Abstract 37: Effect of glucose metabolism-related enzymes on the proliferation of gastric cancer cells in hypoxic microenvironment
Authors: Kitayama Kishu , Yashiro Masakazu , Morisaki Tamami , Masuda Go , Kasashima Hiroakai , Miki Yuichiro , Kinoshita Haruhito , Fukuoka Tastunari , Hasegawa Tsuyoshi , Ohira Masaichi , Hirakawa Kosei 
Published: July 2016

Background: Many kinds of solid tumor have heterogeneously a hypoxic environment. Cancer cells acquire characteristics that allow them to survive and proliferate in the hypoxic environment. However, the mechanisms by which hypoxia affects the aggressive phenotypes remain unclear. “Warburg effect” is known as anaerobic glycolysis of cancer cells, contributes tumorigenesis and tumor development. The aim of this study was to analyze the significance of glucose metabolism-related enzymes on the proliferation of gastric cancer cells in hypoxic microenvironment.

Materials and Methods: Two hypoxia-resistant cancer cell lines, OCUM-12hypo cells and OCUM-2MD3hypo cells, and their parent OCUM-12 cells and OCUM-2MD3 cells were used. OCUM-12hypo cells and OCUM-2MD3hypo cells were cloned from OCUM-12 cells and OCUM-2MD3 cells by continuous exposure to 1% oxygen, respectively. Protein lysates from each cell line were analyzed using QSTAR Elite Liquid Chromatography with Tandem Mass Spectrometry, coupled with isobaric tags for relative and absolute quantitation technology. mRNA expression level of enolase 1 (ENO1), pyruvate kinase isozymes M2 (PKM2), and glutaminase (GLS) were evaluated by RT-PCR. Effects of inhibition of glucose metabolism-related enzymes on the proliferation and apoptosis were examined by CCK assay or FACScan analysis using siRNA (siENO1, siPKM2, and siGLS), or PKM2 inhibitor Shikonin and GLS inhibitor BPTES.

Results: Glucose metabolism-related enzymes, ENO1 and PKM2, were significantly increased in both hypoxia-resistant cancer cell lines in comparison with their parent cell lines. Expression level of ENO1, PKM2, and GLS were also significantly high in both hypoxia-resistant cancer cell lines in comparison with their parent cell lines. The proliferation of OCUM-12hypo cells and OCUM-2MD3hypo cells was significantly inhibited by the knockdown of PKM2 and GLS, but not by the knockdown of ENO1. Cell proliferation was also significantly inhibited by PKM2 inhibitor and/or GLS inhibitor. A synergistic anti-tumor effect for gastric cancer cells in hypoxia was found in combination with PKM2 inhibitor and GLS inhibitor. The combination of PKM2 inhibitor and GLS inhibitor increased apoptosis rates of hypoxic gastric cancer cells.

Conclusion: PKM2 and GLS might be important enzymes for the proliferation of gastric cancer in hypoxic microenvironment. The combined treatment with PKM2 inhibitor and GLS inhibitor produced synergistic anti-tumor effects, and might be a therapeutically promising for the treatment of gastric cancer.

Citation Format: Kishu Kitayama, Masakazu Yashiro, Tamami Morisaki, Go Masuda, Hiroakai Kasashima, Yuichiro Miki, Haruhito Kinoshita, Tastunari Fukuoka, Tsuyoshi Hasegawa, Masaichi Ohira, Kosei Hirakawa. Effect of glucose metabolism-related enzymes on the proliferation of gastric cancer cells in hypoxic microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 37.