Di Paola Floriana Jessica, Cardoso Luiza Hd, Nikitopoulou Efterpi, Kulik Bianca, Rühl Sandra, Eva Alexander, Sommer Natascha, Linn Thomas, Gnaiger Erich, Failing Klaus, Büttner Kathrin, Frezza Christian, Mazurek Sybille
Institute of Veterinary Physiology and Biochemistry, Justus Liebig University of Giessen, Giessen, Germany.
IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy.
PLoS One. 2025 Jun 9;20(6):e0325509. doi: 10.1371/journal.pone.0325509. eCollection 2025.
Mitochondrial glycerol 3-P dehydrogenase (mtG3PDH) plays a significant role in cellular bioenergetics by serving as a rate-limiting element in the glycerophosphate shuttle, which connects cytosolic glycolysis to mitochondrial oxidative metabolism. mtG3PDH was identified as an important site of electron leakage leading to ROS production to the mitochondrial matrix and intermembrane space. Our research focused on the role of two published mtG3PDH inhibitors (RH02211 and iGP-1) on the proliferation and metabolism of PC-3 and DU145 prostate cancer cells characterized by different mtG3PDH activities. Since pyruvate as a substrate of lactate dehydrogenase (LDH) may represent an escape mechanism for the recycling of cytosolic NAD+ via the glycerophosphate shuttle, we investigated the effect of pyruvate on the mode of action of the mtG3PDH inhibitors. Extracellular pyruvate weakened the growth-inhibitory effects of RH02211 and iGP-1 in PC-3 cells but not in DU145 cells, which correlated with higher H-type LDH and lower mitochondrial glutamate-oxaloacetate transaminase in DU145 cells. In the pyruvate-low medium, the strength of inhibition was more pronounced in PC-3 cells, characterized by higher mtG3PDH activities compared to DU145 cells. Pyruvate conversion rates (production in pyruvate-low and consumption in pyruvate-high PC-3 cells) were not impaired by RH02211 and iGP-1, suggesting that the conversion of extracellular pyruvate to lactate was not the primary factor responsible for the weakening effect of extracellular pyruvate on the RH02211-induced inhibition of PC-3 proliferation. In pyruvate-high PC-3 cells, the intracellular glycerol-3-P and dihydroxyacetone-P concentrations were consistent with an inhibition of mtG3PDH. In contrast, in pyruvate-low cells, the concentrations of these metabolites suggested an activation of mtG3PDH in parallel with an impairment of cytosolic G3PDH by RH02211. Of all metabolic characterizations recorded in this study (fluxes, intracellular intermediates, O2 consumption and H2O2 production), the decrease in glutaminolysis correlated best with the RH02211-induced inhibition of proliferation in pyruvate-low and pyruvate-high PC-3 cells.
线粒体甘油-3-磷酸脱氢酶（mtG3PDH）在细胞生物能量学中发挥着重要作用，它是甘油磷酸穿梭途径中的限速因子，该途径将胞质糖酵解与线粒体氧化代谢联系起来。mtG3PDH被确定为电子泄漏的重要位点，会导致线粒体基质和膜间隙产生活性氧（ROS）。我们的研究聚焦于两种已发表的mtG3PDH抑制剂（RH02211和iGP-1）对具有不同mtG3PDH活性的PC-3和DU145前列腺癌细胞增殖和代谢的作用。由于丙酮酸作为乳酸脱氢酶（LDH）的底物可能代表了一种通过甘油磷酸穿梭途径回收胞质NAD+的逃逸机制，我们研究了丙酮酸对mtG3PDH抑制剂作用模式的影响。细胞外丙酮酸减弱了RH02211和iGP-1对PC-3细胞的生长抑制作用，但对DU145细胞没有影响，这与DU145细胞中较高的H型LDH和较低的线粒体谷氨酸-草酰乙酸转氨酶有关。在丙酮酸含量低的培养基中，抑制强度在PC-3细胞中更为明显，与DU145细胞相比，PC-3细胞的mtG3PDH活性更高。RH02211和iGP-1并未损害丙酮酸转化率（丙酮酸含量低时的生成率和丙酮酸含量高时的PC-3细胞中的消耗率），这表明细胞外丙酮酸向乳酸的转化不是细胞外丙酮酸削弱RH02211诱导的PC-3细胞增殖抑制作用的主要因素。在丙酮酸含量高的PC-3细胞中，细胞内甘油-3-磷酸和二羟基丙酮磷酸的浓度与mtG3PDH受到抑制一致。相反，在丙酮酸含量低的细胞中，这些代谢物的浓度表明mtG3PDH被激活，同时RH02211损害了胞质甘油-3-磷酸脱氢酶。在本研究记录的所有代谢特征（通量、细胞内中间体、氧气消耗和过氧化氢生成）中，谷氨酰胺分解的减少与RH02211在丙酮酸含量低和丙酮酸含量高的PC-3细胞中诱导的增殖抑制最为相关。
