RESUMEN
Inflammatory changes are involved in tumor cell proliferation, migration, and invasion. Tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) play important roles in inflammatory regulation during tumor development. Oct4 acts as a transcription factor that modulates inflammatory changes in mesenchymal stem cells. In this study, we explored the role of Oct4 in the invasion and migration of oral squamous cell carcinoma (OSCC) cells. LPS and TNF-α were used to treat the OSCC cell lines HN4 and CAL27 to induce inflammation. The generation of inflammatory cytokines, including TNF-α, interleukin (IL)-1ß, and IL-6, was evaluated by enzyme-linked immunosorbent assay and real-time quantitative PCR. Western blot analysis was employed to detect the expression and phosphorylation of JNK1, p65, and STAT3, which are key modulators of inflammation. Wound scratch healing and transwell invasion assays were further used to determine the role of inflammation in the invasion and migration of OSCC cells. Robust inflammation was observed in HN4 and CAL27 cells treated with LPS and TNF-α. A marked increase in JNK1, p65, and STAT3 phosphorylation levels in OSCC cells was also detected after LPS and TNF-α treatment. The migration and invasion of HN4 and CAL27 cells were significantly boosted by stimulation with LPS and TNF-α. Furthermore, Oct4 mRNA and protein levels were significantly upregulated by stimulation with LPS and TNF-α. Silencing of Oct4 led to reduced inflammation and decreased levels of phosphorylated JNK1, p65, and STAT3 and impaired invasion and migration in LPS- and TNF-α-stimulated OSCC cells. Overall, LPS- and TNF-α-induced inflammation suppressed the migration and invasion of OSCC cells by upregulating Oct4 expression.
Asunto(s)
Células Epiteliales/efectos de los fármacos , Lipopolisacáridos/farmacología , Factor 3 de Transcripción de Unión a Octámeros/genética , Procesamiento Proteico-Postraduccional , Factor de Necrosis Tumoral alfa/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cámaras de Difusión de Cultivos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Inflamación , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/genética , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Modelos Biológicos , Factor 3 de Transcripción de Unión a Octámeros/antagonistas & inhibidores , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Fosforilación/efectos de los fármacos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: To examine the activation and cytotoxicity of human peripheral blood T lymphocyte induced in vitro by human 4-1-BB ligand (4-1-BBL) gene transfected into tumor Tca8113 cells. METHODS: The eukaryotic expression vector pEGFP-h4-1-BBL was transfected into human oral carcinoma cell line Tca8113 by Lipofectamine 2000. The transfected cells were then selected in medium containing G-418, cloned by limited dilution and named as Tca8113-4-1-BBL. Human 4-1-BBL mRNA and protein expression of transfected cells was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting respectively. The tumor cell vaccines (TCV) were obtained by treatment with mitomycin (MMC). Human peripheral blood mononuclear cells (PBMC) were prepared from lymphoprep, and then stimulated with anti-CD-3 mAb and incubated with non-transfected or transfected TCV-Tca8113 cells, respectively. The proliferation of T cells was evaluated by trypan blue exclusion; the CCK-8 was used to detect the cytotoxic effect of T lymphocytes. Meanwhile, the secretion of interferon-gamma (IFN-gamma) and interleukin (IL)-2 in culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: The Tca8113 cells transfected by pEGFP-h4-1-BBL could express human 4-1-BBL efficiently. As compared with wild type Tca8113 cells, the transfected Tca8113 cells could markedly promote proliferation, IL-2 and IFN-gamma production and cytotoxic activity of lymphocytes. CONCLUSIONS: The transfection of human 4-1-BBL gene in Tca8113 cells is effective in enhancing its immunogenicity and inducing antitumor immune response in vitro.