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

被引:135
作者
Giovannetti, Elisa [1 ,2 ]
Lemos, Clara [1 ]
Tekle, Christina [1 ]
Smid, Kees [1 ]
Nannizzi, Sara [2 ]
Rodriguez, Jose A. [1 ]
Ricciardi, Simona [2 ]
Danesi, Romano [2 ]
Giaccone, Giuseppe [3 ]
Peters, Godefridus J. [1 ]
机构
[1] Vrije Univ Amsterdam Med Ctr, Dept Med Oncol, Amsterdam, Netherlands
[2] Univ Pisa, Dept Internal Med, Div Pharmacol & Chemotherapy, I-56126 Pisa, Italy
[3] NCI, Med Oncol Branch, Bethesda, MD 20892 USA
关键词
D O I
10.1124/mol.107.042382
中图分类号
R9 [药学];
学科分类号
1007 ;
摘要
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.
引用
收藏
页码:1290 / 1300
页数:11
相关论文
共 47 条
[1]   Synergistic antitumor activity of ZD6474, an inhibitor of vascular endothelial growth factor receptor and epidermal growth factor receptor signaling, with gemcitabine and ionizing radiation against pancreatic cancer [J].
Bianco, Cataldo ;
Giovannetti, Elisa ;
Ciardiello, Fortunato ;
Mey, Valentina ;
Nannizzi, Sara ;
Tortora, Giampaolo ;
Troiani, Teresa ;
Pasqualetti, Francesco ;
Eckhardt, Gail ;
de Liguoro, Mario ;
Ricciardi, Simona ;
Del Tacca, Mario ;
Raben, David ;
Cionini, Luca ;
Danesi, Romano .
CLINICAL CANCER RESEARCH, 2006, 12 (23) :7099-7107
[2]   Identification of potentially useful combinations of epidermal growth factor receptor tyrosine kinase antagonists with conventional cytotoxic agents using median effect analysis [J].
Budman, Daniel R. ;
Soong, Richie ;
Calabro, Anthony ;
Tai, Julia ;
Diasio, Robert .
ANTI-CANCER DRUGS, 2006, 17 (08) :921-928
[3]   Akt phosphorylation and gefitinib efficacy in patients with advanced non-small-cell lung cancer [J].
Cappuzzo, F ;
Magrini, E ;
Ceresoli, GL ;
Bartolini, S ;
Rossi, E ;
Ludovini, V ;
Gregorc, V ;
Ligorio, C ;
Cancellieri, A ;
Damiani, S ;
Spreafico, A ;
Paties, CT ;
Lombardo, L ;
Calandri, C ;
Bellezza, G ;
Tonato, M ;
Crinò, L .
JNCI-JOURNAL OF THE NATIONAL CANCER INSTITUTE, 2004, 96 (15) :1133-1141
[4]   AUTOREGULATION OF HUMAN THYMIDYLATE SYNTHASE MESSENGER-RNA TRANSLATION BY THYMIDYLATE SYNTHASE [J].
CHU, E ;
KOELLER, DM ;
CASEY, JL ;
DRAKE, JC ;
CHABNER, BA ;
ELWOOD, PC ;
ZINN, S ;
ALLEGRA, CJ .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1991, 88 (20) :8977-8981
[5]   Synergistic effects of gemcitabine and gefitinib in the treatment of head and neck carcinoma [J].
Chun, PY ;
Feng, FY ;
Scheurer, AM ;
Davis, MA ;
Lawrence, TS ;
Nyati, MK .
CANCER RESEARCH, 2006, 66 (02) :981-988
[6]   Epidermal growth factor receptor messenger RNA expression, gene dosage, and gefitinib sensitivity in non-small cell lung cancer [J].
Dziadziuszko, Rafal ;
Witta, Samir E. ;
Cappuzzo, Federico ;
Park, Seongjin ;
Tanaka, Koji ;
Danenberg, Peter V. ;
Baron, Anna E. ;
Crino, Lucio ;
Franklin, Wilbur A. ;
Bunn, Paul A., Jr. ;
Varella-Garcia, Marileila ;
Danenberg, Kathleen D. ;
Hirsch, Fred R. .
CLINICAL CANCER RESEARCH, 2006, 12 (10) :3078-3084
[7]   Mutations in the epidermal growth factor receptor and in KRAS are predictive and prognostic indicators in patients with non-small-cell lung cancer treated with chemotherapy alone and in combination with erlotinib [J].
Eberhard, DA ;
Johnson, BE ;
Amler, LC ;
Goddard, AD ;
Heldens, SL ;
Herbst, RS ;
Ince, WL ;
Jänne, PA ;
Januario, T ;
Johnson, DH ;
Klein, P ;
Miller, VA ;
Ostland, MA ;
Ramies, DA ;
Sebisanovic, D ;
Stinson, JA ;
Zhang, YR ;
Seshagiri, S ;
Hillan, KJ .
JOURNAL OF CLINICAL ONCOLOGY, 2005, 23 (25) :5900-5909
[8]   Role of epidermal growth factor receptor degradation in gemcitabine-mediated cytotoxicity [J].
Feng, F. Y. ;
Varambally, S. ;
Tomlins, S. A. ;
Chun, P. Y. ;
Lopez, C. A. ;
Li, X. ;
Davis, M. A. ;
Chinnaiyan, A. M. ;
Lawrence, T. S. ;
Nyati, M. K. .
ONCOGENE, 2007, 26 (23) :3431-3439
[9]   Phase III study of erlotinib in combination with cisplatin and gemcitabine in advanced non-small-cell lung cancer: The Tarceva Lung Cancer Investigation Trial [J].
Gatzemeier, Ulrich ;
Pluzanska, Anna ;
Szczesna, Aleksandra ;
Kaukel, Eckhard ;
Roubec, Jaromir ;
De Rosa, Flavio ;
Milanowski, Janusz ;
Karnicka-Mlodkowski, Hanna ;
Pesek, Milos ;
Serwatowski, Piotr ;
Ramlau, Rodryg ;
Janaskova, Terezie ;
Vansteenkiste, Johan ;
Strausz, Janos ;
Manikhas, Georgy Moiseevich ;
Von Pawel, Joachim .
JOURNAL OF CLINICAL ONCOLOGY, 2007, 25 (12) :1545-1552
[10]   Gefitinib in combination with gemcitabine and cisplatin in advanced non-small-cell lung cancer: A phase III trial-INTACT1 [J].
Giaccone, G ;
Herbst, RS ;
Manegold, C ;
Scagliotti, G ;
Rosell, R ;
Miller, V ;
Natale, RB ;
Schiller, JH ;
von Pawel, J ;
Pluzanska, A ;
Gatzemeier, M ;
Grous, J ;
Ochs, JS ;
Averbuch, SD ;
Wolf, MK ;
Rennie, P ;
Fandi, A ;
Johnson, DH .
JOURNAL OF CLINICAL ONCOLOGY, 2004, 22 (05) :777-784