Mitochondrial Respiration in Colon Cancer

We studied the mitochondrial respiration, specifically, oxygen consumption, of ex vivo tissue samples and two colon cancer cell lines harboring BRAF mutations (HT-29 and WiDr) using the high-resolution respirometry system Oxygraph-2k (OROBOROS Instruments, Innsbruck, Austria) at 37 °C. The medium for the measurements contained 0.5 mM of EGTA, 3 mM of MgCl₂, 60 mM of K-lactobionate, 20 mM of taurine, 10 mM of KH₂PO₄, 20 mM of HEPES, and 110 mM of sucrose, adjusted to pH 7.1 at 37 °C. To permeabilize the cell membranes, 16 μg/mL of digitonin was added. We documented the non-phosphorylating state 2 (V0) respiration rate in the medium containing the tissue samples, mitochondrial complex I substrates (5 mM of glutamate + 2 mM of malate), and the complex II substrate succinate (12 mM). After adding 1 mM of ADP, we assessed the state 3 respiration rate (VADP). All respiration rates were normalized to the dry weight of the tissue in milligrams.