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Analytical, clinical and medical applications of luminescence

Use of a novel pH-sensing luciferase to determine pH changes associated with the switch from mitochondrial respiration to glycolysis in colon cancer cells

Vanessa R. Bevilaqua1, Angela P.1, Alessia S.2, Eliana A. R. Duek1, Aldo Roda3, Vadim Viviani4, Cristiana Caliceti5

1Department of Biomedical and Neuromotor Sciences, University of Bologna, 40138 Bologna, Italy., 2Department for Life Quality Studies, University of Bologna, 47921 Rimini, Italy., 3National Institute of Biostructure and Biosystems (INBB), 00136 Rome, Italy., 4Department of Physics, Chemistry and Mathematics, Federal University of São Carlos (UFSCar), Sorocaba 18052-780, Brazil., 5Department of Biomedical and Neuromotor Sciences, University of Bologna, 40138 Bologna, Italy. National Institute of Biostructure and Biosystems (INBB), 00136 Rome, Italy.

E-mail: vani_bevilaqua8@hotmail.com

Cancer cells generally exhibit increased glycolysis for ATP generation (the Warburg effect) due in part to mitochondrial respiration injury and hypoxia, which are frequently associated with resistance to therapeutic agents. This switching induces cancer cells to plunder more glucose than normal ones from microenvironment, thus secreting more lactic acid to meet energy requirements (doi: 10.1016/j.gendis.2017.02.003). During this process, at the initial stage a large amount of protons is produced, so mechanisms to expel protons from the cell are activated to keep an alkaline intracellular pH (doi: 10.1038/nrd3554). As a result, the tumor microenvironment (TME) becomes acidic. Furthermore, extracellular acidic pH and intracellular alkaline pH of cancer cells are known to induce malignant behaviors, such as increased invasion and metastasis, multi-drug resistance, and suppression of immune surveillance. To investigate the pH changes associated with the switching to glycolysis in cancer cells, we used a novel pH-sensing luciferase reporter gene (AmyLuc), which is thermally more stable at 37°C, and intentionally broke down mitochondria respiration in human colorectal adenocarcinoma cells (Caco-2) and monitored the intracellular pH by measuring the ratio of red and green light intensities. Considering that this luciferase emits more red light in an acidic environment while at an alkaline pH produces more green light, we measured BL spectra of AmyLuc transfected Caco-2 cells seeded in cells media at pH 6.0 and 8.0, and determined the BL spectral peaks (pH 6.0: 593 nm; pH 8.0: 548 nm). Based on the BL peak at these two pHs, we then calculated the red/green light intensity ratio, allowing to estimate the intracellular physiological pH of Caco2 cells and to monitor the intracellular acidification or alkalinization upon different treatments. Indeed, the AmyLuc transfected cells were treated with the mitochondrial uncoupler FCCP (10 µM), the respiratory chain inhibitor antimycin (10 µM), and the proton antiporter K+/H+ nigericin (2 µg/mL). Upon treatment with these drugs, the BL spectra showed a gradual increase of the ratio red/green light intensities during the first 20-30 min, indicating an acidification of the cytoplasm, especially in the case of antimycin, and then a slower decrease of this ratio indicating recovering of buffering capacity. Such initial intracellular acidification upon mitochondria respiratory chain inhibition or uncoupling could be caused by the switch from mitochondrial respiratory catabolism to fermentative glycolysis, which initially results in a burst of lactate production, temporarily acidifying the cytoplasmic environment. Altogether, these results indicate that the Amydetes vivianii color-tuning luciferase could be a new promising bioimaging tool to investigate the switch from aerobic respiration to anaerobic glycolysis in cancer cells.

Keywords: Bioluminescence, mitochondrial inhibitors, FCCP, Antimycin

Acknowledgments: FAPESP 2023/02482-4, 2022/04800-0, PRIN 2017 (Prot. 017Y2PAB8) and Fondazione Cassa di Risparmio di Bologna (ID 19045- COD SME 2020.0399)

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