title: Molecular mechanisms underlying the synergistic interaction of erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor, with the multitargeted antifolate pemetrexed in non-small-cell lung cancer cells.
abstract: Because the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib and the multitargeted antifolate pemetrexed are registered in the treatment of second-line non-small-cell lung cancer (NSCLC), empirical combinations of these drugs are being tested. This study investigated molecular mechanisms underlying their combination in six NSCLC cell lines. Cells were characterized by heterogeneous expression of pemetrexed determinants, including thymidylate synthase (TS) and dihydrofolate reductase (DHFR), and mutations potentially affecting chemosensitivity. Pharmacological interaction was studied using the combination index (CI) method, whereas cell cycle, apoptosis induction, and EGFR, extracellular signal-regulated kinases 1 and 2, and Akt phosphorylation were studied by flow cytometry, fluorescence microscopy, and enzyme-linked immunosorbent assays. Reverse-transcriptase polymerase chain reaction (RT-PCR), Western blot, and activity assays were performed to assess whether erlotinib influenced TS. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assays demonstrated that EGFR and k-Ras mutations were related to erlotinib sensitivity, whereas TS and DHFR expression were related to pemetrexed sensitivity. Synergistic cytotoxicity was found in all cells, most pronounced with pemetrexed + erlotinib (24 h) --> erlotinib (48 h) sequence (CI, 0.09-0.40), which was associated with a significant induction of apoptosis. Pemetrexed increased EGFR phosphorylation and reduced Akt phosphorylation, which was additionally reduced by drug combination (-70.6% in H1650). Erlotinib significantly reduced TS expression and activity, possibly via E2F-1 reduction, as detected by RT-PCR and Western blot, and the combination decreased TS in situ activity in all cells. Erlotinib and pemetrexed showed a strong synergism in NSCLC cells, regardless of their genetic characteristics. Induction of apoptosis, modulation of EGFR and Akt phosphorylation, and changes in the expression of critical genes involved in pemetrexed activity contribute to this synergistic interaction and support the clinical investigation of these markers.
authors.affiliation: Division of Pharmacology and Chemotherapy, Department of Internal Medicine, 55, Via Roma, 56126 Pisa, Italy. elisa.giovannetti@gmail.com
authors.forename: Elisa
authors.initials: E
authors.lastname: Giovannetti
authors.forename: Clara
authors.initials: C
authors.lastname: Lemos
authors.forename: Christina
authors.initials: C
authors.lastname: Tekle
authors.forename: Kees
authors.initials: K
authors.lastname: Smid
authors.forename: Sara
authors.initials: S
authors.lastname: Nannizzi
authors.forename: José A
authors.initials: JA
authors.lastname: Rodriguez
authors.forename: Simona
authors.initials: S
authors.lastname: Ricciardi
authors.forename: Romano
authors.initials: R
authors.lastname: Danesi
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Giaccone
authors.forename: Godefridus J
authors.initials: GJ
authors.lastname: Peters
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D002289:Carcinoma, Non-Small-Cell Lung / Q000201:enzymology / Q000235:genetics / Q000473:pathology*; D002384:Catalysis / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D002457:Cell Extracts; D045744:Cell Line, Tumor; D002867:Chromones / Q000494:pharmacology; D004354:Drug Screening Assays, Antitumor; D004357:Drug Synergism; D050687:E2F1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D066246:ErbB Receptors / Q000037:antagonists & inhibitors*; D000069347:Erlotinib Hydrochloride; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005971:Glutamates / Q000494:pharmacology*; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology / Q000235:genetics / Q000473:pathology*; D009025:Morpholines / Q000494:pharmacology; D000068437:Pemetrexed; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011799:Quinazolines / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D013940:Thymidylate Synthase / Q000235:genetics / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D002457:Cell Extracts; D002867:Chromones; D050687:E2F1 Transcription Factor; D005493:Folic Acid Antagonists; D005971:Glutamates; D009025:Morpholines; D011799:Quinazolines; D012333:RNA, Messenger; D000068437:Pemetrexed; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D006147:Guanine; D000069347:Erlotinib Hydrochloride; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D066246:ErbB Receptors; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases