Alpha-Ketoglutarate and 5-HMF: A Potential Anti-Tumoral Combination against Leukemia Cells

Abstract

We have recently shown that a combined solution containing alpha-ketoglutarate (aKG) and 5-hydroxymethyl-furfural (5-HMF) might have anti-tumoral potential due to its antioxidative activities. The question arises if these substances have caspase-3- and apoptosis-activating effects on the cell proliferation in Jurkat and HF-SAR cells. Antioxidative capacity of several combined aKG + 5-HMF solution was estimated by cigarette smoke radical oxidized proteins of fetal calf serum (FCS) using the estimation of carbonylated proteins. The usage of 500 µg/mL aKG + 166.7 µg/mL 5-HMF showed the best antioxidative capacity to inhibit protein modification of more than 50% compared to control measurement. A Jurkat cell line and human fibroblasts (HF-SAR) were cultivated in the absence or presence of combined AKG + 5-HMF solutions between 0 µg/mL aKG + 0 µg/mL 5-HMF and different concentrations of 500 µg/mL aKG + 166.7 µg/mL 5-HMF. Aliquots of Jurkat cells were tested for cell proliferation, mitochondrial activity, caspase activity, apoptotic cells and of the carbonylated protein content as marker of oxidized proteins in cell lysates after 24, 48, and 72 h of incubation. The combined solutions of aKG + 5-HMF were shown to cause a reduction in Jurkat cell growth that was dependent on the dose and incubation time, with the greatest reductions using 500 µg/mL aKG + 166.7 µg/mL 5-HMF after 24 h of incubation compared to 24 h with the control (22,832 cells vs. 32,537 cells), as well as after 48 h (21,243 vs. 52,123 cells) and after 72 h (23,224 cells). Cell growth was totally inhibited by the 500 µg/mL AKG + 166.7 µg/mL solution between 0 and 72 h of incubation compared to 0 h of incubation for the control. The mitochondrial activity measurements supported the data on cell growth in Jurkat cells: The highest concentration of 500 µg/mL aKG + 166.7 µg/mL 5-HMF was able to reduce the mitochondrial activity over 24 h (58.9%), 48 h (28.7%), and 72 h (9.9%) of incubation with Jurkat cells compared not only to the control incubation, but also to the concentrations of 500 µg/mL aKG + 166.7 µg/mL 5-HMF or 375 µg/mL aKG 125 µg/mL 5-HMF, which were able to significantly reduce the mitochondrial activity after 48 h (28.7% or 35.1%) and 72 h (9.9% or 18.2%) compared to 24 h with the control (100%). A slight increase in cell proliferation was found in HF-SAR using the highest concentration (500 µg/mL aKG + 166.7 µg/mL 5-HMF) between 0 h and 72 h incubation of 140%, while no significant differences were found in the mitochondrial activity of HF-SAR in the absence or presence of several combined aKG + 5-HMF solutions. The solutions with 500 µg/mL aKG + 166.7 µg/mL 5-HMF or 250 µg/mL aKG + 83.3 µg/mL 5-HMF showed a significantly higher caspase activity (51.6% or 13.5%) compared to the control (2.9%) in addition to a higher apoptosis rate (63.2% or 31.4% vs. control: 14.9%). Cell lysate carbonylated proteins were significantly higher in Jurkat cells compared to HF-SAR cells (11.10 vs. 2.2 nmol/mg). About 72 h incubation of Jurkat cells with 500 µg/mL aKG + 166.7 µg/mL 5-HMF or 250 µg/mL aKG + 83.3 µg/mL 5-HMF reduced significantly the carbonylated protein content down to 5.55 or 7.44 nmol/mg whereas only the 500 µg/mL aKG + 166.7 µg/mL 5-HMF solution showed a significant reduction of carbonylated proteins of HF-SAR (1.73 nmol/mg).

Alpha-Ketoglutarate or 5-HMF: Single Compounds Effectively Eliminate Leukemia Cells via Caspase-3 Apoptosis and Antioxidative Pathways

“…The aKG was shown to induce apoptosis via caspase-9 and JNK mechanism by 7% and 12.1% using 5 and 200 mM [ 4 ], and we also observed apoptosis effects, which were not significantly different compared to the control using 0–3.4 mM solutions (3.4 mM = 500 µg/mL). Caspase-3 activity was not increased by all used aKG concentrations, indicating that the JNK and caspase pathways JNK and caspase-9 were used.…”

“…Furthermore, significance was found between 6 and 12 mM ( p < 0.01). Measurements of nitrated tyrosine in the presence of several aKG concentrations were recently presented elsewhere [ 4 ] describing a significant but lower antinitrating effect using (0.61 ± 0.017), 24 (0.137 ± 0.014) and 36 mM aKG (0.162 ± 0.029) compared to equimolar 5-HMF concentrations.…”

“…Even the combined substances showed a better reduction at the highest used concentration compared to the single compounds indicating again the different mechanism of aKG and 5-HMF. The same synergistic effect was also estimated using other radical induced measurements such as cigarette smoke radicals on proteins generating carbonyl proteins [ 4 ]. In addition to more than 4000 radicals in cigarette smoke, there is peroxynitrite, which can oxidize and/or nitrate lipids, proteins and nucleic acids.…”

“…We have recently shown that addition of a combined solution containing aKG + 5-HMF to leukemic cells exerted antitumoral, antiproliferative and apoptotic effects compared to HF-SAR [ 4 ]. This seems to be associated with the antioxidative capacity of both substances, including the prevention of oxidative protein modifications, but also protein nitration and oxidative DNA damage [ 5 ].…”

“…Overall, MDA, a lipid peroxidation end product, and 8-OHdG, a nucleic acid oxidation marker, were markedly higher in a cancer group that also included CLL patients, whereas all antioxidative enzymes levels were lower compared to the healthy population. We estimated that the Jurkat cell line expresses higher carbonylated proteins in the cell membrane compared to normal cells [ 4 ]. Malondialdehyde is among the most expressed subgroups of these carbonyl groups on membrane proteins.…”