title: Prolonged pemetrexed pretreatment augments persistence of cisplatin-induced DNA damage and eliminates resistant lung cancer stem-like cells associated with EMT.
abstract: BACKGROUND
  Lung cancer is the leading cause of cancer-related mortality, and new therapeutic options are urgently needed. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers, with the current standard regimen of care for NSCLC including chemotherapy with pemetrexed as a single agent or in combination with platinum-based agents, e.g. cisplatin. Pemetrexed is a folic acid antagonist that inhibits the synthesis of precursor nucleotides, whereas cisplatin directly induces DNA adducts, the repair of which is dependent on sufficiently high nucleotide levels. In the clinical setting, the pemetrexed-cisplatin combination therapy is administered concomitantly. We hypothesized that prolonged pretreatment with pemetrexed could be beneficial, as prior depletion of nucleotide pools could sensitize cancer cells to subsequent treatment with cisplatin.
  
  
  METHODS
  NSCLC A549 and H460 cells were treated with pemetrexed for 72 h. In addition, 24 h of cisplatin treatment was initiated at day 1, 2 or 3 resulting in either simultaneous pemetrexed application or pemetrexed pretreatment for 24 or 48 h, respectively. Cell growth and colony formation as well as senescence induction were quantified after treatment. Cell cycle distribution and phosphorylation of histone variant H2AX as a surrogate marker for DNA damage was quantified by flow cytometry. Relative changes in gene expression were determined by quantitative real time PCR.
  
  
  RESULTS
  Prolonged pemetrexed pretreatment for 48 h prior to cisplatin treatment maximally delayed long-term cell growth and significantly reduced the number of recovering clones. Moreover, apoptosis and senescence were augmented and recovery from treatment-induced DNA damage was delayed. Interestingly, a cell population was identified that displayed an epithelial-to-mesenchymal transition (EMT) and which had a stem cell phenotype. This population was highly resistant to concomitant pemetrexed-cisplatin treatment but was sensitized by pemetrexed pretreatment.
  
  
  CONCLUSIONS
  Adaptation of the standard treatment schedule to include pretreatment with pemetrexed optimizes the anticancer efficiency of pemetrexed-cisplatin combination therapy, which correlates with a persistence of treatment-induced DNA damage. Therefore, this study warrants further investigations to elucidate whether such an adaptation could enhance the effectiveness of the standard clinical treatment regimen. In addition, a subpopulation of therapy resistant cells with EMT and cancer stem cell features was identified that was resistant to the standard treatment regimen but sensitive to pemetrexed pretreatment combined with cisplatin.
authors.affiliation: Division of General Thoracic Surgery, Inselspital, Bern University Hospital, Department of Clinical Research, University of Bern, Murtenstrasse 50, 3008, Bern, Switzerland. colin.tieche@insel.ch.
authors.forename: Colin Charles
authors.initials: CC
authors.lastname: Tièche
authors.affiliation: Division of General Thoracic Surgery, Inselspital, Bern University Hospital, Department of Clinical Research, University of Bern, Murtenstrasse 50, 3008, Bern, Switzerland. renwang.peng@insel.ch.
authors.forename: Ren-Wang
authors.initials: RW
authors.lastname: Peng
authors.affiliation: Division of General Thoracic Surgery, Inselspital, Bern University Hospital, Department of Clinical Research, University of Bern, Murtenstrasse 50, 3008, Bern, Switzerland. Patrick.dorn@insel.ch.
authors.forename: Patrick
authors.initials: P
authors.lastname: Dorn
authors.affiliation: Division of General Thoracic Surgery, Inselspital, Bern University Hospital, Department of Clinical Research, University of Bern, Murtenstrasse 50, 3008, Bern, Switzerland. laurene.froment@insel.ch.
authors.forename: Laurène
authors.initials: L
authors.lastname: Froment
authors.affiliation: Division of General Thoracic Surgery, Inselspital, Bern University Hospital, Department of Clinical Research, University of Bern, Murtenstrasse 50, 3008, Bern, Switzerland. ralph.schmid@insel.ch.
authors.forename: Ralph Alexander
authors.initials: RA
authors.lastname: Schmid
authors.affiliation: Division of General Thoracic Surgery, Inselspital, Bern University Hospital, Department of Clinical Research, University of Bern, Murtenstrasse 50, 3008, Bern, Switzerland. thomas.marti@insel.ch.
authors.forename: Thomas Michael
authors.initials: TM
authors.lastname: Marti
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D016922:Cellular Senescence / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology*; D004249:DNA Damage; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D004357:Drug Synergism; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D014411:Neoplastic Stem Cells / Q000187:drug effects; D000068437:Pemetrexed / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D000068437:Pemetrexed; D002945:Cisplatin