Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081HV, Amsterdam, Netherlands.
Department of Pharmacy, University of Pisa, Pisa, Italy.
Br J Cancer. 2014 Jan 7;110(1):172-82. doi: 10.1038/bjc.2013.681. Epub 2013 Oct 31.
Hypoxia is a driving force in pancreatic-ductal-adenocarcinoma (PDAC) growth, metastasis and chemoresistance. The muscle-isoform of lactate dehydrogenase (LDH-A) constitutes a major checkpoint for the switch to anaerobic glycolysis, ensuring supply of energy and anabolites in hypoxic-environments. Therefore, we investigated the molecular mechanisms underlying the pharmacological interaction of novel LDH-A inhibitors in combination with gemcitabine in PDAC cells.
Lactate dehydrogenase A levels were studied by quantitative RT-PCR, western blot, immunofluorescence and activity assays in 14 PDAC cells, including primary-cell-cultures and spheroids, in normoxic and hypoxic conditions. Cell proliferation, migration and key determinants of drug activity were evaluated by sulforhodamine-B-assay, wound-healing assay, PCR and LC-MS/MS.
Lactate dehydrogenase A was significantly increased under hypoxic conditions (1% O2), where the novel LDH-A inhibitors proved to be particularly effective (e.g., with IC50 values of 0.9 vs 16.3 μM for NHI-1 in LPC006 in hypoxia vs normoxia, respectively). These compounds induced apoptosis, affected invasiveness and spheroid-growth, reducing expression of metalloproteinases and cancer-stem-like-cells markers (CD133+). Their synergistic interaction with gemcitabine, with combination index values <0.4 in hypoxia, might also be attributed to modulation of gemcitabine metabolism, overcoming the reduced synthesis of phosphorylated metabolites.
Lactate dehydrogenase A is a viable target in PDAC, and novel LDH-A inhibitors display synergistic cytotoxic activity with gemcitabine, offering an innovative tool in hypoxic tumours.
缺氧是胰腺导管腺癌（PDAC）生长、转移和化疗耐药的驱动力。肌肉同工型乳酸脱氢酶（LDH-A）构成了向无氧糖酵解转变的主要检查点，确保在缺氧环境中供应能量和代谢物。因此，我们研究了新型 LDH-A 抑制剂与吉西他滨联合在 PDAC 细胞中相互作用的分子机制。
在常氧和缺氧条件下，通过定量 RT-PCR、western blot、免疫荧光和活性测定，在 14 种 PDAC 细胞（包括原代细胞培养物和球体）中研究 LDH-A 水平。通过磺酰罗丹明 B 测定法、划痕愈合测定法、PCR 和 LC-MS/MS 评估细胞增殖、迁移和药物活性的关键决定因素。
在缺氧条件下（1% O2），LDH-A 显著增加，新型 LDH-A 抑制剂在此条件下特别有效（例如，在缺氧条件下，NHI-1 的 IC50 值为 0.9 μM，而 LPC006 为 16.3 μM）。这些化合物诱导细胞凋亡，影响侵袭性和球体生长，降低金属蛋白酶和癌症干细胞样细胞标志物（CD133+）的表达。它们与吉西他滨的协同相互作用，在缺氧条件下的组合指数值<0.4，也可能归因于调节吉西他滨代谢，克服磷酸化代谢物合成减少。
LDH-A 是 PDAC 的一个可行靶点，新型 LDH-A 抑制剂与吉西他滨具有协同细胞毒性作用，为缺氧肿瘤提供了一种创新工具。
