RESUMO
Triple-negative breast cancers are the most aggressive subtypes with poor prognosis due to lack of targeted cancer therapy. Recently, we reported an association of A-kinase anchor protein 4 expression with various clinico-pathological parameters of breast cancer patients. In this context, we examined the effect of knockdown of A-kinase anchor protein 4 on cell cycle, apoptosis, cellular proliferation, colony formation, migration, and invasion in triple-negative breast cancer cells. We also examined the synergistic cytotoxic effect of paclitaxel on A-kinase anchor protein 4 downregulated triple-negative breast cancer cells. Knockdown of A-kinase anchor protein 4 resulted in significant reduction in cellular growth and migratory abilities. Interestingly, we also observed enhanced cell death in A-kinase anchor protein 4 downregulated cells treated with paclitaxel. Knockdown of A-kinase anchor protein 4 in cell cycle resulted in G0/G1 phase arrest. Knockdown of A-kinase anchor protein 4 also led to increased reactive oxygen species generation as a result of upregulation of NOXA and CHOP. In addition, levels of cyclins, cyclin-dependent kinases, anti-apoptotic molecules, and mesenchymal markers were reduced in A-kinase anchor protein 4 downregulated cells. Moreover, downregulation of A-kinase anchor protein 4 also caused tumor growth reduction in in vivo studies. These data together suggest that A-kinase anchor protein 4 downregulation inhibits various malignant properties and enhances the cytotoxic effect of paclitaxel, and this combinatorial approach could be useful for triple-negative breast cancer treatment.
Assuntos
Proteínas de Ancoragem à Quinase A/deficiência , Neoplasias de Mama Triplo Negativas/genética , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Imunofenotipagem , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Paclitaxel/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
SPAG9 is a novel tumor associated antigen, expressed in variety of malignancies. However, its role in ovarian cancer remains unexplored. SPAG9 expression was validated in ovarian cancer cells by real time PCR and Western blot. SPAG9 involvement in cell cycle, DNA damage, apoptosis, paclitaxel sensitivity and epithelial- mesenchymal transition (EMT) was investigated employing RNA interference approach. Combinatorial effect of SPAG9 ablation and paclitaxel treatment was evaluated in in vitro. Quantitative PCR and Western blot analysis revealed SPAG9 expression in A10, SKOV-3 and Caov3 compared to normal ovarian epithelial cells. SPAG9 ablation resulted in reduced cellular proliferation, colony forming ability and enhanced cytotoxicity of chemotherapeutic agent paclitaxel. Effect of ablation of SPAG9 on cell cycle revealed S phase arrest and showed decreased expression of CDK1, CDK2, CDK4, CDK6, cyclin B1, cyclin D1, cyclin E and increased expression of tumor suppressor p21. Ablation of SPAG9 also resulted in increased apoptosis with increased expression of various pro- apoptotic molecules including BAD, BID, PUMA, caspase 3, caspase 7, caspase 8 and cytochrome C. Decreased expression of mesenchymal markers and increased expression of epithelial markers was found in SPAG9 ablated cells. Combinatorial effect of SPAG9 ablation and paclitaxel treatment was evaluated in in vitro assays which showed that ablation of SPAG9 resulted in increased paclitaxel sensitivity and caused enhanced cell death. In vivo ovarian cancer xenograft studies showed that ablation of SPAG9 resulted in significant reduction in tumor growth. Present study revealed therapeutic potential of SPAG9 in ovarian cancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Adenocarcinoma/tratamento farmacológico , Vetores Genéticos/uso terapêutico , Terapia de Alvo Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias Ovarianas/tratamento farmacológico , Interferência de RNA , RNA Interferente Pequeno/uso terapêutico , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma Papilar/tratamento farmacológico , Adenocarcinoma Papilar/metabolismo , Adenocarcinoma Papilar/patologia , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Cistadenocarcinoma Papilar/tratamento farmacológico , Cistadenocarcinoma Papilar/patologia , Cistadenocarcinoma Seroso/tratamento farmacológico , Cistadenocarcinoma Seroso/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/administração & dosagem , Humanos , Injeções Intralesionais , Camundongos Nus , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Paclitaxel/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Breast cancer is one of the leading cause of cancer-related deaths in women worldwide and increasing rapidly in developing countries. In the present study, we investigated the potential role and association of HSP70-2 with breast cancer. METHODS: HSP70-2 expression was examined in 154 tumor and 103 adjacent non-cancerous tissue (ANCT) specimens and breast cancer cell lines (MCF7, BT-474, SK-BR-3 and MDA-MB-231) by RT-PCR, quantitative-PCR, immunohistochemistry, Western blotting, flow cytometry and indirect immunofluorescence. Plasmid driven short hairpin RNA approach was employed to validate the role of HSP70-2 in cellular proliferation, senescence, migration, invasion and tumor growth. Further, we studied the effect of HSP70-2 protein ablation on signaling cascades involved in apoptosis, cell cycle and Epithelial-Mesenchymal-Transition both in culture as well as in-vivo human breast xenograft mouse model. RESULTS: HSP70-2 expression was detected in majority of breast cancer patients (83 %) irrespective of various histotypes, stages and grades. HSP70-2 expression was also observed in all breast cancer cells (BT-474, MCF7, MDA-MB-231 and SK-BR-3) used in this study. Depletion of HSP70-2 in MDA-MB-231 and MCF7 cells resulted in a significant reduction in cellular growth, motility, onset of apoptosis, senescence, cell cycle arrest as well as reduction of tumor growth in the xenograft model. At molecular level, down-regulation of HSP70-2 resulted in reduced expression of cyclins, cyclin dependent kinases, anti-apoptotic molecules and mesenchymal markers and enhanced expression of CDK inhibitors, caspases, pro-apoptotic molecules and epithelial markers. CONCLUSIONS: HSP70-2 is over expressed in breast cancer patients and was involved in malignant properties of breast cancer. This suggests HSP70-2 may be potential candidate molecule for development of better breast cancer treatment.
