ABSTRACT
Emodin, a tyrosine kinase inhibitor, is a natural anthraquinone derivative found in the roots and rhizomes of numerous plants; it exhibits an anticancer effect on many malignancies. The most important chemotherapeutic agent for patients with advanced non-small cell lung cancer (NSCLC) is a platinum-containing compound such as cisplatin or carboplatin. The molecular mechanism underlying decreased NSCLC cell viability after treatment with emodin and cisplatin is unclear. Therefore, the aim of this study was to assess the cytotoxic effect of combined emodin and cisplatin on NSCLC cell lines and to clarify underlying molecular mechanisms. Exposure of human NSCLC cells to emodin decreased cisplatin-elicited ERK1/2 activation and ERCC1 protein induction by increasing instability of ERCC1 protein. Cisplatin alone did not affect expression of ERCC1 mRNA. However, emodin alone or combined with cisplatin significantly decreased expression of ERCC1 mRNA levels. Enhancement of ERK1/2 activation by transfection with constitutively active MKK1/2 (MKK1/2-CA) vector increased ERCC1 protein levels and protein stability, as well as increasing viability of NSCLC cells treated with emodin and cisplatin. In contrast, blocking ERK1/2 activation by U0126 (an MKK1/2 inhibitor) decreased cisplatin-elicited ERCC1 expression and enhanced cisplatin-induced cytotoxicity. Depletion of endogenous ERCC1 expression by si-ERCC1 RNA transfection significantly enhanced cisplatin's cytotoxic effect. In conclusion, ERCC1 protein protects NSCLC cells from synergistic cytotoxicity induced by emodin and platinum agents. Further investigation of combined emodin and cisplatin may lead to novel therapy in the future for NSCLC through down-regulating expression of ERCC1.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Non-Small-Cell Lung/metabolism , DNA-Binding Proteins/metabolism , Endonucleases/metabolism , Lung Neoplasms/metabolism , Mitogen-Activated Protein Kinase 3/biosynthesis , Apoptosis/drug effects , Butadienes/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/physiopathology , Cell Line, Tumor , Cell Survival/drug effects , Cisplatin/administration & dosage , Cisplatin/adverse effects , DNA-Binding Proteins/genetics , Drug Synergism , Emodin/administration & dosage , Emodin/adverse effects , Endonucleases/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lung Neoplasms/physiopathology , MAP Kinase Kinase 1/biosynthesis , MAP Kinase Kinase 1/genetics , Mitogen-Activated Protein Kinase 3/genetics , Nitriles/pharmacology , RNA, Small Interfering/geneticsABSTRACT
Emodin, a tyrosine kinase inhibitor, is a natural anthraquinone derivative found in the roots and rhizomes of numerous plants. Emodin exhibits anticancer effects against a variety of cancer cells, including lung cancer cells. ERCC1 and Rad51 proteins are essential for nucleotide excision repair and homologous recombination, respectively. Furthermore, ERCC1 and Rad51 overexpression induces resistance to DNA-damaging agents that promote DNA double-strand breaks. Accordingly, the aim of this study was to determine the role of ERCC1 and Rad51 in emodin-mediated cytotoxicity in human non-small cell lung cancer (NSCLC) cells. Both ERCC1 and Rad51 protein levels as well as mRNA levels were decreased in four different NSCLC cell lines after exposure to emodin. These decreases correlated with the inactivation of the MKK1/2-ERK1/2 pathway. Moreover, cellular ERCC1 and Rad51 protein and mRNA levels were specifically inhibited by U0126, a MKK1/2 inhibitor. We found that transient transfection of human NSCLC cells with si-ERCC1 or si-Rad51 RNA and cotreatment with U0126 could enhance emodin-induced cytotoxicity. In contrast, overexpression of constitutively active MKK1/2 vectors (MKK1/2-CA) was shown to significantly recover reduced phospho-ERK1/2, ERCC1, and Rad51 protein levels and to rescue cell viability upon emodin treatment. These results demonstrate that activation of the MKK1/2-ERK1/2 pathway is the upstream signal regulating the expressions of ERCC1 and Rad51, which are suppressed by emodin to induce cytotoxicity in NSCLC cells.
Subject(s)
Carcinoma, Non-Small-Cell Lung/metabolism , DNA-Binding Proteins/biosynthesis , Endonucleases/biosynthesis , Lung Neoplasms/metabolism , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Rad51 Recombinase/biosynthesis , Carcinoma, Non-Small-Cell Lung/drug therapy , Cell Line, Tumor , DNA Repair/physiology , DNA-Binding Proteins/genetics , Dose-Response Relationship, Drug , Emodin/therapeutic use , Emodin/toxicity , Endonucleases/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lung Neoplasms/drug therapy , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Rad51 Recombinase/geneticsABSTRACT
Erlotinib (Tarceva) is a selective epidermal growth factor receptor tyrosine kinase inhibitor in the treatment of human non-small cell lung cancer (NSCLC). In this study, we investigated the roles of ERK1/2 and AKT signaling pathways in regulating Rad51 expression and cytotoxic effects in different NSCLC cell lines treated with erlotinib. Erlotinib decreased cellular levels of phosphorylated ERK1/2, phosphorylated AKT, Rad51 protein, and mRNA in erlotinib-sensitive H1650, A549, and H1869 cells, leading to cell death via apoptosis, but these results were not seen in erlotinib-resistant H520 and H1703 cells. Erlotinib decreased Rad51 protein levels by enhancing Rad51 mRNA and protein instability. Enforced expression of constitutively active MKK1 or AKT vectors could restore Rad51 protein levels, which were inhibited by erlotinib, and decrease erlotinib-induced cytotoxicity. Knocking down endogenous Rad51 expression by si-Rad51 RNA transfection significantly enhanced erlotinib-induced cytotoxicity. In contrast, overexpression of Rad51 by transfection with Rad51 vector could protect the cells from cytotoxic effects induced by erlotinib. Blocking the activations of ERK1/2 and AKT by MKK1/2 inhibitor (U0126) and phosphoinositide 3-kinase inhibitor (wortmannin) suppressed the expression of Rad51 and enhanced the erlotinib-induced cell death in erlotinib-resistant cells. In conclusion, suppression of Rad51 may be a novel therapeutic modality in overcoming drug resistance of erlotinib in NSCLC.
Subject(s)
Carcinoma, Non-Small-Cell Lung/enzymology , Extracellular Signal-Regulated MAP Kinases/metabolism , Mitogen-Activated Protein Kinase Kinases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Quinazolines/pharmacology , Rad51 Recombinase/metabolism , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Cell Death/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Resistance, Neoplasm/drug effects , Enzyme Activation/drug effects , Erlotinib Hydrochloride , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , Humans , Lung Neoplasms/enzymology , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Phosphorylation/drug effects , Proteasome Endopeptidase Complex/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Processing, Post-Translational/drug effects , Protein Stability/drug effects , Rad51 Recombinase/genetics , Signal Transduction/drug effects , TransfectionABSTRACT
Emodin, a tyrosine kinase inhibitor, is a natural anthraquinone derivative found in the roots and rhizomes of numerous plants. It reportedly exhibits an anticancer effect on lung cancer. Gefitinib (Iressa) is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor for human non-small cell lung cancer (NSCLC). However, the molecular mechanism of how emodin combined with gefitinib decreases NSCLC cell viability is unclear. The recombinase protein Rad51 is essential for homologous recombination repair, and Rad51 overexpression is resistant to DNA double-strand break-inducing cancer therapies. In this study, we found that emodin enhanced the cytotoxicity induced by gefitinib in two NSCLC cells lines, A549 and H1650. Emodin at low doses of 2-10 microM did not affect ERK1/2 activation, mRNA, and Rad51 protein levels; however, it enhanced a gefitinib-induced decrease in phospho-ERK1/2 and Rad51 protein levels by enhancing Rad51 protein instability. Expression of constitutively active MKK1/2 vectors (MKK1/2-CA) significantly rescued the reduced phospho-ERK1/2 and Rad51 protein levels as well as cell viability on gefitinib and emodin cotreatment. Blocking of ERK1/2 activation by U0126 (an MKK1/2 inhibitor) lowered Rad51 protein levels and cell viability in emodin-treated H1650 and A549 cells. Knockdown of Rad51 expression by transfection with si-Rad51 RNA enhanced emodin cytotoxicity. In contrast, Rad51 overexpression protected the cells from the cytotoxic effects induced by emodin and gefitinib. Consequently, emodin-gefitinib cotreatment may serve as the basis for a novel and better therapeutic modality in the management of advanced lung cancer.
Subject(s)
Antineoplastic Agents , Emodin/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Protein Kinase Inhibitors , Quinazolines/toxicity , Rad51 Recombinase/metabolism , Animals , Antineoplastic Agents/metabolism , Antineoplastic Agents/toxicity , Apoptosis/physiology , Butadienes/metabolism , Cell Line, Tumor , Gefitinib , Humans , MAP Kinase Kinase 1/antagonists & inhibitors , MAP Kinase Kinase 1/genetics , MAP Kinase Kinase 1/metabolism , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/genetics , Nitriles/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/toxicity , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rad51 Recombinase/genetics , Signal Transduction/physiologyABSTRACT
Celecoxib (Celebrex) is a cyclooxygenase-2 (COX-2) selective inhibitor and gefitinib (Iressa(R), ZD1839) is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor for human non-small cell lung cancer (NSCLC). The addition of celecoxib to gefitinib to prolong the survival of patients with NSCLC still remains controversial and needs to be investigated. The Rad51 protein is essential for homologous recombination repair, and is overexpressed in chemo- or radioresistant carcinomas. In this study, we characterize the role of celecoxib in the cytotoxicity, ERK1/2 activation and Rad51 expression affected by gefitinib in NSCLC cells. We show that celecoxib can enhance the cytotoxicity induced by gefitinib in NSCLC cells. Treatment with celecoxib alone has no effect on the ERK1/2 activation, Rad51 mRNA and protein levels, however, combined treatment with gefitinib results in a significant reduction of phospho-ERK1/2 and Rad51 protein levels, and triggers the degradation of Rad51 via a 26S proteasome-dependent pathway. Expression of constitutively active MKK1/2 vectors (MKK1/2-CA) significantly rescues the decreased ERK1/2 activity, and restores Rad51 protein levels and cell survival under co-treatment with gefitinib and celecoxib. Furthermore, blocking ERK1/2 activation by U0126 (MKK1/2 inhibitor) and knocking down Rad51 expression by transfection with small interfering RNA of Rad51 can enhance the cytotoxicity of celecoxib.
