Circumventing the Crabtree effect: forcing oxidative phosphorylation (OXPHOS) via galactose medium increases sensitivity
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Circumventing the Crabtree effect: forcing oxidative phosphorylation (OXPHOS) via galactose medium increases sensitivity of HepG2 cells to the purine derivative kinetin riboside Marta Orlicka‑Płocka1 · Dorota Gurda‑Wozna1 · Agnieszka Fedoruk‑Wyszomirska1 · Eliza Wyszko1 Accepted: 4 September 2020 © The Author(s) 2020
Abstract Small-molecule compound-based therapies have provided new insights into cancer treatment against mitochondrial impairment. N6-furfuryladenosine (kinetin riboside, KR) is a purine derivative and an anticancer agent that selectively affects the molecular pathways crucial for cell growth and apoptosis by interfering with mitochondrial functions and thus might be a potential mitotoxicant. Metabolism of cancer cells is predominantly based on the Crabtree effect that relies on glucoseinduced inhibition of cell respiration and thus on oxidative phosphorylation (OXPHOS), which supports the survival of cancer cells in metabolic stress conditions. The simplest way to circumvent this phenomenon is to replace glucose with galactose in the culture environment. Consequently, cells become more sensitive to mitochondrial perturbations caused by mitotoxicants. In the present study, we evaluated several cellular parameters and investigated the effect of KR on mitochondrial functions in HepG2 cells forced to rely mainly on OXPHOS. We showed that KR in the galactose environment is a more potent apoptosisinducing agent. KR decreases the mitochondrial membrane potential, reduces glutathione level, depletes cellular ATP, and induces reactive oxygen species (ROS) production in the OXPHOS state, leading to the loss of cell viability. Taken together, these results demonstrate that KR directly acts on the mitochondria to limit their function and that the sensitivity of cells is dependent on their ability to cope with energetic stress. Keywords Purine derivative · Kinetin riboside · Mitochondria · Crabtree effect · Metabolism · Cancer cells Abbreviations 2-DG 2-Deoxy-D-glucose 2-NBDG 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose 7-AAD 7-Aminoactinomycin D A172 Human glioblastoma cell line ADK Adenosine kinase ADP Adenosine diphosphate AICAR 5-Aminoimidazole-4-carboxamide 1-β-d-ribofuranoside ATP Adenosine triphosphate BAX Bcl-2-associated X protein BCl-2 B cell lymphoma 2 Marta Orlicka-Płocka and Dorota Gurda-Wozna contributed equally to this work. * Eliza Wyszko [email protected] 1
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61‑704 Poznan, Poland
CCCP Carbonyl cyanide 3-chlorophenylhydrazone CCND2 Cyclin D2 DAPI 2-(4-Amidinophenyl)-1H -indole-6-carboxamidine DMEM Dulbecco’s Modified Eagle Medium DPBS Dulbecco’s phosphate-buffered saline ECAR Extracellular acidification rate EMEM Eagle’s Minimum Essential Medium FBS Fetal bovine serum FCCP Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone FDA Food and Drug Administration GSH (2S) 2-Amino-4-{[(1R)-1-[(carboxymethyl) carbamoyl]-2-sulfanylethyl]carbamoyl}butanoic acid; glutathion
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