvate kinase activity assay of PKM2 in the presence of PA-12. The activity was assessed by an in-vitro ATP assay. (B) Effect of PA-12 on lung cancer cell growth in an NEAA-depleted medium. Cell growth was analyzed using the CellTiter-Blue® assay. (C) PA-12 inhibits anchorage-independent growth of lung cancer cells in an NEAA-depleted medium. Data shown are three different areas randomly selected to represent the total population; similar results were obtained from three independent experiments. (D) PA-12 induces pyruvate kinase activity in A549 lung cancer cells.
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f2-molce-38-4-373: PA-12 suppresses lung cancer cell growth. (A) In-vitro pyruvate kinase activity assay of PKM2 in the presence of PA-12. The activity was assessed by an in-vitro ATP assay. (B) Effect of PA-12 on lung cancer cell growth in an NEAA-depleted medium. Cell growth was analyzed using the CellTiter-Blue® assay. (C) PA-12 inhibits anchorage-independent growth of lung cancer cells in an NEAA-depleted medium. Data shown are three different areas randomly selected to represent the total population; similar results were obtained from three independent experiments. (D) PA-12 induces pyruvate kinase activity in A549 lung cancer cells.

Mentions: For further validation of the efficacy of PA-12 as a PKM2 activator suppressing cancer cell growth, we examined the binding characteristics between PA-12 and PKM2 protein. We predicted a binding model for the two molecules by docking PA-12 in silico to a selected allosteric pocket in the PKM2 protein structure. The results indicated that PA-12 forms favorable interactions at numerous points and docked precisely within the allosteric site of tetrameric PKM2 (Supplementary Fig. 2A). We then quantitatively determined the PKM2 activating activity of PA-12 by a dose-responsive pyruvate kinase assay in vitro. PA-12 showed an effective PKM2 activation with a half-maximal activity concentration of 4.92 μM, which was comparable to that of the reference compound (Fig. 2A). We also confirmed that PA-12 increases pyruvate kinase activity in cancer cell lysates (Supplementary Fig. 2B). These data indicate that PA-12 is a potent activator 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)

PA-12 suppresses lung cancer cell growth. (A) In-vitro pyruvate kinase activity assay of PKM2 in the presence of PA-12. The activity was assessed by an in-vitro ATP assay. (B) Effect of PA-12 on lung cancer cell growth in an NEAA-depleted medium. Cell growth was analyzed using the CellTiter-Blue® assay. (C) PA-12 inhibits anchorage-independent growth of lung cancer cells in an NEAA-depleted medium. Data shown are three different areas randomly selected to represent the total population; similar results were obtained from three independent experiments. (D) PA-12 induces pyruvate kinase activity in A549 lung cancer cells.
© Copyright Policy
Related In: Results  -  Collection

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

f2-molce-38-4-373: PA-12 suppresses lung cancer cell growth. (A) In-vitro pyruvate kinase activity assay of PKM2 in the presence of PA-12. The activity was assessed by an in-vitro ATP assay. (B) Effect of PA-12 on lung cancer cell growth in an NEAA-depleted medium. Cell growth was analyzed using the CellTiter-Blue® assay. (C) PA-12 inhibits anchorage-independent growth of lung cancer cells in an NEAA-depleted medium. Data shown are three different areas randomly selected to represent the total population; similar results were obtained from three independent experiments. (D) PA-12 induces pyruvate kinase activity in A549 lung cancer cells.
Mentions: For further validation of the efficacy of PA-12 as a PKM2 activator suppressing cancer cell growth, we examined the binding characteristics between PA-12 and PKM2 protein. We predicted a binding model for the two molecules by docking PA-12 in silico to a selected allosteric pocket in the PKM2 protein structure. The results indicated that PA-12 forms favorable interactions at numerous points and docked precisely within the allosteric site of tetrameric PKM2 (Supplementary Fig. 2A). We then quantitatively determined the PKM2 activating activity of PA-12 by a dose-responsive pyruvate kinase assay in vitro. PA-12 showed an effective PKM2 activation with a half-maximal activity concentration of 4.92 μM, which was comparable to that of the reference compound (Fig. 2A). We also confirmed that PA-12 increases pyruvate kinase activity in cancer cell lysates (Supplementary Fig. 2B). These data indicate that PA-12 is a potent activator 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