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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

The anticancer effects of PA-12 are dependent on PKM2 expression. (A and B) PKM2 depletion desensitizes cancer cells to PA-12 treatment in an NEAA-depleted medium in normoxia (A) and under hypoxia (B). (C) The inhibitory effect of PA-12 on HRE reporter activity is dependent on PKM2 expression. The cells were pretreated with PA-12 or PKIII for 2 h and then cultured for 12 h under hypoxia. HRE reporter activity was measured by a luciferase assay. (D) The inhibitory effect of PA-12 on the hypoxic expression of HIF target gene, GLUT3, is dependent on PKM2 expression. Stable shPKM2 and shControl cells were pretreated with PA-12 or PKIII for 2 h and then cultured for 12 h under hypoxia. Gene expression was analyzed by RT-PCR. Numbers under the graphic RT-PCR data are quantitative estimations of the band intensity made using Image J program. Similar results were obtained from three independent experiments, and representative data are shown.
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f4-molce-38-4-373: The anticancer effects of PA-12 are dependent on PKM2 expression. (A and B) PKM2 depletion desensitizes cancer cells to PA-12 treatment in an NEAA-depleted medium in normoxia (A) and under hypoxia (B). (C) The inhibitory effect of PA-12 on HRE reporter activity is dependent on PKM2 expression. The cells were pretreated with PA-12 or PKIII for 2 h and then cultured for 12 h under hypoxia. HRE reporter activity was measured by a luciferase assay. (D) The inhibitory effect of PA-12 on the hypoxic expression of HIF target gene, GLUT3, is dependent on PKM2 expression. Stable shPKM2 and shControl cells were pretreated with PA-12 or PKIII for 2 h and then cultured for 12 h under hypoxia. Gene expression was analyzed by RT-PCR. Numbers under the graphic RT-PCR data are quantitative estimations of the band intensity made using Image J program. Similar results were obtained from three independent experiments, and representative data are shown.

Mentions: In order to study the influence of PKM2 expression on the PA-12-mediated inhibition of lung cancer cell viability, we constructed A549 cell lines stably expressing short hairpin RNA (shRNA) for PKM2 (shPKM2) or mock (shControl). The cells were treated with PA-12 or PKIII in NEAA-depleted medium for 48 h in normoxia, and cell viability was assessed by counting cell numbers. Results indicated that shPKM2 cells were less sensitive to the inhibitory effect of PA-12 (30 μM) than shControl cells (Fig. 4A). Since PA-12 could inhibit HIF signaling, we also determined the effect of PA-12 on cell viability under hypoxia. A549 lung cancer cells were pretreated with PA-12 or PKIII for 2 h and cultured further for 48 h under hypoxia. Results showed that the PA-12-mediated inhibitory effect on cell viability was significantly blocked in the PKM2-depleted cells (Fig. 4B). In accordance, the hypoxic HRE reporter activity suppressed by PA-12 was dramatically restored in the PKM2-depleted cells (Fig. 4C). We then examined the effect of PA-12 on the hypoxia-induced expression of the HIF-1α target gene, GLUT3. Cells were pretreated with PA-12 or PKIII for 2 h and then incubated under hypoxia for 12 h. The hypoxic induction of GLUT3 was much weaker in shPKM2 cells than in control cells (Fig. 4D). In addition, the inhibitory effect of PA-12 on the hypoxia-induced expression of GLUT3 seemed to be relatively weaker in shPKM2 cells compared to that in shControl cells. Taken together, these results imply that the inhibitory effect of PA-12 on lung cancer cell viability is dependent on PKM2 protein expression.


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)

The anticancer effects of PA-12 are dependent on PKM2 expression. (A and B) PKM2 depletion desensitizes cancer cells to PA-12 treatment in an NEAA-depleted medium in normoxia (A) and under hypoxia (B). (C) The inhibitory effect of PA-12 on HRE reporter activity is dependent on PKM2 expression. The cells were pretreated with PA-12 or PKIII for 2 h and then cultured for 12 h under hypoxia. HRE reporter activity was measured by a luciferase assay. (D) The inhibitory effect of PA-12 on the hypoxic expression of HIF target gene, GLUT3, is dependent on PKM2 expression. Stable shPKM2 and shControl cells were pretreated with PA-12 or PKIII for 2 h and then cultured for 12 h under hypoxia. Gene expression was analyzed by RT-PCR. Numbers under the graphic RT-PCR data are quantitative estimations of the band intensity made using Image J program. Similar results were obtained from three independent experiments, and representative data are shown.
© Copyright Policy
Related In: Results  -  Collection

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

f4-molce-38-4-373: The anticancer effects of PA-12 are dependent on PKM2 expression. (A and B) PKM2 depletion desensitizes cancer cells to PA-12 treatment in an NEAA-depleted medium in normoxia (A) and under hypoxia (B). (C) The inhibitory effect of PA-12 on HRE reporter activity is dependent on PKM2 expression. The cells were pretreated with PA-12 or PKIII for 2 h and then cultured for 12 h under hypoxia. HRE reporter activity was measured by a luciferase assay. (D) The inhibitory effect of PA-12 on the hypoxic expression of HIF target gene, GLUT3, is dependent on PKM2 expression. Stable shPKM2 and shControl cells were pretreated with PA-12 or PKIII for 2 h and then cultured for 12 h under hypoxia. Gene expression was analyzed by RT-PCR. Numbers under the graphic RT-PCR data are quantitative estimations of the band intensity made using Image J program. Similar results were obtained from three independent experiments, and representative data are shown.
Mentions: In order to study the influence of PKM2 expression on the PA-12-mediated inhibition of lung cancer cell viability, we constructed A549 cell lines stably expressing short hairpin RNA (shRNA) for PKM2 (shPKM2) or mock (shControl). The cells were treated with PA-12 or PKIII in NEAA-depleted medium for 48 h in normoxia, and cell viability was assessed by counting cell numbers. Results indicated that shPKM2 cells were less sensitive to the inhibitory effect of PA-12 (30 μM) than shControl cells (Fig. 4A). Since PA-12 could inhibit HIF signaling, we also determined the effect of PA-12 on cell viability under hypoxia. A549 lung cancer cells were pretreated with PA-12 or PKIII for 2 h and cultured further for 48 h under hypoxia. Results showed that the PA-12-mediated inhibitory effect on cell viability was significantly blocked in the PKM2-depleted cells (Fig. 4B). In accordance, the hypoxic HRE reporter activity suppressed by PA-12 was dramatically restored in the PKM2-depleted cells (Fig. 4C). We then examined the effect of PA-12 on the hypoxia-induced expression of the HIF-1α target gene, GLUT3. Cells were pretreated with PA-12 or PKIII for 2 h and then incubated under hypoxia for 12 h. The hypoxic induction of GLUT3 was much weaker in shPKM2 cells than in control cells (Fig. 4D). In addition, the inhibitory effect of PA-12 on the hypoxia-induced expression of GLUT3 seemed to be relatively weaker in shPKM2 cells compared to that in shControl cells. Taken together, these results imply that the inhibitory effect of PA-12 on lung cancer cell viability is dependent on PKM2 protein expression.

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