RESUMO
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 µM) down-regulates 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.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Curcumina/farmacologia , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/efeitos dos fármacos , Endonucleases/metabolismo , Timidina Fosforilase/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Proteínas de Ligação a DNA/genética , Sinergismo Farmacológico , Endonucleases/genética , Humanos , RNA Mensageiro/genética , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timidina Fosforilase/genéticaRESUMO
Chemotherapy for advanced human non-small-cell lung cancer (NSCLC) includes platinum-containing compound such as cisplatin in combination with a second- or third-generation cytotoxic agent. 5-Fluorouracil (5-FU) belongs to antimetabolite chemotherapeutics, and its mechanism of cytotoxicity is involved in the inhibition of thymidylate synthase (TS). TS and thymidine phosphorylase (TP) are key enzymes of the pyrimidine salvage pathway. In this study, we have examined the molecular mechanism of TS and TP in regulating drug sensitivity to cisplatin in NSCLC cell lines. Cisplatin could increase the phosphorylation of mitogen-activated protein kinase kinase 1/2 (MKK1/2)-extracellular signal-regulated kinase 1/2 (ERK1/2) and the protein levels of TS and TP through enhancing the protein stability in A549 and H1975 cells. Blocking ERK1/2 activation by MKK1/2 inhibitor [U0126; 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene)] decreased TS and TP protein levels in both cell lines treated with cisplatin. Depletion of endogenous TS or TP expression by specific small interfering RNA transfection significantly increased cisplatin-induced cell death and growth inhibition. Combined treatment with 5-FU could decrease cisplatin-induced ERK1/2 activation and the induction of TS and TP, which subsequently resulted in synergistic cytotoxic effects. Enforced expression of constitutive active MKK1/2 vectors rescued the protein levels of phospho-ERK1/2, TS, and TP, and the cell viability that were decreased by cisplatin and 5-FU combination. In contrast, U0126 enhanced drug sensitivity to cisplatin and/or 5-FU in lung cancer cells. In conclusion, the up-regulation of ERK1/2-dependent TS and TP can protect human lung cancer cells from cisplatin-induced cytotoxicity.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/enzimologia , Cisplatino/uso terapêutico , Neoplasias Pulmonares/enzimologia , Proteína Quinase 1 Ativada por Mitógeno/biossíntese , Proteína Quinase 3 Ativada por Mitógeno/biossíntese , Timidina Fosforilase/biossíntese , Timidilato Sintase/biossíntese , Regulação para Cima/fisiologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Técnicas de Silenciamento de Genes/métodos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Regulação para Cima/efeitos dos fármacosRESUMO
Curcumin (diferuloylmethane), a major active component of turmeric (Curcuma longa), has been reported to suppress the proliferation of a wide variety of tumor cells. Rad51 is a key protein in the homologous recombination (HR) pathway of DNA double-strand break repair, and HR represents a novel target for cancer therapy. A high expression of Rad51 has been reported in chemo- or radio-resistant carcinomas. Therefore, in the current study, we will examine whether curcumin could enhance the effects of mitomycin C (MMC), a DNA interstrand cross-linking agent, to induce cytotoxicity by decreasing Rad51 expression. Exposure of two human non-small lung cancer (NSCLC) cell lines (A549 and H1975) to curcumin could suppress MMC-induced MKK1/2-ERK1/2 signal activation and Rad51 protein expression. Enhancement of ERK1/2 activation by constitutively active MKK1/2 (MKK1/2-CA) increased Rad51 protein levels in curcumin and MMC co-treated human lung cells. Moreover, the synergistic cytotoxic effect induced by curcumin combined with MMC was decreased by MKK1-CA-mediated enhancement of ERK1/2 activation by a significant degree. In contrast, MKK1/2 inhibitor, U0126 was shown to augment the cytotoxicity of curcumin and MMC through downregulation of ERK1/2 activation and Rad51 expression. Depletion of endogenous Rad51 expression by siRad51 RNA transfection significantly enhanced MMC and/or curcumin induced cell death and cell growth inhibition. In contrast, an overexpression of Rad51 protected lung cancer cells from synergistic cytotoxic effects induced by curcumin and MMC. We concluded that Rad51 inhibition may be an additional action mechanism for enhancing the chemosensitization of MMC by curcumin in NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/enzimologia , Curcumina/farmacologia , Neoplasias Pulmonares/enzimologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mitomicina/farmacologia , Rad51 Recombinase/biossíntese , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Curcumina/uso terapêutico , Citotoxinas/farmacologia , Citotoxinas/uso terapêutico , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Mitomicina/uso terapêutico , RNA Interferente Pequeno/fisiologia , Rad51 Recombinase/antagonistas & inibidores , Rad51 Recombinase/genéticaRESUMO
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.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Emodina/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Mitomicina/farmacologia , Rad51 Recombinase/antagonistas & inibidores , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Regulação para Baixo , Sinergismo Farmacológico , Humanos , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Complexo de Endopeptidases do Proteassoma/fisiologia , RNA Mensageiro/análise , Rad51 Recombinase/química , Rad51 Recombinase/genética , Rad51 Recombinase/fisiologiaRESUMO
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.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Desoxicitidina/análogos & derivados , Regulação para Baixo/genética , Resistencia a Medicamentos Antineoplásicos/genética , Expressão Gênica/genética , Rad51 Recombinase/genética , Antimetabólitos Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Desoxicitidina/farmacologia , Sinergismo Farmacológico , Cloridrato de Erlotinib , Expressão Gênica/efeitos dos fármacos , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/genética , MAP Quinase Quinase 2/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinazolinas/farmacologia , RNA Interferente Pequeno/genética , Rad51 Recombinase/metabolismo , Transfecção , Ubiquitina/metabolismo , GencitabinaRESUMO
Pancreatic adenocarcinoma is an aggressive disease with a high mortality rate. In this study, we have newly generated a monoclonal antibody (mAb), Pa65-2, which specifically binds to pancreatic cancer cells and tumor blood vessels. The target protein of Pa65-2 is identified as human clathrin heavy chain (CHC). In vitro and In vivo study showed that suppression of CHC either by shRNA or by Pa65-2 inhibited tumor growth and angiogenesis. One of the key functions of CHC was to bind with the hypoxia-inducing factor (HIF)-1α protein, increasing the stability of this protein and facilitating its nuclear translocation, thereby regulating the expression of VEGF. Taken together, our findings indicate that CHC plays a role in the processes of tumorigenesis and angiogenesis. Pa65-2 antibody or CHC shRNA can potentially be used for pancreatic cancer therapy.
Assuntos
Adenocarcinoma/patologia , Proteínas de Ciclo Celular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neovascularização Patológica , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adenocarcinoma/irrigação sanguínea , Adenocarcinoma/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Apoptose , Western Blotting , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proliferação de Células , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Técnicas Imunoenzimáticas , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Neoplasias Pancreáticas/irrigação sanguínea , Neoplasias Pancreáticas/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Curcumin (diferuloylmethane), a phenolic compound obtained from the rhizome of Curcuma longa, has been found to inhibit cell proliferation in various human cancer cell lines, including non-small cell lung cancer (NSCLC). Thymidine phosphorylase (TP) is considered an attractive therapeutic target, because increased TP expression can suppress cancer cell death induced by DNA-damaging agents. Mitomycin C (MMC), a chemotherapeutic agent used to treat NSCLC, inhibits tumour growth through DNA cross-linking and breaking. Whether MMC can affect TP expression in NSCLC is unknown. Therefore, in this study, we suggested that curcumin enhances the effects of MMC-mediated cytotoxicity by decreasing TP expression and ERK1/2 activation. Exposure of human NSCLC cell lines H1975 and H1650 to curcumin decreased MMC-elicited phosphorylated MKK1/2-ERK1/2 protein levels. Moreover, curcumin significantly decreased MMC-induced TP protein levels by increasing TP mRNA and protein instability. Enhancement of ERK1/2 activation by constitutively active MKK1/2 (MKK1/2-CA) increased TP protein levels and cell viability in curcumin- and MMC-co-treated cells. In contrast, U0126, a MKK1/2 inhibitor, augmented the cytotoxic effect and the down-regulation of TP by curcumin and MMC. Specific inhibition of TP by siRNA significantly enhanced MMC-induced cell death and cell growth inhibition. Our results suggest that suppression of TP expression or administration of curcumin along with MMC may be a novel lung cancer therapeutic modality in the future.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Curcumina/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Mitomicina/farmacologia , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Curcuma/química , Curcumina/administração & dosagem , Curcumina/isolamento & purificação , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Neoplasias Pulmonares/enzimologia , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Sistema de Sinalização das MAP Quinases , Mitomicina/administração & dosagem , Fosforilação/efeitos dos fármacos , Timidina Fosforilase/genéticaRESUMO
Etoposide (VP-16), a topoisomerase II inhibitor, is an effective anticancer drug currently used for the treatment of a wide range of cancers. Excision repair cross-complementary 1 (ERCC1) is a key protein involved in the process of nucleotide excision repair. High level of ERCC1 expression in cancers is associated with resistance to DNA damage-based chemotherapy. In this study, the effects of p38 mitogen-activated protein kinase (MAPK) signal on the ERCC1 expression induced by etoposide in non-small cell lung cancer (NSCLC) cell lines was investigated. Etoposide increased phosphorylated MAPK kinase 3/6 (MKK3/6)-p38 MAPK and ERCC1 protein and mRNA levels in A549 and H1975 cells. Moreover, SB202190, a p38 inhibitor, or knockdown of p38 expression by specific short interfering RNA (siRNA) significantly decreased the etoposide-induced ERCC1 protein levels and DNA repair capacity in etoposide-exposed NSCLC cells. Enhancement of p38 activation by constitutively active MKK6 (MKK6E) increased ERCC1 protein levels. Specific inhibition of ERCC1 by siRNA significantly enhanced the etoposide-induced cytotoxicity and hypoxanthine guanine phosphoribosyltransferase (hprt) gene mutation rate. Moreover, the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) could decrease the etoposide-induced p38 MAPK-mediated ERCC1 expression and augment the cytotoxic effect and growth inhibition by etopsoside. 17-AAG and etoposide-induced synergistic cytotoxic effect and DNA repair capacity decrease could be abrogated in lung cancer cells with MKK6E or HA-p38 MAPK expression vector transfection. Our results suggest that in human NSCLC cells, ERCC1 is induced by etoposide through the p38 MAPK pathway, and this phenomenon is required for NSCLC survival and resistant DNA damage.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Etoposídeo/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Benzoquinonas/farmacologia , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Endonucleases/genética , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Lactamas Macrocíclicas/farmacologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Thymidine phosphorylase (TP) is the rate-limiting enzyme for the activation of capecitabine (pro-drug of fluorouracil), and as a useful predictor of tumor response to capecitabine-based chemotherapy. Overexpression of Rad51 and ERCC1 induce resistance to chemotherapeutic agents. Emodin, one of the main bioactive anthraquinone derivatives in the roots and rhizomes of numerous plants, possesses potent antitumor effects. Accordingly, we aimed to explore the molecular mechanism of emodin enhances the capecitabine-induced cytotoxicity through controlling Rad51, ERCC1, and TP expression in human non-small cell lung cancer (NSCLC). The results show that capecitabine increases the phosphorylation of MKK1/2-ERK1/2 and protein levels of Rad51 and ERCC1 through enhancing the protein stability. Depletion of endogenous Rad51 or ERCC1 expression by specific small interfering RNA transfection significantly increases capecitabine-induced cell death and growth inhibition. Emodin enhances the capecitabine-induced cytotoxic effects through ERK1/2 inactivation and decreasing the Rad51 and ERCC1 protein levels induced by capecitabine. Enhancement of ERK1/2 signaling by constitutively active MKK1/2 (MKK1/2-CA) results in increasing Rad51 and ERCC1 protein levels and cell viability in NSCLC cell lines treated with emodin and capecitabine. Interestingly, emodin enhances TP mRNA and protein expression in capecitabine treated NSCLC cell lines, and depletion of the TP expression decreases the cytotoxic effects induced by capecitabine and emodin. We conclude that enhancing the cytotoxicity to capecitabine by emodin is mediated by down-regulation the expression of Rad51 and ERCC1 and up-regulation TP expression.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Ligação a DNA/biossíntese , Desoxicitidina/análogos & derivados , Emodina/farmacologia , Endonucleases/biossíntese , Fluoruracila/análogos & derivados , Rad51 Recombinase/biossíntese , Timidina Fosforilase/biossíntese , Capecitabina , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Desoxicitidina/farmacologia , Sinergismo Farmacológico , Endonucleases/genética , Fluoruracila/farmacologia , Técnicas de Silenciamento de Genes , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , RNA Mensageiro/biossíntese , Rad51 Recombinase/genética , Timidina Fosforilase/genéticaRESUMO
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.