CC BY
2Effect of methylene blue on the NADH metabolic index of tumor
cells before and after PDT
A.V. Ryabova12*, D.V. Pominova12, I.V. Markova2, I.D. Romanishkin1, V.B. Loschenov12
1-Prokhorov General Physics Institute of the Russian Academy of Sciences, 119991 Russia, Moscow, Vavilova
st. 38
2- National research nuclear university MEPHI, 115409 Russia, Moscow, Kashirskoye sh. 31
Hypoxia plays an important role in the interaction of cancer cells, stroma and immune cells and contributes to tumor resistance to treatment [1]. Strategies to overcome hypoxia include oxygen delivery or in situ generation, reduction of oxygen consumption by the tumor, normalization of the tumor vasculature [2]. Our previous studies showed that MB leads to increased tumor oxygenation in a mouse model of Lewis lung carcinoma [3]. The effect of the methylene blue (MB) photosensitizer on the tumor cells' metabolism is due to, among other things, interaction with NADH, while MB is reduced to the leuco form, and NADH is oxidized to NAD+, providing an increase in the ratios of pyruvate:lactate and reactivation of the electron transport chain [4].
This paper presents studies of the effect of PDT with MB and the combination of MB/chlorin e6 photosensitizers in terms of metabolic changes in tumor cells. The studies were performed on HeLa tumor cell culture in vitro and on murine Ehrlich adenocarcinoma in vivo. PDT on HeLa cells in vitro included 1 hour incubation of cells with 5 mg/kg of chlorin e6 and 1-20 mg/kg of MB and their combination; 660 nm, 30-100 mW/cm2. In vivo PDT with 5 mg/kg of chlorin e6, 10 mg/kg of MB and their combination; 660 nm, 60 J/cm2. Control animals were animals with and without MB/chlorin e6, without irradiation. Using confocal microscopy, mitochondrial stress and generation of reactive oxygen species in cells in response to laser irradiation with MB were studied. The changes in cellular metabolism in response to PDT were assessed by analyzing the fluorescence life-time imaging microscopy (FLIM) of NADH.
Oxidative stress from chlorin e6 incubation in combination with 1-20 mg/kg MB or for 10-20 mg/kg MB without chlorin e6 leads to an elongation of the NADH fluorescence distribution on the FLIM phasor diagram along the NADH metabolic trajectory 0.4-2.5 ns.
After PDT with MB, NADH fluorescence lifetime becomes shorter (closer to anaerobic glycolysis). After PDT with MB and chlorin e6, however, it shifts towards longer lifetimes (closer to oxidative phosphorylation). With a large dose of PDT (strong oxidative stress), the NADH fluorescence distribution on the phasor diagram becomes rounded; dead cells a day after PDT also retain a rounded distribution of the NADH fluorescence cloud on the phasor. The obtained data will make it possible to optimize hypoxic tumor treatment, increasing the sensitivity of cancer and immune cells triggering the process of immunogenic cell death.
The study was funded by a grant from the Russian Science Foundation (project N 22-72-10117).
[1] V. Petrova, M. Annicchiarico-Petruzzelli, G. Melino, et al, The hypoxic tumour microenvironment. Oncogenesis, 7(10), 1-13 (2018).
[2] Z. Shen, Q. Ma, X. Zhou, et al, Strategies to improve photodynamic therapy efficacy by relieving the tumor hypoxia environment. NPG Asia Mater, 13(39), 1-19 (2021).
[3] D. Pominova, A. Ryabova, A. Skobeltsin, I. Markova, K. Linkov, I. Romanishkin, The use of methylene blue to control the tumor oxygenation level, Photodiagnosis and Photodynamic Therapy, 46, 104047:1-8 (2024).
[4] T. Komlodi and L.Tretter, Methylene blue stimulates substrate-level phosphorylation catalysed by succinyl-CoA ligase in the citric acid cycle, Neuropharmacology, 123, 287-298, (2017).
