Greilberger Joachim, Erlbacher Katharina, Stiegler Philipp, Wintersteiger Reinhold, Herwig Ralf
Institut für Laborwissenschaften Dr. Greilberger, Schwarzl Medical Center, 8301 Lassnitzhoehe, Austria.
HG Pharma GmbH, 6365 Kirchberg in Tirol, Austria.
Curr Issues Mol Biol. 2023 Aug 7;45(8):6503-6525. doi: 10.3390/cimb45080410.
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.
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.
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.
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.
羰基化蛋白质（CPs）是癌细胞中活性氧和氮物种（RONS）生成增加的特异性指标，这归因于被称为瓦伯格效应的线粒体能量代谢失调。本研究的目的是研究α-酮戊二酸（aKG）、5-羟甲基糠醛（5-HMF）及其组合作为线粒体靶向抗氧化剂在MTC-SK或NCI-H23癌细胞中的潜力。
将MTC-SK和NCI-H23细胞分别在不存在或存在不同浓度（0-500μg/mL）的aKG、5-HMF以及aKG + 5-HMF组合溶液的情况下进行培养。在0、24、48和72小时后，对MTC-SK和NCI-H23细胞的等分试样进行线粒体活性、癌细胞膜CP水平、细胞生长和半胱天冬酶-3活性的评估。
用aKG、5-HMF或aKG + 5-HMF组合处理后，MTC-SK细胞的线粒体活性呈现出浓度和时间依赖性降低。72小时后，线粒体活性的半数最大抑制浓度（IC50%）在500μg/mL aKG、200μg/mL 5-HMF以及200μg/mL aKG + 66.7μg/mL 5-HMF时达到。相比之下，即使在aKG + 5-HMF的最高组合浓度下，NCI-H23细胞的线粒体活性也仅出现最小程度的降低（10%）。MTC-SK细胞中的CP水平测定为8.7 nmol/mg蛋白质，而NCI-H23细胞的CP水平为1.4 nmol/mg蛋白质。aKG + 5-HMF的组合导致CP水平特别是在MTC-SK细胞中降低。在MTC-SK细胞中存在不同浓度的aKG + 5-HMF组合时，线粒体活性与CP水平之间的相关性表明CP水平和线粒体活性呈线性且浓度依赖性下降。相反，在NCI-H23细胞中这种效应不太明显。当用500μg/mL aKG（IC50%）处理时，MTC-CK细胞的细胞生长在48小时后降至60%，并在72小时孵育后维持在50%。添加500μg/mL 5-HMF无论孵育时间如何都能完全抑制细胞生长。5-HMF对MTC-CK细胞生长的IC50%在孵育24小时后计算为375μg/mL，72小时后为200μg/mL 5-HMF。用500μg/mL aKG + 167μg/mL 5-HMF处理的MTC-SK细胞未显示细胞生长。组合物质的计算IC50%为250μg/mL aKG + 83.3μg/mL 5-HMF（48小时孵育）和200μg/mL aKG + 66.7μg/mL 5-HMF（72小时孵育）。在任何孵育时间，所测试的aKG、5-HMF或组合溶液的浓度均对NCI-H23细胞生长没有任何影响。在500μg/mL aKG存在下，MTC-CK细胞中的半胱天冬酶-3活性增加到21%，而使用500μg/mL 5-HMF时观察到增加到59.6%。500μg/mL aKG + 167.7μg/mL 5-HMF的组合导致半胱天冬酶-3活性为55.2%。当用aKG、5-HMF或组合溶液处理时，在NCI-H23细胞中未观察到半胱天冬酶-3的激活。
CPs可能作为区分受RONS调节的癌细胞的潜在标志物。与低RONS生成的癌细胞相比，aKG + 5-HMF的组合在高RONS生成的癌细胞中显示出诱导细胞死亡的作用。
