Abstract
Gemcitabine (2′,2′-difluorodeoxycytidine), a deoxycytidine analog, and erlotinib, an epidermal growth factor receptor-tyrosine kinase inhibitor, are used clinically to treat patients with non–small-cell lung cancer (NSCLC). However, the molecular mechanisms for the drug resistance of gemcitabine in NSCLC cells are poorly understood. In this study, we used constructs containing human Rad51 cDNA or specific Rad51 small interfering RNA (siRNA) to examine the role of Rad51 in chemoresistance of gemcitabine in three different human NSCLC cell lines. Exposure of human NSCLC cell lines to gemcitabine increased the phosphorylation levels of mitogen-activated protein kinase kinase (MKK) 1/2-extracellular signal-regulated kinase (ERK) 1/2 and AKT in a time- and dose-dependent manner, which was accompanied by an induction of Rad51 mRNA and protein expression. Gemcitabine increased the expression of Rad51 by increasing its mRNA and protein stability. Blockage of ERK1/2 or AKT activation by 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126; MKK1/2 inhibitor) or 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002; phosphatidyl inositol 3-kinase inhibitor), respectively, decreased the gemcitabine-induced Rad51 expression. Gemcitabine-induced cytotoxicity was significantly increased using siRNA depletion of Rad51 or blockage of ERK1/2 and AKT activation. Erlotinib enhanced the gemcitabine-induced cytotoxicity via the inactivation of ERK1/2 and AKT and the down-regulation of Rad51. Enforced expression of constitutively active MKK1/2 or AKT recovered cell viability and Rad51 protein levels that were decreased by the combination of erlotinib and gemcitabine. Suppression of Rad51 expression or the inactivation of ERK1/2 or AKT signaling may be considered potential therapeutic modalities for gemcitabine-resistant lung cancer.
Footnotes
This work was supported by the National Science Council of Taiwan [Grant NSC 97-2311-B-415-001-MY3].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.173146.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- NSCLC
- non–small-cell lung cancer
- MAPK
- mitogen-activated protein kinase
- ERK
- extracellular signal-regulated kinase
- EGFR
- epidermal growth factor receptor
- TKI
- tyrosine kinase inhibitor
- AKT
- protein kinase B
- HR
- homologous recombination
- PD-321852
- 4-(2,6-dichloro-phenyl)-9-hydroxy-6-(3-methylamino-propyl)-6H-pyrrolo[3,4-c]carbazole-1,3-dione
- LY294002
- 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
- U0126
- 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene
- MG132
- N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal
- ALLN
- N-acetyl-Leu-Leu-norleucinal
- siRNA
- small interfering RNA
- MKK
- MAPK kinase
- MKK1/2-CA
- constitutively active form of MKK1/2
- myr-AKT
- constitutively active form of AKT
- MTT
- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- CI
- combination index
- RT-PCR
- reverse transcriptase-polymerase chain reaction
- PI3K
- phosphatidyl inositol 3-kinase.
- Received July 20, 2010.
- Accepted September 17, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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