RESUMEN
MYC overexpression is a common phenomenon in gastric carcinogenesis. In this study, we identified genes differentially expressed with a downregulated profile in gastric cancer (GC) cell lines with silenced MYC. The TTLL12, CDKN3, CDC16, PTPRA, MZT2B, UBE2T genes were validated using qRT-PCR, western blot and immunohistochemistry in tissues of 213 patients with diffuse and intestinal GC. We identified high levels of TTLL12, MZT2B, CDC16, UBE2T, associated with early and advanced stages, lymph nodes, distant metastases and risk factors such as H. pylori. Our results show that in the diffuse GC the overexpression of CDC16 and UBE2T indicate markers of poor prognosis higher than TTLL12. That is, patients with overexpression of these two genes live less than patients with overexpression of TTLL12. In the intestinal GC, patients who overexpressed CDC16 had a significantly lower survival rate than patients who overexpressed MZT2B and UBE2T, indicating in our data a worse prognostic value of CDC16 compared to the other two genes. PTPRA and CDKN3 proved to be important for assessing tumor progression in the early and advanced stages. In summary, in this study, we identified diagnostic and prognostic biomarkers of GC under the control of MYC, related to the cell cycle and the neoplastic process.
Asunto(s)
Adenocarcinoma/genética , Proteínas Proto-Oncogénicas c-myc/genética , Neoplasias Gástricas/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidad , Subunidad Apc6 del Ciclosoma-Complejo Promotor de la Anafase/genética , Subunidad Apc6 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Línea Celular Tumoral , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/genética , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación hacia Abajo , Fosfatasas de Especificidad Dual/genética , Fosfatasas de Especificidad Dual/metabolismo , Femenino , Silenciador del Gen , Humanos , Masculino , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Persona de Mediana Edad , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Pronóstico , ARN Interferente Pequeño , RNA-Seq , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidad , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
We investigated the role of protein tyrosine phosphatase-alpha (PTPα) expression in the cell death profile of the A431 human carcinoma cell line that was induced by cytotoxic concentrations of the nitric oxide (NO) donors sodium nitroprusside (SNP) and 3,3-bis-(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18). Both NO donors promoted extensive cell detachment in A431 parental cells as compared to the detachment observed for A431 cells that ectopically expressed PTPα (A431 (A27B(PTPα)) cells). The NO-induced cell death characteristics for both cell lines were examined. After incubation for 10 hours with 2.0 mM SNP, attached or detached A431 cells underwent apoptosis. Cells were highly positive for Annexin-V, featured increased cleavage of procaspase-8, activation of downstream caspase-3, and activation of poly-ADP-ribose polymerase 1 (PARP-1). In contrast, exposure of A431 (A27B(PTPα)) cells to 2.0 mM SNP produced an increase in the release of lactate dehydrogenase and enhanced incorporation of propidium iodide. In addition, A431 (A27B(PTPα)) cells showed partial inhibition of the activities of caspase-8, caspase-3, and PARP-1 upon detachment and cell death induced by SNP treatment. Results indicate that necrotic cell damage was induced, characterized by cellular swelling and lysis. We conclude from these results that PTPα regulates the A431 tumor cell death profile mediated by NO donors. Expression of PTPα or its absence may determine the occurrence of NO-induced cell death with necrotic or apoptotic features, respectively.
Asunto(s)
Apoptosis , Donantes de Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Compuestos Nitrosos/farmacología , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores/metabolismo , Caspasas/efectos de los fármacos , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Células Epiteliales/metabolismo , Humanos , L-Lactato Deshidrogenasa/efectos de los fármacos , Óxido Nítrico/metabolismo , Fosfatidilserinas/análisis , Poli(ADP-Ribosa) Polimerasas/efectos de los fármacos , Propidio/análisis , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores/genética , TransfecciónRESUMEN
The role of NO in regulating the focal adhesion proteins, Src, FAK, p130 Cas, and PTP-alpha, was investigated. Fibroblasts expressing PTP-alpha (PTP-alpha(WT) cells), fibroblasts "knockout" for PTP-alpha (PTP-alpha(-/-) cells), and "rescued" "knockout" fibroblasts (PTP-alpha A5/3 cells) were stimulated with either S-nitroso-N-acetylpenicillamine (SNAP) or fetal bovine serum (FBS). FBS increased inducible NO synthase in both cell lines. Activation of Src mediated either by SNAP or by FBS occurred independent of dephosphorylation of Tyr527 in PTP-alpha(-/-) cells. Both stimuli promoted dephosphorylation of Tyr527 and activation of Src kinase in PTP-alpha(WT) cells. NO-mediated activation of Src kinase affected the activities of FAK and p130Cas and was dependent on the expression of PTP-alpha. Analogous to tyrosine phosphorylation, SNAP and FBS stimulated differential generation of NO and S-nitrosylation of Src kinase in both cell lines. Incubation with SNAP resulted in higher levels of NO and S-nitrosylation of immunoprecipitated Src in PTP-alpha(-/-) cells (oxidizing redox environment) as compared with the levels of NO and S-nitrosylated Src in PTP-alpha(WT) cells (reducing redox environment). SNAP differentially stimulated cell proliferation of both cell lines is dependent on the intracellular redox environment, Src activity, and PTP-alpha expression. This dependence also is observed with FBS-stimulated cell migration.