Gemcitabine (2Ј,2Ј-difluorodeoxycytidine), a deoxycytidine analog, and erlotinib, an epidermal growth factor receptor-tyrosine kinase inhibitor, are used clinically to treat patients with nonsmall-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.

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Curcumin (diferuloylmethane), a phenolic compound obtained from the rhizome of Curcuma longa, is known to have antiproliferative and antitumor properties. Thymidine phosphorylase (TP), an enzyme of the pyrimidine salvage pathway, is considered an attractive therapeutic target, and its expression could suppress cancer cell death induced by DNA damage agents. Excision repair cross-complementary 1 (ERCC1) is a protein involved the process of nucleotide excision repair. The ERCC1 gene is expressed at high levels in cancers and has been associated with resistance to platinum-based chemotherapy. In this study, the effects of curcumin on TP and ERCC1 expression induced by cisplatin in non-small-cell lung cancer (NSCLC) cell lines was investigated. Exposure of the NSCLC cells to various concentrations of curcumin (5-40 M) downregulates the mRNA and protein levels of TP and ERCC1 through destabilization of the mRNA and proteins via a mechanism involving inactivation of MKK1/2-extracellular signal-regulated kinase (ERK1/2). Depletion of endogenous TP or ERCC1 expression by transfection with specific small interfering RNAs significantly decreases cell viability in curcumin-exposed NSCLC cells. Curcumin enhances the sensitivity of cisplatin treatment for NSCLC through inactivation of ERK1/2 and by decreasing the TP and ERCC1 protein levels. Enhancement of ERK1/2 signaling by constitutively active MKK1/2 causes an increase in TP and ERCC1 protein levels and promotes cell viability after cotreatment with curcumin and cisplatin. Enhancement of the cytotoxicity to cisplatin by administration of curcumin is mediated by down-regulation of the expression levels of TP and ERCC1 and by inactivation of ERK1/2.

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Emodin (1,3,8-trihydroxy-6-methyl-anthraquinone) is a natural anthraquinone derivative found in the roots and rhizomes of numerous plants. It is a tyrosine kinase inhibitor and has anticancer effects on lung cancer. Rad51 plays a central role in homologous recombination, and high levels of Rad51 expression are observed in chemo-or radioresistant carcinomas. Our previous studies have shown that the mitogen-activated protein kinase kinase (MKK) 1/2-extracellular signal-regulated kinase (ERK) 1/2 signal pathway maintains the expression of Rad51. Therefore, in this study, we hypothesized that emodin could enhance the effects of the antitumor antibiotic mitomycin C (MMC)-mediated cytotoxicity by decreasing the expression of Rad51 and the phosphorylation of ERK1/2. Exposure of the human non-small-cell lung cancer H1703 or A549 cell lines to emodin decreased the MMC-elicited phosphorylated ERK1/2 and Rad51 levels. Moreover, emodin significantly decreased the MMC-elicited Rad51 mRNA and protein levels by increasing the instability of Rad51 mRNA and protein. In emodin-and MMC-cotreated cells, ERK1/2 phosphorylation was enhanced by constitutively active MKK1/2 (MKK1/2-CA), thus increasing Rad51 protein levels and protein stability. The synergistic cytotoxic effects induced by emodin combined with MMC were remarkably decreased by MKK1-CA-mediated enhancement of ERK1/2 activation. Depletion of endogenous Rad51 expression by small interfering Rad51 RNA transfection significantly enhanced MMC-induced cell death and cell growth inhibition. In contrast, overexpression of Rad51 protects lung cancer cells from the synergistic cytotoxic effects induced by emodin and MMC. We conclude that suppression of Rad51 expression or a combination of emodin with chemotherapeutic agents may be considered as potential therapeutic modalities for lung cancer.Lung cancer is the leading cause of cancer-related death in the world and can be broadly classified into small cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) (Bhattacharjee et al., 2001). NSCLC accounts for approximately 85% of all lung cancers (Landis et al., 1999), and unlike SCLC, NSCLC is less sensitive to chemotherapeutic agents; the average 5-year survival rates are 10 to 15% . Mitomycin C (MMC) is an anticancer drug that forms monoadducts and intrastrand cross-links between the N-2 guanines of the d(CpG) sequence in the minor groove of DNA (Warren and Hamilton, 1996;Dronkert and Kanaar, 2001). MMC is typically used as a first-or second-line regimen to treat NSCLC and is often combined with other chemotherapeutic agents during advanced NSCLC treatment (Babiak et al., 2007). The therapeutic value of MMC depends on the ability of cells to remove DNA damage (McHugh et al., 2001).