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1.
Gastroenterology ; 145(6): 1312-22.e1-8, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23993973

RESUMO

BACKGROUND & AIMS: Chronic inflammation contributes to the pathogenesis of gastric tumorigenesis. The aurora kinase A (AURKA) gene is frequently amplified and overexpressed in gastrointestinal cancers. We investigated the roles of AURKA in inflammation and gastric tumorigenesis. METHODS: We used quantitative real-time reverse transcription polymerase chain reaction, immunofluorescence, immunohistochemistry, luciferase reporter, immunoblot, co-immunoprecipitation, and in vitro kinase assays to analyze AGS and MKN28 gastric cancer cells. We also analyzed Tff1(-/-) mice, growth of tumor xenografts, and human tissues. RESULTS: We correlated increased expression of AURKA with increased levels of tumor necrosis factor-α and inflammation in the gastric mucosa of Tff1(-/-) mice (r = 0.62; P = .0001). MLN8237, an investigational small-molecule selective inhibitor of AURKA, reduced nuclear staining of nuclear factor-κB (NF-κB) p65 in human gastric cancer samples and mouse epithelial cells, suppressed NF-κB reporter activity, and reduced expression of NF-κB target genes that regulate inflammation and cell survival. Inhibition of AURKA also reduced growth of xenograft tumors from human gastric cancer cells in mice and reversed the development of gastric tumors in Tff1(-/-) mice. AURKA was found to regulate NF-κB activity by binding directly and phosphorylating IκBα in cells. Premalignant and malignant lesions from the gastric mucosa of patients had increased levels of AURKA protein and nuclear NF-κB, compared with healthy gastric tissue. CONCLUSIONS: In analyses of gastric cancer cell lines, human tissue samples, and mouse models, we found AURKA to be up-regulated during chronic inflammation to promote activation of NF-κB and tumorigenesis. AURKA inhibitors might be developed as therapeutic agents for gastric cancer.


Assuntos
Adenocarcinoma/metabolismo , Aurora Quinase A/metabolismo , Carcinogênese/metabolismo , Inflamação/metabolismo , Neoplasias Gástricas/metabolismo , Regulação para Cima , Adenocarcinoma/patologia , Animais , Aurora Quinase A/antagonistas & inibidores , Aurora Quinase A/efeitos dos fármacos , Azepinas/farmacologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Xenoenxertos , Humanos , Técnicas In Vitro , Camundongos Knockout , Camundongos Nus , NF-kappa B/metabolismo , Peptídeos/deficiência , Peptídeos/genética , Peptídeos/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Neoplasias Gástricas/patologia , Fator Trefoil-1
2.
Anticancer Drugs ; 25(3): 296-302, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24201306

RESUMO

Pancreatic cancer has dismally low mean survival rates worldwide. Only a few chemotherapeutic agents including gemcitabine have been shown to improve the survival of pancreatic cancer patients. Biochanin A, an isoflavone, is known to exert an anticancer effect on various cancer types. In this study, we examined the anticancer properties of biochanin A on pancreatic cancer cells. The effect of biochanin A on cellular survival, apoptosis, and proliferation was analyzed using MTT, flow cytometry, and colony formation assay. The effect of biochanin A on pancreatic cancer's mitogenic signaling was determined using western blot analysis. Migration assay and zymography were used to determine biochanin A's effect on pancreatic cancer progression. Biochanin A induced dose-dependent toxicity on pancreatic cancer cells (Panc1 and AsPC-1). It reduced colony formation ability of Panc1 cells and induced dose-dependent apoptosis. Activation of Akt and MAPK was inhibited. Furthermore, the migratory and invasive potential of the cancer cells was also reduced. The results suggest that biochanin A is effective in reducing pancreatic cancer cell survival by inhibiting their proliferation and inducing apoptosis. It affects mitogenic, migratory, and invasive processes involved in cancer progression. These findings may lead to novel approaches to treat pancreatic cancer using isoflavones in combination with other therapeutic drugs.


Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Genisteína/farmacologia , Neoplasias Pancreáticas/patologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Humanos , Invasividade Neoplásica , Transdução de Sinais
3.
Cancer ; 119(4): 904-14, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22972611

RESUMO

BACKGROUND: Upper gastrointestinal adenocarcinomas (UGCs) respond poorly to current chemotherapeutic regimes. The authors and others have previously reported frequent Aurora kinase A (AURKA) gene amplification and mRNA and protein overexpression in UGCs. The objective of the current study was to determine the therapeutic potential of alisertib (MLN8237) alone and in combination with docetaxel in UGCs. METHODS: After treatment with alisertib and/or docetaxel, clonogenic cell survival, cell cycle analyses, Western blot analyses, and tumor xenograft growth assays were carried out to measure cell survival, cell cycle progression, apoptotic protein expression, and tumor xenograft volumes, respectively. RESULTS: By using the AGS, FLO-1, and OE33 UGC cell lines, which have constitutive AURKA overexpression and variable tumor protein 53 (p53) status, significantly enhanced inhibition of cancer cell survival was observed with alisertib and docetaxel treatment in combination (P < .001), compared with single-agent treatments. Cell cycle analyses, after 48 hours of treatment with alisertib, produced a significant increase in the percentage of polyploidy in UGC cells (P < .01) that was further enhanced by docetaxel (P < .001). In addition, an increase in the percentage of cells in sub-G1-phase observed with alisertib (P < .01) was significantly enhanced with the combination treatment (P < .001). Western blot analysis demonstrated higher induction of cleaved caspase 3 protein expression with the combined treatment compared with single-agent treatments. In addition, FLO-1 and OE33 cell xenograft models demonstrated enhanced antitumor activity for the alisertib and docetaxel combination compared with single-agent treatments (P < .001). CONCLUSIONS: The current study demonstrated that alisertib combined with docetaxel can mediate a better therapeutic outcome in UGC cell lines.


Assuntos
Adenocarcinoma/tratamento farmacológico , Azepinas/farmacologia , Neoplasias Gastrointestinais/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Taxoides/farmacologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Aurora Quinase A , Aurora Quinases , Azepinas/administração & dosagem , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Docetaxel , Feminino , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/patologia , Humanos , Camundongos , Camundongos Nus , Poliploidia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/administração & dosagem , Taxoides/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Macromol Biosci ; 17(8)2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28485094

RESUMO

Upper Gastrointestinal Cancers (UGCs) are a leading cause of cancer-related deaths worldwide. Paclitaxel (PTX) is frequently used for the treatment of UGCs; however, low bioavailability, reduced solubility, and dose-dependent toxicity impede its therapeutic use. PAMAMG4.0 -NH2 -DHA is synthesized by linking amine-terminated fourth-generation poly(amidoamine) (PAMAMG4.0 -NH2 ) dendrimers with omega-3 fatty acid docosahexaenoic acid (DHA). Next, PAMAMG4.0 -NH2 -DHA-PTX (DHATX) and PAMAMG4.0 -NH2 -PTX (PAX) conjugates are synthesized by subsequent covalent binding of PTX with PAMAMG4.0 -NH2 -DHA and PAMAMG4.0 -NH2 , respectively. 1 H-NMR and MALDI-TOF analyses are performed to confirm conjugation of DHA to PAMAMG4.0 -NH2 and PTX to PAMAMG4.0 -NH2 -DHA. The cell viability, clonogenic cell survival, and flow cytometry analyses are used to determine the anticancer activity of PTX, PAX, and DHATX in UGC cell lines. The in vitro data indicate that treatment with DHATX is significantly more potent than PTX or PAX at inhibiting cellular proliferation, suppressing long-term survival, and inducing cell death in UGC cells.


Assuntos
Dendrímeros , Sistemas de Liberação de Medicamentos , Ácidos Graxos Ômega-3 , Neoplasias Gastrointestinais/tratamento farmacológico , Paclitaxel , Linhagem Celular Tumoral , Dendrímeros/síntese química , Dendrímeros/química , Dendrímeros/farmacologia , Ácidos Graxos Ômega-3/química , Ácidos Graxos Ômega-3/farmacologia , Neoplasias Gastrointestinais/metabolismo , Neoplasias Gastrointestinais/patologia , Humanos , Paclitaxel/química , Paclitaxel/farmacologia
5.
Clin Cancer Res ; 20(1): 76-86, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24240108

RESUMO

PURPOSE: Suppression of P53 (tumor protein 53) transcriptional function mediates poor therapeutic response in patients with cancer. Aurora kinase A (AURKA) and human double minute 2 (HDM2) are negative regulators of P53. Herein, we examined the role of AURKA in regulating HDM2 and its subsequent effects on P53 apoptotic function in gastric cancer. EXPERIMENTAL DESIGN: Primary tumors and in vitro gastric cancer cell models with overexpression or knockdown of AURKA were used. The role of AURKA in regulating HDM2 and cell survival coupled with P53 expression and activity were investigated. RESULTS: Overexpression of AURKA enhanced the HDM2 protein level; conversely, knockdown of endogenous AURKA decreased expression of HDM2 in AGS and SNU-1 cells. Dual co-immunoprecipitation assay data indicated that AURKA was associated with HDM2 in a protein complex. The in vitro kinase assay using recombinant AURKA and HDM2 proteins followed by co-immunoprecipitation revealed that AURKA directly interacts and phosphorylates HDM2 protein in vitro. The activation of HDM2 by AURKA led to induction of P53 ubiquitination and attenuation of cisplatin-induced activation of P53 in gastric cancer cells. Inhibition of AURKA using an investigational small-molecule specific inhibitor, alisertib, decreased the HDM2 protein level and induced P53 transcriptional activity. These effects markedly decreased cell survival in vitro and xenograft tumor growth in vivo. Notably, analysis of immunohistochemistry on tissue microarrays revealed significant overexpression of AURKA and HDM2 in human gastric cancer samples (P < 0.05). CONCLUSION: Collectively, our novel findings indicate that AURKA promotes tumor growth and cell survival through regulation of HDM2-induced ubiquitination and inhibition of P53. Clin Cancer Res; 20(1); 76-86. ©2013 AACR.


Assuntos
Adenocarcinoma/enzimologia , Aurora Quinase A/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Neoplasias Gástricas/enzimologia , Ubiquitinação , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Animais , Antineoplásicos/farmacologia , Aurora Quinase A/antagonistas & inibidores , Azepinas/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Humanos , Camundongos , Camundongos Nus , Fosforilação , Proteólise , Pirimidinas/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Cancer Res ; 73(1): 331-40, 2013 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23117882

RESUMO

Esophageal adenocarcinoma (EAC) is characterized by resistance to chemotherapy and poor outcome. Although cisplatin (CDDP) has been used as a first-line therapy in patients with EAC, resistance remains a major clinical problem. The AXL receptor tyrosine kinase, originally isolated as a transforming gene from leukemia, is overexpressed in several solid tumors. Herein, we assessed AXL protein expression in human EACs and examined its role in CDDP resistance in human EAC cells. AXL overexpression was detected in more than 50% of tumors examined. Elevating AXL in nonoverexpressing cells doubled the CDDP IC(50) and increased cell survival three-fold, while attenuating AXL in overexpressing cells reduced survival two-fold. The effects of AXL modulation on cell survival were associated with changes in cellular and molecular markers of apoptosis. Mechanistic investigations revealed that AXL blocked CDDP-induced activation of endogenous p73ß (TP73), reducing its protein half-life, and inhibited CDDP-induced levels of p-c-ABL(Y412) and p-p73ß(Y99). These changes were associated with a disruption of c-ABL/p73ß protein interactions due to association with c-ABL in the cytoplasm, thereby blocking nuclear accumulation of c-ABL and phosphorylation of p73ß in response to DNA damage. Together, our results establish that AXL promotes CDDP resistance in esophageal adenocarcinoma and argue that therapeutic targeting of AXL may sensitize these cancers to DNA-damaging drugs.


Assuntos
Adenocarcinoma/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Neoplasias Esofágicas/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Adenocarcinoma/genética , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Cisplatino/farmacologia , Neoplasias Esofágicas/genética , Humanos , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Receptor Tirosina Quinase Axl
7.
Mol Cancer Res ; 11(1): 86-94, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23160836

RESUMO

Although Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) is overexpressed in two-thirds of gastric cancers, its impact on molecular functions has not been fully characterized. In this study, we examined the role of DARPP-32 in gastric cancer cell invasion. Using matrigel-coated Boyden chamber invasion assay, DARPP-32-overexpressing AGS cells showed a three-fold increase in invasion relative to the vector control (P < 0.01). We also tested the transendothelial cell invasion as a measure of cell aggressiveness using the impedance-based human umbilical vein endothelial cells invasion assay and obtained similar results (P < 0.001). Western blot analysis indicated that overexpression of DARPP-32 mediated an increase in the membrane-type 1 matrix metalloproteinase (MT1-MMP) and CXCR4 protein levels. Consistent with the role of MT1-MMP in cleaving extracellular matrix proteins initiating the activation of soluble MMPs, we detected a robust increase in MMP-2 activity in DARPP-32-overexpressing cells. The knockdown of endogenous DARPP-32 in the MKN-45 cells reversed these signaling events and decreased cell invasive activity. We tested whether the invasive activity mediated by DARPP-32 might involve sustained signaling via CXCR4-dependent activation of the MT1-MMP/MMP-2 pathway. The small-molecule CXCR4 antagonist (AMD3100) and CXCR4-siRNA blocked DARPP-32-induced cell invasion. We further examined our hypothesis that DARPP-32 could interact with CXCR4 and stabilize its levels following stimulation with its ligand, CXCL12. Using reciprocal coimmunoprecipitation and immunofluorescence experiments, we found that DARPP-32 and CXCR4 coexist in the same protein complex. DARPP-32 prolonged the CXCR4 protein half-life and reduced ubiquitination of the CXCR4 protein, following treatment with its ligand, CXCL12. In conclusion, these findings show a novel mechanism by which DARPP-32 promotes cell invasion by regulating CXCR4-mediated activation of the MT1-MMP/MMP-2 pathway.


Assuntos
Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Receptores CXCR4/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Linhagem Celular Tumoral , Fosfoproteína 32 Regulada por cAMP e Dopamina/biossíntese , Fosfoproteína 32 Regulada por cAMP e Dopamina/genética , Células HEK293 , Humanos , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Invasividade Neoplásica , Receptores CXCR4/biossíntese , Receptores CXCR4/genética , Transdução de Sinais
8.
Mol Cancer Ther ; 11(3): 763-74, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22302096

RESUMO

Esophageal adenocarcinomas are poorly responsive to chemotherapeutics. This study aimed to determine the levels of Aurora kinase A (AURKA) and the therapeutic potential of MLN8237, an investigational AURKA inhibitor, alone and in combination with cisplatin. Using quantitative real-time PCR, we detected frequent AURKA gene amplification (15 of 34, 44%) and mRNA overexpression (37 of 44, 84%) in esophageal adenocarcinomas (P < 0.01). Immunohistochemical analysis showed overexpression of AURKA in more than two-thirds of esophageal adenocarcinoma tissue samples (92 of 132, 70%; P < 0.001). Using FLO-1, OE19, and OE33 esophageal adenocarinoma cell lines, with constitutive AURKA overexpression and mutant p53, we observed inhibition of colony formation with a single treatment of 0.5 µmol/L MLN8237 (P < 0.05). This effect was further enhanced in combination with 2.5 µmol/L cisplatin (P < 0.001). Twenty-four hours after treatment with the MLN8237 or MLN8237 and cisplatin, cell-cycle analyses showed a sharp increase in the percentage of polyploid cells (P < 0.001). This was followed by an increase in the percentage of cells in the sub-G(1) phase at 72 hours, concordant with the occurrence of cell death (P < 0.001). Western blot analysis showed higher induction of TAp73ß, PUMA, NOXA, cleaved caspase-3, and cleaved PARP with the combined treatment, as compared with a single-agent treatment. Using xenograft models, we showed an enhanced antitumor role for the MLN8237 and cisplatin combination, as compared with single-agent treatments (P < 0.001). In conclusion, this study shows frequent overexpression of AURKA and suggests that MLN8237 could be an effective antitumor agent, which can be combined with cisplatin for a better therapeutic outcome in esophageal adenocarcinomas.


Assuntos
Azepinas/farmacologia , Cisplatino/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Aurora Quinase A , Aurora Quinases , Western Blotting , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Esôfago/efeitos dos fármacos , Esôfago/metabolismo , Esôfago/patologia , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ensaios Antitumorais Modelo de Xenoenxerto
9.
J Oncol ; 2009: 121458, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-20169097

RESUMO

Overexpression of HER-2 receptor is associated with poor prognosis and aggressive forms of breast cancer. Scientific literature indicates a preventive role of isoflavones in cancer. Since activation of HER-2 receptor initiates growth-promoting events in cancer cells, we studied the effect of biochanin A (an isoflavone) on associated signaling events like receptor activation, downstream signaling, and invasive pathways. HER-2-positive SK-BR-3 breast cancer cells, MCF-10A normal breast epithelial cells, and NIH-3T3 normal fibroblast cells were treated with biochanin A (2-100 muM) for 72 hours. Subsequently cell viability assay, western blotting and zymography were carried out. The data indicate that biochanin A inhibits cell viability, signaling pathways, and invasive enzyme expression and activity in SK-BR-3 cancer cells. Biochanin A did not inhibit MCF-10A and NIH-3T3 cell viability. Therefore, biochanin A could be a unique natural anticancer agent which can selectively target cancer cells and inhibit multiple signaling pathways in HER-2-positive breast cancer cells.

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