RESUMO
AIM: To study the role of PARP-1 in EMT of non-small cell lung carcinoma. MATERIALS AND METHODS: We used H1299 and H460 lung cancer cells for knockdown study of PARP-1 using shPARP-1 lentiviral particle. We performed western blotting, confocal microscopy, semi-quantitative PCR, wound healing and colony formation assays. BACKGROUND AND KEY FINDINGS: PARP-1 (poly-ADP ribose polymerase-1) is a multi-domain protein having DNA binding, auto-modification and catalytic domain, that participates in many biological processes including DNA damage detection and repair, transcription regulation, apoptosis, necrosis, cancer progression and metastasis. Metastasis is a leading cause of death in cancer patients, which starts in epithelial tumors via initiating epithelial to mesenchymal transition. There are various transcription factors involved in EMT including Snail-1, Smads, p65, ZEB1 and Twist1. We studied the effect of PARP-1 knockdown on EMT in non-small cell lung cancer cell line H1299. We found a significant increase in epithelial marker including ZO1 and ß-catenin, while prominent decrease in the mesenchymal marker vimentin after PARP-1 knockdown in H1299 cells. Transcription factors including p65, Smad4 and ZEB1 showed significant decrease with concurrent expression of EMT markers. Cell migration and colony formation decreased after PARP-1 knockdown in H1299 cells. SIGNIFICANCE: Overall, the shRNA mediated knockdown of PARP-1 in H1299 cells resulted in reversal of EMT or mesenchymal to epithelial transition (MET) characterized by an increase in epithelial markers and a decrease in mesenchymal markers, via down-regulating transcription factors including Smad4, p65 and ZEB1. Thus PARP-1 has a role in EMT in lung cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares/patologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteína Smad4/metabolismo , Fator de Transcrição RelA/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular/genética , Regulação para Baixo/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Lentivirus/metabolismo , Neoplasias Pulmonares/genética , Modelos Biológicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Junções Íntimas/metabolismo , Ensaio Tumoral de Célula-Tronco , beta Catenina/metabolismoRESUMO
Epithelial-mesenchymal transition (EMT) is a programed course of developmental changes resulting in the acquisition of invasiveness and mobility in cells. In cancer, this course is used by epithelial cells to attain movability. Translationally controlled tumor protein (TCTP) has been extensively characterized following the observation on tumor reversion ensuing its depletion. However, the role of TCTP in cancer progression is still elusive. Here, we demonstrate for the first time that TCTP is a target of transforming growth factor-ß1 (TGF-ß1), a key regulator of EMT in A549 cells. We here present changes in expression patterns of intermediate filament markers (vimentin and cytokeratin 18a) of EMT following TCTP knockdown or over expression. The TCTP over-expression in cancer cells is associated with mesenchymal characters, while downregulation promotes the epithelial markers in the cells. Interaction of TCTP with ß-catenin seems to stabilize ß-catenin, preparative to its nuclear localization highlighting a role for ß-catenin signaling in EMT. Moreover, the induction of urokinase plasminogen activator (uPA) following ectopic expression of TCTP leads to destabilization of ECM. The cells knocked down for TCTP show diminished invasiveness and migration under TGF-ß1 treatment. The present results for the first time demonstrate that TGF-ß1 dependent TCTP expression is required for EMT in cells.
Assuntos
Biomarcadores Tumorais/fisiologia , Citoesqueleto/metabolismo , Transição Epitelial-Mesenquimal , Fator de Crescimento Transformador beta1/farmacologia , Células A549 , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Citoesqueleto/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Células HEK293 , Humanos , Células MCF-7 , Metástase Neoplásica , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator de Crescimento Transformador beta1/metabolismo , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
Microbial communities in rhizosphere interact with each other and form a basis of a cumulative impact on plant growth. Rhizospheric microorganisms like Piriformospora indica and Azotobacter chroococcum are well known for their beneficial interaction with plants. These features make P. indica/A. chroococcum co-inoculation of crops most promising with respect to sustainable agriculture and to understanding the transitions in the evolution of rhizospheric microbiome. Here, we investigated interactions of P. indica with A. chroococcum in culture. Out of five Azotobacter strains tested, WR5 exhibited growth-promoting while strain M4 exerted growth-inhibitory effect on the fungus in axenic culture. Electron microscopy of co-culture indicated an intimate association of the bacterium with the fungus. 2-D gel electrophoresis followed by mass spectrometry of P. indica cellular proteins grown with or without WR5 and M4 showed differential expression of many metabolic proteins like enolase-I, ureaseD, the GTP binding protein YPT1 and the transmembrane protein RTM1. Fungal growth as influenced by bacterial crude metabolites was also monitored. Taken together, the results conform to a model where WR5 and M4 influence the overall growth and physiology of P. indica which may have a bearing on its symbiotic relationship with plants.