title: Molecular mechanism implicated in Pemetrexed-induced apoptosis in human melanoma cells.
abstract: BACKGROUND
  Metastatic melanoma is a lethal skin cancer and its incidence is rising every year. It represents a challenge for oncologist, as the current treatment options are non-curative in the majority of cases; therefore, the effort to find and/or develop novel compounds is mandatory. Pemetrexed (Alimta®, MTA) is a multitarget antifolate that inhibits folate-dependent enzymes: thymidylate synthase, dihydrofolate reductase and glycinamide ribonucleotide formyltransferase, required for de novo synthesis of nucleotides for DNA replication. It is currently used in the treatment of mesothelioma and non-small cell lung cancer (NSCLC), and has shown clinical activity in other tumors such as breast, colorectal, bladder, cervical, gastric and pancreatic cancer. However, its effect in human melanoma has not been studied yet.
  
  
  RESULTS
  In the current work we studied the effect of MTA on four human melanoma cell lines A375, Hs294T, HT144 and MeWo and in two NSCLC cell lines H1299 and Calu-3. We have found that MTA induces DNA damage, S-phase cell cycle arrest, and caspase- dependent and -independent apoptosis. We show that an increment of the intracellular reactive oxygen species (ROS) and p53 is required for MTA-induced cytotoxicity by utilizing N-Acetyl-L-Cysteine (NAC) to blockage of ROS and p53-defective H1299 NSCLC cell line. Pretreatment of melanoma cells with NAC significantly decreased the DNA damage, p53 up-regulation and cytotoxic effect of MTA. MTA was able to induce p53 expression leading to up-regulation of p53-dependent genes Mcl-1 and PIDD, followed by a postranscriptional regulation of Mcl-1 improving apoptosis.
  
  
  CONCLUSIONS
  We found that MTA induced DNA damage and mitochondrial-mediated apoptosis in human melanoma cells in vitro and that the associated apoptosis was both caspase-dependent and -independent and p53-mediated. Our data suggest that MTA may be of therapeutic relevance for the future treatment of human malignant melanoma.
authors.affiliation: Medical Oncology Department, Hospital Universitario Cruces, Barakaldo, Bizkaia, Spain.
authors.forename: Aitziber
authors.initials: A
authors.lastname: Buqué
authors.forename: Jangi Sh
authors.initials: JSh
authors.lastname: Muhialdin
authors.forename: Alberto
authors.initials: A
authors.lastname: Muñoz
authors.forename: Begoña
authors.initials: B
authors.lastname: Calvo
authors.forename: Sergio
authors.initials: S
authors.lastname: Carrera
authors.forename: Unai
authors.initials: U
authors.lastname: Aresti
authors.forename: Aintzane
authors.initials: A
authors.lastname: Sancho
authors.forename: Itziar
authors.initials: I
authors.lastname: Rubio
authors.forename: Guillermo
authors.initials: G
authors.lastname: López-Vivanco
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics*; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005971:Glutamates / Q000494:pharmacology*; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D000068437:Pemetrexed; D015398:Signal Transduction / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
chemical_list: D000964:Antimetabolites, Antineoplastic; D004791:Enzyme Inhibitors; D005971:Glutamates; D016159:Tumor Suppressor Protein p53; D000068437:Pemetrexed; D006147:Guanine; D020169:Caspases