Different RONS Generation in MTC-SK and NSCL Cells Lead to Varying Antitumoral Effects of Alpha-Ketoglutarate + 5-HMF

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Abstract
Background: Carbonylated proteins (CPs) serve as specific indicators of increased reactive oxygen and nitrogen species (RONS) production in cancer cells, attributed to the dysregulated mitochondrial energy metabolism known as the Warburg effect. The aim of this study was to investigate the potential of alpha-ketoglutarate (aKG), 5-hydroxymethylfurfural (5-HMF), and their combination as mitochondrial-targeting antioxidants in MTC-SK or NCI-H23 cancer cells. Methods: MTC-SK and NCI-H23 cells were cultured in the absence or presence of varying concentrations (0–500 µg/mL) of aKG, 5-HMF, and the combined aKG + 5-HMF solutions. After 0, 24, 48, and 72 h, mitochondrial activity, cancer cell membrane CP levels, cell growth, and caspase-3 activity were assessed in aliquots of MTC-SK and NCI-H23 cells. Results: The mitochondrial activity of MTC-SK cells exhibited a concentration- and time-dependent reduction upon treatment with aKG, 5-HMF, or the combined aKG + 5-HMF. The half-maximal inhibitory concentration (IC50%) for mitochondrial activity was achieved at 500 µg/mL aKG, 200 µg/mL 5-HMF, and 200 µg/mL aKG + 66.7 µg/mL 5-HMF after 72 h. In contrast, NCI-H23 cells showed a minimal reduction (10%) in mitochondrial activity even at the highest combined concentration of aKG + 5-HMF. The CP levels in MTC-SK cells were measured at 8.7 nmol/mg protein, while NCI-H23 cells exhibited CP levels of 1.4 nmol/mg protein. The combination of aKG + 5-HMF led to a decrease in CP levels specifically in MTC-SK cells. The correlation between mitochondrial activity and CP levels in the presence of different concentrations of combined aKG + 5-HMF in MTC-SK cells demonstrated a linear and concentration-dependent decline in CP levels and mitochondrial activity. Conversely, the effect was less pronounced in NCI-H23 cells. Cell growth of MTC-CK cells was reduced to 60% after 48 h and maintained at 50% after 72 h incubation when treated with 500 µg/mL aKG (IC50%). Addition of 500 µg/mL 5-HMF inhibited cell growth completely regardless of the incubation time. The IC50% for 5-HMF on MTC-CK cell growth was calculated at 375 µg/mL after 24 h incubation and 200 µg/mL 5-HMF after 72 h. MTC-SK cells treated with 500 µg/mL aKG + 167 µg/mL 5-HMF showed no cell growth. The calculated IC50% for the combined substances was 250 µg/mL aKG + 83.3 µg/mL 5-HMF (48 h incubation) and 200 µg/mL aKG + 66.7 µg/mL 5-HMF (72 h incubation). None of the tested concentrations of aKG, 5-HMF, or the combined solution had any effect on NCI-H23 cell growth at any incubation time. Caspase-3 activity increased to 21% in MTC-CK cells in the presence of 500 µg/mL aKG, while an increase to 59.6% was observed using 500 µg/mL 5-HMF. The combination of 500 µg/mL aKG + 167.7 µg/mL 5-HMF resulted in a caspase-3 activity of 55.2%. No caspase-3 activation was observed in NCI-H23 cells when treated with aKG, 5-HMF, or the combined solutions. Conclusion: CPs may serve as potential markers for distinguishing between cancer cells regulated by RONS. The combination of aKG + 5-HMF showed induced cell death in high-RONS-generating cancer cells compared to low-RONS-generating cancer cells.

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