Preclinical emergence of novel lactate dehydrogenase inhibitors as potent antitumor agents in hypoxic models of pancreatic cancer: molecular mechanisms underlying their synergistic interaction with gemcitabine

Introduction: Hypoxia plays a key role in pancreatic cancer metastasis and chemoresistance. Since LDH-A constitutes a major checkpoint to ensure sufficient energy supply in hypoxic environments, we investigated the pharmacological activity of a series of novel LDH-A inhibitors (Granchi et al, Eur J Med Chem 2011). Methods: In vitro studies were performed in 7 pancreatic cancer cell lines and 7 primary cultures, characterized by differential expression of LDH-A. Cytotoxicy was evaluated with sulforhodamine-B assay, whereas LDH -A modulation was investigated by RT-PCR, Western-blot and activity assays, using also specific siRNA. Cell cycle perturbation and apoptosis were studied with flow-cytometry, while pharmacological interaction with gemcitabine was investigated with the combination index (CI) method. Furthermore, we examined if LDH-A inhibition modulated invasiveness, expression of cancer stem cell (CSC) markers and EMT phenotype, in adherent-cells and spheroids. All these experiments were performed in both normoxic and hypoxic conditions (1% O2). Results: LDH-A correlated with HIF-1alpha expression and significantly increased under hypoxic conditions. The novel LDH-A inhibitors proved to be particularly effective under hypoxic conditions, with IC50s ranging from 0.1 to 0.8 μM. These compounds induced apoptosis, affected invasiveness and spheroid growth, reducing CSC markers and EMT. Their synergistic interaction with gemcitabine (CI values<0.8) might be attributed to modulation of gemcitabine metabolism, with increased synthesis of phosphorylated-metabolites as detected by LC-MS/MS. Conclusions: These data provide evidence that LDH -A is a viable target in pancreatic cancer cells, and novel LDH-A inhibitors display synergistic cytotoxic activity with gemcitabine, offering an innovative tool for optimizing chemotherapy in hypoxic tumors.