title: Synergistic interaction of novel lactate dehydrogenase inhibitors with gemcitabine against pancreatic cancer cells in hypoxia.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081HV, Amsterdam, Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: Maftouh
authors.affiliation: Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081HV, Amsterdam, Netherlands.
authors.forename: A
authors.initials: A
authors.lastname: Avan
authors.affiliation: Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081HV, Amsterdam, Netherlands.
authors.forename: R
authors.initials: R
authors.lastname: Sciarrillo
authors.affiliation: Department of Pharmacy, University of Pisa, Pisa, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Granchi
authors.affiliation: Center for Biomedical Research of the Canary Islands, Instituto de Tecnologias Biomedicas, University of La Laguna, La Laguna, Spain.
authors.forename: L G
authors.initials: LG
authors.lastname: Leon
authors.affiliation: Department of Pharmacy, University of Pisa, Pisa, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Rani
authors.affiliation: Department of Surgery, University of Pisa, Pisa, Italy.
authors.forename: N
authors.initials: N
authors.lastname: Funel
authors.affiliation: Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081HV, Amsterdam, Netherlands.
authors.forename: K
authors.initials: K
authors.lastname: Smid
authors.affiliation: Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081HV, Amsterdam, Netherlands.
authors.forename: R
authors.initials: R
authors.lastname: Honeywell
authors.affiliation: Department of Surgery, University of Pisa, Pisa, Italy.
authors.forename: U
authors.initials: U
authors.lastname: Boggi
authors.affiliation: Department of Pharmacy, University of Pisa, Pisa, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Minutolo
authors.affiliation: Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081HV, Amsterdam, Netherlands.
authors.forename: G J
authors.initials: GJ
authors.lastname: Peters
authors.affiliation: Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081HV, Amsterdam, Netherlands.
authors.forename: E
authors.initials: E
authors.lastname: Giovannetti
mesh_terms: D000071916:AC133 Antigen; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis / Q000235:genetics; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D021441:Carcinoma, Pancreatic Ductal; D015687:Cell Hypoxia / Q000502:physiology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D003841:Deoxycytidine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000494:pharmacology; D015536:Down-Regulation; D004357:Drug Synergism; D000071221:Enhancer of Zeste Homolog 2 Protein; D004791:Enzyme Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D000080864:Lactate Dehydrogenase 5; D045726:Metalloproteases / Q000096:biosynthesis / Q000235:genetics; D010190:Pancreatic Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D010455:Peptides / Q000235:genetics; D063151:Polycomb Repressive Complex 2 / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018874:Spheroids, Cellular; D014407:Tumor Cells, Cultured; D000093542:Gemcitabine
chemical_list: D000071916:AC133 Antigen; D015703:Antigens, CD; D004791:Enzyme Inhibitors; D006023:Glycoproteins; D007527:Isoenzymes; C000606023:PROM1 protein, human; D010455:Peptides; D012333:RNA, Messenger; D003841:Deoxycytidine; D007770:L-Lactate Dehydrogenase; D000080864:Lactate Dehydrogenase 5; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2; D045726:Metalloproteases; D000093542:Gemcitabine