HIF-1-mediated suppression of mitochondria electron transport chain function confers resistance to lidocaine-induced cell death

Abstract

The local anesthetic lidocaine induces cell death by altering reactive oxygen species (ROS) generation and mitochondrial electron transport chain function. Because hypoxia-inducible factor 1 (HIF-1) is involved in determining oxygen metabolism and mitochondria function, we investigated the involvement of HIF-1 activity in lidocaine-induced cell death. We investigated the role of HIF activation on lidocaine-induced caspase activation and cell death in renal cell-derived RCC4 cells lacking functional von Hippel-Lindau (VHL) protein. We demonstrate that HIF-1 suppressed oxygen consumption and facilitated glycolysis in a pyruvate dehydrogenase kinase-1-dependent manner and that activation of HIF-1 conferred resistance to lidocaine-induced cell death. We also demonstrated that exogenous HIF-1 activation, through HIFα-hydroxylase inhibition or exposure to hypoxic conditions, alleviates lidocaine toxicity by suppressing mitochondria function and generating ROS, not only in RCC4 cells, but also in the neuronal SH-SY5Y cells. In conclusion, we demonstrate that HIF-1 activation due to VHL deletion, treatment with small molecule HIFα-hydroxylase inhibitors, and exposure to hypoxic conditions suppresses mitochondrial respiratory chain function and confers resistance to lidocaine toxicity.Local anesthetics, including lidocaine, affect the intra-and extra-cellular signaling pathways of both neuronal and non-neuronal cells, resulting in long-term modulation of biological functions such as cell growth and death 1 . Although the primary target of lidocaine is voltage-gated sodium channels, the targets and mechanisms in the context of cell growth and death are unknown. Studies indicate that mitochondria are one of the critical targets of lidocaine [2][3][4] . Similarly, we previously reported that reactive oxygen species (ROS) derived from mitochondria play an essential role in lidocaine-induced apoptosis and treatment with the antioxidants N-acetyl cysteine (NAC) and Trolox effectively prevents apoptosis 4 . The mitochondrial oxidative phosphorylation (OXPHOS) system plays a critical role in modulating ATP supply in the human body. Deficits derived from genetic and pharmacologic treatments cause functional disorders and cell death. The OXPHOS machinery consists of four enzyme complexes in the respiratory chain that transport electrons obtained from the oxidation of carbohydrates and fats to molecular oxygen (O 2 ); a fifth enzyme complex uses the energy derived from this process to drive ATP synthesis. O 2 is the electron acceptor, resulting in the production of H 2 O in a process that is catalyzed by complex IV of the mitochondrial electron transport chain (ETC). Because the process is not completely efficient, electron transport to O 2 may occur in complexes I or III, resulting in generation of ROS that can oxidize cellular proteins, lipids, and nucleic acids [5][6][7] . The generated ROS causes cell dysfunction or death when cells are exposed to drugs affecting electron transport in the ETC.

Propofol induces a metabolic switch to glycolysis and cell death in a mitochondrial electron transport chain-dependent manner

“…In addition to biguanides, other clinically used drugs, including chloramphenicol 43,44 , aspirin 45,46 , statins 18 , and local anesthetics 47 , also inhibit mitochondrial functions. Our results indicate that pre-exposure to mitochondrial inhibitors may increase the toxicity of propofol.…”

“…Cell growth was assessed using a CellTiter 96 AQueous One Solution Cell Proliferation Assay™ with MTS (Promega, Madison, WI, USA) 47,49 . Briefly, cells were seeded into 96-well plates (2 × 10 4 cells/well) and cultured overnight.…”

“…Activity levels of caspase-3/7 and caspase-9 were assessed using an Apo-ONE™ homogeneous caspase-3/7 assay kit (Promega) and a Caspase-Glo™ 9 assay kit (Promega), respectively, according to the manufacturer's protocols 47,49 . Briefly, cells were seeded into 96-well plates (2 × 10 4 cells/well) and incubated overnight.…”

“…Cytotoxicity was measured using a CytoTox-ONE™ kit (Promega) as described previously 47,49 . Briefly, cells were cultured overnight in 96-well plates (2 × 10 4 cells/well) and treated with the indicated drug(s) for varying lengths of time.…”

“…Cellular OCR and ECAR were determined with the XF Cell Mito Stress Test™ and XF Glycolysis Stress Test™, respectively, using an XFp Extracellular Flux Analyzer™ (Seahorse Bioscience, USA) 47 . Cells (2 × 10 5 cells/well) were seeded into an XFp cell culture microplate, and OCR was assessed in glucose-containing XF base medium according to the manufacturer's instructions.…”