Anaerobic Cancer Cell Metabolism and Viability

Abstract

IntroductionNormal internal human physiologic oxygen levels are hypoxic, ranging from about 0.5 to 14%. However, in solid tumors, there are intra‐tumor niches, which like stem cell niches, are anaerobic. A historical roadblock to studying anaerobic cancer cell metabolism is the inability to maintain cell viability in an oxygen‐free environment for longer than 36 hrs. We previously showed that anaerobic cancerous and non‐cancerous cells, which survive at least 14d of anaerobic culture, exhibit an altered cell phenotype, i.e. diminished attachment capacity, slowed growth rate, and inverse hypoxia inducible factor‐1α mRNA and protein expression relative to anaerobic endogenous reactive oxygen species production. Since bacteria (mitochondrial precursors) use Nitric Oxide (NO) and Hydrogen Sulfide (H2S) as terminal electron acceptors, we focused on determining the role NO and H2S generating pathways play in sustaining anaerobic cancer cell viability.MethodsHeLa 229 cells were grown to 80% confluence in 24 well plates (normoxic conditions, 5% CO2 in air; 10% FBS; DMEM medium) then transferred to degassed PS‐74656 medium (Whitley A35, anaerobic gas mixture: H2, CO2, N2; 37°C). Viability was determined by trypan blue dye exclusion. Anoxic nitric oxide production was determined by fluorescent microscopy (DAF‐2; excitation 491 nm; emission 513 nm), with NO positive cells counted (10 random fields; Mean ± SEM; p < 0.05). Controls consisted of cells from the same lot processed in parallel but anaerobically incubated in control PS‐74656 medium without additional NO and H2S pathway precursors or inhibitors.ResultsUpon anaerobic incubation, NO was detected at days 3 and 10 of culture. NO is a by‐product of the Xanthine Oxidase (XO) and Nitric Oxide Synthase (NOS) pathways. Addition of pathway precursors xanthine (4 × 10−5 M) or arginine (50 mM), respectively, significantly (p < 0.05) increased HeLa cell viability by 3 and 20.9 fold, respectively, at day 10 of anaerobic incubation. Allopurinol (200 μM), an XO pathway inhibitor, inhibited the xanthine induced increase in viability while L‐NAME had a similar effect on arginine‐mediated viability, which was also reduced to control level.To determine if H2S supports HeLa cell anaerobic viability, the effects of H2S pathway precursor cysteine (0.4 mM), and H2S donors JK‐2 (300 μM) and GYY4137 (400 μM) were tested. Overall, any treatment that should increase cellular H2S levels caused significantly (p < 0.05) enhanced viability, as compared to medium alone (cysteine, 3.6; JK‐2, 6.3; and GYY4137, 7.6 fold increases).These results confirm our hypothesis that similar to bacteria, terminal electron donors other than oxygen can function to support tumor cell anaerobic respiration.Support or Funding InformationThis project was supported by research funds from Office of Research and Sponsored Programs (ORSP), Midwestern University, IL, College of Osteopathic Medicine (CCOM) at Midwestern University and College of Graduate Studies (CGS) at Midwestern University with assistance from the MWU Core Facility.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.