Background/Aim: Pancreatic ductal adenocarcinoma (PDAC) is an extremely scirrhous and hypoxic tumor. Intratumoral hypoxia is a driving force in pancreatic cancer growth and metastasis, implicated as a major cause of drug resistance to conventional chemotherapy and radiotherapy. Another major consequence effect of intratumoral hypoxia is cell metabolic switch, which is required for tumor proliferation under low oxygen and nutrient.
Since the muscle-isoform of lactate-dehydrogenase (LDH-A) constitutes a major checkpoint for the switch to anaerobic-glycolysis, ensuring a sufficient energy supply even in hypoxic environment, we investigated the molecular mechanisms underlying the pharmacological interaction of a series of novel LDH-A inhibitors, PI-FLY21 and PI-FLY124, with gemcitabine in PDAC.
Methods: In vitro studies were performed in 7 pancreatic cancer cell lines and 7 primary cultures (PP), characterized by differential expression of LDH-A and HIF-1α. Cytotoxicity was evaluated with sulforhodamine-B assay, whereas LDH-A modulation was investigated by RT-PCR, Western-blot and enzymatic-activity-assay, 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. Total cytosolic adenosine and phosphorylated deoxynucleosides were evaluated using Liquid chromatography-mass spectrometry (LC-MS/MS). 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 (1% O2) conditions.
Results: The mRNA expression of LDH-A and HIF-1α was detected by quantitative-RT-PCR in all the cells, showing that LDH-A had a large range of mRNA levels (from 27 to 338 arbitrary unit [a.u.] in Capan-1 and PP109 cells, respectively), and correlated with HIF-1α expression.
LDH-A expression significantly increased in PANC-1, PP78 and PP109 under hypoxic condition, as detected by qRT-PCR, and Western blotting, while it was specifically reduced by siRNA-anti-LDH-A. The anti-proliferative activity of gemcitabine was significantly reduced in hypoxic conditions, most probably through reduction of the active phosphorylated gemcitabine metabolites, as detected by LC-MS/MS. Conversely, the novel LDH-A-inhibitors proved to be particularly effective under hypoxia, with IC50s ranging from 0.1 to 0.8 μM, and synergistically enhanced the antiproliferative activity of gemcitabine in PANC-1 and PP78 (CI values <0.8). PI-FLY21 and PI-FLY124 significantly decreased LDH activity in PANC-1 and PP78, also in comparison with siRNA-anti-LDH-A. In addition, these compounds caused a slight cell cycle arrest in the G1-S boundary, while the drug combination reduced the percentages of cells in the G2/M phase (e.g. in PANC-1 from 21 to 10% under hypoxia, p<0.05) and significantly enhanced apoptosis induction. PI-FLY compounds reduced cell migration, the volume of PDAC spheroids growing in CSC-selective-medium under hypoxia, and the expression of the CSC markers CD133 and CD44.
Conclusions: These data provide evidence that LDH-A is a viable target in pancreatic cancer, and novel LDH-A-inhibitors display synergistic cytotoxic activity with gemcitabine, offering an innovative tool for optimizing chemotherapy in hypoxic pancreatic tumors.
Note: This abstract was not presented at the conference.
Citation Format: Amir Avan, Rocco Sciarrillo, Carlotta Granchi, Niccola Funel, Richard J. Honeywell, Marco Macchia, Filippo Minutolo, Godefridus J. Peters, Elisa Giovannetti. Targeting metabolic reprogramming in hypoxic models of pancreatic cancer: Preclinical emergence of novel LDH inhibitors, molecular mechanisms underlying their synergistic interaction with gemcitabine. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B53.