RESUMEN
OBJECTIVES: SNCG in breast cancer is a marker for advanced and aggressive disease thereby correlating with a poor prognosis in patients. We set out to determine if SNCG expression in UPSC correlates with aggressive cellular properties, poor prognosis, and chemoresistance, and if silencing SNCG can reverse these attributes in vitro. METHODS: A focused, real time PCR array was performed comparing a papillary serous (SPEC2) and an endometrioid (Ishikawa) endometrial cancer cell line. SNCG was the most differentially expressed gene. SNCG expression was confirmed by real time PCR, Western blot, and immunohistochemistry (IHC) and correlated with outcomes in a pilot set of 20 UPSC patients. A stably transfected SPEC2 cell line was created using shSNCG oligonucleotides. The effect of SNCG knockdown in SPEC2 cells on cell proliferation and sensitivity to paclitaxel-induced apoptosis was measured using a cell viability assay, BrdU incorporation assay, as well as cleaved PARP analyses. RESULTS: SNCG mRNA as well as protein was highly expressed in SPEC2 cells while minimally to undetectable in several endometrioid endometrial cancer and normal endometrial cell lines. IHC also confirmed unique SNCG expression in UPSC tumors compared to low grade endometrial cancers. In UPSC patients, SNCG expression by IHC correlated with advanced stage and decreased progression-free survival. Knockdown of SNCG in SPEC2 cells caused a significant decrease in cell proliferation and increased sensitivity to paclitaxel-induced apoptosis. CONCLUSIONS: SNCG is a novel biomarker for aggressive disease and chemoresistance in UPSC and merits further investigation both as a prognostic tool and as a therapeutic target.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Carcinoma Papilar/metabolismo , Cistadenocarcinoma Seroso/metabolismo , Proteínas de Neoplasias/biosíntesis , Neoplasias Uterinas/metabolismo , gamma-Sinucleína/biosíntesis , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Biomarcadores de Tumor/genética , Carcinoma Papilar/tratamiento farmacológico , Carcinoma Papilar/genética , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Cistadenocarcinoma Seroso/tratamiento farmacológico , Cistadenocarcinoma Seroso/genética , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Paclitaxel/farmacología , ARN Interferente Pequeño/genética , Transfección , Neoplasias Uterinas/tratamiento farmacológico , Neoplasias Uterinas/genética , gamma-Sinucleína/antagonistas & inhibidores , gamma-Sinucleína/genéticaRESUMEN
OBJECTIVE: Endometrial cancer is the most common type of gynecologic cancer in the United States. In this study, we propose that a marine sponge compound, psammaplysene A (PsA) induces apoptosis in endometrial cancer cells through forced nuclear expression of FOXO1. METHODS: Ishikawa and ECC1 cells were treated with varying doses of PsA. FOXO1 protein localization was observed using immunofluorescent staining of cells. The effects of PsA on cell viability and proliferation were assessed using a cell viability assay and a BrdU incorporation assay respectively. Cell cycle analysis was performed using flow cytometry. To assess the role of FOXO1 in PsA-induced apoptosis, FOXO1 was silenced in ECC1 cells using siRNA technique, and overexpressed in Ishikawa cells using an adenovirus containing FOXO1 cDNAs. Western blots were used to measure levels of FOXO1 and cleaved PARP proteins. RESULTS: Treatment of both ECC1 and Ishikawa cells with PsA caused an increase in nuclear FOXO1 protein, a dramatic decrease in cell viability of approximately 5-fold (p<0.05) and minimal effect on proliferation. Furthermore, treatment of cells with PsA doubled the percentage of cells in the G2/M phase (p<0.05). PsA induced apoptosis in endometrial cancer cells. When FOXO1 was silenced in ECC1 cells and treated with PsA, the incidence of apoptosis decreased. In addition, overexpression of FOXO1 with PsA treatment increased apoptosis. CONCLUSIONS: Increasing nuclear FOXO1 function is important for the induction of apoptosis of endometrial cancer cells by PsA.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias Endometriales/tratamiento farmacológico , Factores de Transcripción Forkhead/biosíntesis , Tirosina/análogos & derivados , División Celular/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Núcleo Celular/metabolismo , Neoplasias Endometriales/genética , Neoplasias Endometriales/metabolismo , Neoplasias Endometriales/patología , Femenino , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Fase G2/efectos de los fármacos , Humanos , ARN Interferente Pequeño/genética , Transfección , Tirosina/farmacologíaRESUMEN
OBJECTIVE: Endometrial cancer is the most common type of gynecologic cancer in the United States. In this study, we propose that inhibition of the AKT pathway sensitizes cells to chemotherapeutic agents by increasing FOXO1 expression. METHODS: Ishikawa and RL95 cells were treated with the AKT inhibitor (API-59CJ-OMe) alone and in combination with carboplatin or paclitaxel. Cells were counted using a hemocytometer and cell cycle analysis done with flow cytometry. Apoptosis was measured with TUNEL and Annexin V/DAPI staining. FOXO1 protein expression and localization was done using immunofluorescent staining of cells. Finally, the adenovirus containing triple mutant FOXO1 was used to overexpress the constitutively active FOXO1 in Ishikawa cells and its effects on cell viability were studied. RESULTS: Treatment with 6 microM API-59CJ-OME resulted in preferential cell death in Ishikawa and RL95 cells compared to another endometrial cancer cell line, ECC1 after 48 h of treatment. API-59CJ-OME treatment of Ishikawa cells resulted in cell cycle arrest in the G2/M phase. The addition of API-59CJ-OME to carboplatin resulted in a synergistic increase in cell death by apoptosis compared to the responses to each agent separately. Treatment with API-59CJ-OME, carboplatin, paclitaxel or the combinations for 24 h increased nuclear expression of FOXO1 in Ishikawa cells. Overexpression of FOXO1 caused 37% of the cells to die within 24 h. Addition of carboplatin to the AD-FOXO1 expressing cells further increased cell death to 71%. CONCLUSIONS: Inhibition of AKT signaling potentiates cell death in Ishikawa and RL95 cells when combined with carboplatin through mechanisms involving FOXO1 activation.