RESUMEN
Rho GDP dissociation inhibitor 2 (RhoGDI2) was initially identified as a regulator of the Rho family of GTPases. Our recent works suggest that RhoGDI2 promotes tumor growth and malignant progression, as well as enhances chemoresistance in gastric cancer. Here, we delineate the mechanism by which RhoGDI2 promotes gastric cancer cell invasion and chemoresistance using two-dimensional gel electrophoresis (2-DE) on proteins derived from a RhoGDI2-overexpressing SNU-484 human gastric cancer cell line and control cells. Differentially expressed proteins were identified using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). In total, 47 differential protein spots were identified; 33 were upregulated, and 14 were downregulated by RhoGDI2 overexpression. Upregulation of SAE1, Cathepsin D, Cofilin1, CIAPIN1, and PAK2 proteins was validated by Western blot analysis. Loss-of-function analysis using small interference RNA (siRNA) directed against candidate genes reveals the need for CIAPIN1 and PAK2 in RhoGDI2-induced cancer cell invasion and Cathepsin D and PAK2 in RhoGDI2-mediated chemoresistance in gastric cancer cells. These data extend our understanding of the genes that act downstream of RhoGDI2 during the progression of gastric cancer and the acquisition of chemoresistance.
Asunto(s)
Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Electroforesis en Gel Bidimensional , Humanos , Péptidos y Proteínas de Señalización Intracelular/análisis , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Metástasis de la Neoplasia , Proteoma/análisis , Proteoma/metabolismo , Proteómica/métodos , Reproducibilidad de los Resultados , Neoplasias Gástricas/tratamiento farmacológico , Regulación hacia Arriba , Quinasas p21 Activadas/análisis , Quinasas p21 Activadas/metabolismo , Inhibidor beta de Disociación del Nucleótido Guanina rho , Inhibidores de la Disociación del Nucleótido Guanina rho-EspecíficoRESUMEN
Gadd45b has been known as a positive mediator of apoptosis induced by certain cytokines and oncogenes. Here, we identified Gadd45b as an effector of Fas-induced apoptosis and found that p38-mediated Rb hyperphosphorylation is one of the mechanisms of Fas-induced apoptosis in murine hepatocyte AML12 cells. Gadd45b has been shown to activate p38 through its physical interaction with MTK1 and induce apoptosis. However, in this study, we have showed that the function of Gadd45b during Fas-induced apoptosis in AML12 cells is different from that reported in previous studies. Depletion of Gadd45b expression did not inhibit the phosphorylation of p38, but it suppressed p38-mediated Rb phosphorylation and apoptosis in response to Fas stimulation by reducing the interaction between p38 and Rb. Ectopic expression of Gadd45b was sufficient to enhance this interaction. These findings suggest that Gadd45b mediates p38-induced Rb phosphorylation by enhancing the interaction between p38 and Rb during Fas-induced apoptosis in murine hepatocytes.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Proteína de Retinoblastoma/metabolismo , Receptor fas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Células HeLa , Humanos , Imidazoles/farmacología , Inmunoprecipitación , Etiquetado Corte-Fin in Situ , Ratones , Unión Proteica/genética , Unión Proteica/fisiología , Piridinas/farmacología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor fas/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Rho GDP dissociation inhibitor 2 (RhoGDI2) is a regulator of the Rho family GTPases. Recent work from our laboratory suggests that RhoGDI2 expression potentially enhances resistance to cisplatin as well as promotes tumor growth and malignant progression in gastric cancer. In this study, we demonstrate that phospholipase C-gamma (PLCγ) is required for RhoGDI2-mediated cisplatin resistance and cancer cell invasion in gastric cancer. The levels of phosphorylated PLCγ are markedly enhanced in RhoGDI2-overexpressing SNU-484 cells and, by contrast, repressed in RhoGDI2-depleted MKN-28 cells. Depletion of PLCγ expression or inhibition of its activity not only significantly increases cisplatin-induced apoptosis but also suppresses the invasive ability of RhoGDI2-overexpressing SNU-484 cells. Taken together, our results suggest that PLCγ plays a key role in RhoGDI2-mediated cisplatin resistance and cell invasion in gastric cancer cells.
Asunto(s)
Antineoplásicos/uso terapéutico , Cisplatino/uso terapéutico , Resistencia a Antineoplásicos/genética , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Fosfolipasa C gamma/fisiología , Neoplasias Gástricas/tratamiento farmacológico , Proteínas Supresoras de Tumor/metabolismo , Línea Celular Tumoral , Activación Enzimática , Inhibidores de Disociación de Guanina Nucleótido/genética , Humanos , Invasividad Neoplásica , Fosfolipasa C gamma/genética , Fosforilación , Neoplasias Gástricas/patología , Proteínas Supresoras de Tumor/genética , Inhibidor beta de Disociación del Nucleótido Guanina rho , Inhibidores de la Disociación del Nucleótido Guanina rho-EspecíficoRESUMEN
Rho GDP dissociation inhibitor 2 (RhoGDI2) expression correlates with tumor growth, metastasis, and chemoresistance in gastric cancer. Here, we show that RhoGDI2 functions in the epithelial-mesenchymal transition (EMT), which is responsible for invasiveness during tumor progression. This tumorigenic activity is associated with repression of E-cadherin by RhoGDI2 via upregulation of Snail. Overexpression of RhoGDI2 induced phenotypic changes consistent with EMT in gastric cancer cells, including abnormal epithelial cell morphology, fibroblast-like properties, and reduced intercellular adhesion. RhoGDI2 overexpression also resulted in decreased expression of the epithelial markers E-cadherin and ß-catenin and increased expression of the mesenchymal markers vimentin and fibronectin. Importantly, RhoGDI2 overexpression also stimulated the expression of Snail, a repressor of E-cadherin and inducer of EMT, but not other family members such as Slug or Twist. RNA interference-mediated knockdown of Snail expression suppressed RhoGDI2-induced EMT and invasion, confirming that the effect was Snail-specific. These results indicate that RhoGDI2 plays a critical role in tumor progression in gastric cancer through induction of EMT. Targeting RhoGDI2 may thus be a useful strategy to inhibit gastric cancer cell invasion and metastasis.
Asunto(s)
Cadherinas/metabolismo , Movimiento Celular , Proliferación Celular , Transición Epitelial-Mesenquimal , Neoplasias Gástricas/patología , Factores de Transcripción/metabolismo , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismo , Antineoplásicos/farmacología , Apoptosis , Western Blotting , Cadherinas/genética , Resistencia a Antineoplásicos , Técnica del Anticuerpo Fluorescente , Humanos , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción de la Familia Snail , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Células Tumorales Cultivadas , Inhibidor beta de Disociación del Nucleótido Guanina rho/genéticaRESUMEN
Rho GDP dissociation inhibitor (RhoGDI)2 has been identified as a regulator of Rho family GTPase. Recently, we suggested that RhoGDI2 could promote tumor growth and malignant progression in gastric cancer. In this study, we demonstrate that RhoGDI2 contributes to another important feature of aggressive cancers, i.e., resistance to chemotherapeutic agents such as cisplatin. Forced expression of RhoGDI2 attenuated cisplatin-induced apoptosis, whereas RhoGDI2 depletion showed opposite effects in vitro. Moreover, the increased anti-apoptotic effect of RhoGDI2 on cisplatin was further validated in RhoGDI2-overexpressing SNU-484 xenograft model in nude mice. Furthermore, we identified Bcl-2 as a major determinant of RhoGDI2-mediated cisplatin resistance in gastric cancer cells. Depletion of Bcl-2 expression significantly increased cisplatin-induced apoptosis in RhoGDI2-overexpressing gastric cancer cells, whereas overexpression of Bcl-2 blocked cisplatin-induced apoptosis in RhoGDI2-depleted gastric cancer cells. Overall, these findings establish RhoGDI2 as an important therapeutic target for simultaneously enhancing chemotherapy efficacy and reducing metastasis risk in gastric cancer.