Singlet Oxygen-Induced Mitochondrial Reset in Cancer: A Novel Approach for Ovarian Cancer Therapy

Abstract

Background/Objectives: This study explores the generation of singlet oxygen (SO) through methylene blue (MB) activation as a metabolic intervention for ovarian cancer. We aimed to examine the role of SO in modulating mitochondrial function, cellular metabolism, and proliferation in ovarian cancer cell lines compared to control cells. Methods: The study utilized two ovarian cancer cell lines, OV1369-R2 and TOV1369, along with ARPE-19 control cells. Following MB treatment and light activation, mitochondrial function and ATP synthesis were assessed. Metabolomic analyses were performed to evaluate changes in central carbon metabolism, particularly focusing on markers of the Warburg effect. Results: TOV1369 cells exhibited a pronounced sensitivity to MB treatment, resulting in significant inhibition of ATP synthesis and reduced proliferation. Metabolomic analysis indicated that MB-induced SO production partially reversed the Warburg effect, suggesting a shift from glycolysis to oxidative phosphorylation. These effects were less pronounced in OV1369-R2 and ARPE-19 cells, correlating with their lower MB sensitivity. Conclusions: MB-generated SO selectively modulates mitochondrial energetics in ovarian cancer cells, driving a metabolic reorganization that curtails their proliferative capacity. This approach, leveraging the bacterial-like features of cancer metabolism, offers a promising therapeutic avenue to induce apoptosis and enhance treatment outcomes in ovarian cancer.

Keywords: metabolic bistability; methylene blue; mitochondrial involution; ovarian cancer; singlet oxygen.

Figure 1

Methylene blue-induced mitochondrial singlet oxygen generation reduces ovarian cancer cell proliferation. (A) Singlet oxygen (SO) levels are monitored before and after laser radiation in cell cultures. Basal SO production (“before radiation”) is observed in all cell cultures in the presence of methylene blue (MB). Subsequent radiation rounds consistently result in the generation of SO to a level of 1900 a.u. (B) The effect of MB on ARPE-19 cell proliferation at a concentration of 10 µM (MB10) shows minimal impact, with no significant difference observed with the addition of SO. (C) The carboplatin-resistant ovarian cancer cell line OV1369-R2 displays limited sensitivity to low concentrations of MB. However, a slight inhibition in proliferation is observed at 10 µM of MB (MB10) when combined with SO. (D) The TOV1369 cell line, which is also carboplatin-resistant, exhibits a pronounced sensitivity to MB. The overflow of SO has a significantly greater effect on the culture condition with MB at a concentration of 10 µM (MB10). The p-values of ** (p ≤ 0.01) and *** (p ≤ 0.001) were considered as significant.

Figure 2

Singlet oxygen-induced (¹O₂) apoptosis in ovarian cancer cells. To assess the impact of singlet oxygen on ovarian cancer cells, we examined the occurrence of apoptosis under different radiation conditions using a 10 µM concentration of methylene blue (MB10) in a culture medium. The results demonstrate a significantly higher proportion of apoptotic cells in the OV1369-R2 and TOV1369 cancer cell lines compared to the normal ARPE-19 line (p ≤ 0.01 and p ≤ 0.001, respectively). This indicates the heightened sensitivity of the cancer cell lines to mitochondrial singlet oxygen generation. The observed increase in apoptotic cells supports the notion that ¹O₂ plays a crucial role in inducing programmed cell death in ovarian cancer cells. These findings underscore the potential therapeutic implications of targeting ¹O₂, a pivotal metabolite in the electron transfer chain (ETF), to selectively eliminate cancer cells while sparing normal cells. The p-values of ** (p ≤ 0.01) and *** (p ≤ 0.001) were considered as significant.

Figure 3

Methylene blue-induced singlet oxygen generation reduces aerobic glycolysis in ovarian cancer cells. (A,D) ARPE-19 exhibits a Warburg effect-like phenotype characterized by high glucose consumption and increased lactate production in the culture media. Treatment with MB10, with or without radiation, leads to reduced lactate production, indicating decreased aerobic glycolysis. (B,C,E,F) The MB10 treatment effectively attenuates lactate production in cancer cells, with a pronounced impact on TOV1369′s aerobic glycolysis. The OV1369-R2 line demonstrates higher glutamine consumption than the other two cell lines. The ”T0”condition refers to the initial metabolite’s concentration prior to cell incubation. The p-values of * (p ≤ 0.05), ** (p ≤ 0.01), and *** (p ≤ 0.001) were considered as significant.

Figure 4

Singlet oxygen-mediated mitochondrial membrane potential stimulation rewires oxidative phosphorylation (OxPhos) in ovarian cancer cells. (A) Singlet oxygen increases the mitochondrial membrane potential (ΔΨm), measured in relative fluorescence units (R.F.U) of the JC-1 cationic dye. (B) Mitochondrial ROS index (Δp), determined by the JC-1 dye red/green fluorescence intensity ratio and measuring relative mitochondrial membrane polarization and depolarization, shows that the MB10 treatment increases mitochondrial ROS levels in cancer cell lines while having the opposite effect in the normal ARPE-19 cell line. (C) Mitochondrial horsepower (h), estimated based on ROS and MMP, is significantly increased in cancer cells under the MB10 conditions. With radiation, mitochondrial horsepower tends to approach values observed in the normal condition (MB0). (D–F) Free cellular ATP levels were assessed in the normal ARPE-19 cell line (D) as well as in the ovarian cancer cell lines OV1369-R2 and TOV1369. (E,F) In general, TOV1369 cells showed higher free ATP levels with a more pronounced response to MB10, indicating greater sensitivity. The p-values of * (p ≤ 0.05), ** (p ≤ 0.01), and *** (p ≤ 0.001) were considered significant.