1. Department of Biochemistry, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan;
2. Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 80756, Taiwan;
3. Department of Surgery, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan;
4. Division of General and Digestive and Pancreatic Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung Medical University 80756, Taiwan;
5. Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan;
6. Department of Medical Laboratory Science and Biotechnology, College of Health Sciences, Kaohsiung Medical University, Kaohsiung 80708, Taiwan;
7. Institute of Medical Science and Technology, College of Sciences, National Sun Yat-sen University, Kaohsiung 80424, Taiwan;
8. Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 80756, Taiwan.
Background: Cancer cells proliferate rapidly and are resistant to cell death, relying on aggravated glycolysis to satisfy their increased demand for energy and biosynthetic precursors. However, this process may create unfavorable microenvironments, such as increased acidity, leading to cytotoxicity. Our previous study demonstrated that arecoline induces anoikis of HA22T/VGH hepatoma cells. The present study aimed to examine if arecoline induced anoikis is related to the glycolytic pathway and explore the underlying mechanisms.
Methods: HA22T/VGH cells were treated with arecoline and changes in the glycolytic end products lactate and ATP, glycolytic-related gene expression, intracellular and extracellular pH, pH-regulating gene expression, reactive oxygen species (ROS) levels, intracellular Ca2+ concentration ([Ca2+]i) and mitochondrial membrane potential were examined, relative to untreated cells. Cell viability and morphology were also assessed.
Results: Arecoline increased lactate and ATP production through induction of glycolytic genes, including glucose transporter 3 (Glut3), hexokinase 1 (HK1), hexokinase 2 (HK2), and pyruvate kinase (PK). The intracellular pH was not changed, despite increased lactate levels, implying that intracellular H+ was exported out of the cells. mRNA expression of pH regulators including monocarboxylate transporter 1 and 4 (MCT 1 and 4), sodium bicarbonate cotransporter 1 (NBC1), carbonic anhydrases (CA) IX and XII and vacuolar ATPase (V-ATPase) were down-regulated. Na+/H+ exchanger 1 (NHE1) mRNA levels remained unchanged while Na+/Ca2+ exchanger (NCX) was up-regulated and eventually [Ca2+]i was increased. ROS generation was increased and mitochondrial membrane potential was decreased followed by cell detachment and death. Addition of 2-deoxy-d-glucose, a glucose competitor that interferes with glycolysis, attenuated arecoline induction of lactate [Ca2+]i, ROS and cell detachment. Similarly, ROS scavengers could block the effects of arecoline.
Conclusions: This study demonstrated that arecoline induced glycolysis and modulated the mRNA expression of pH-regulator genes in HA22T/VGH cells. This phenomenon led to the elevation of [Ca2+]i, ROS generation, and subsequent cell detachment.
Keywords: Arecoline, Glycolysis, ROS, Calcium ion, Anoikis.