Dataset Information

Analysis of tumor necrosis factor ?-induced and nuclear factor ?B-silenced LNCaP prostate cancer cells by RT-qPCR.

ABSTRACT: Prostate cancer is the second leading cause of morbidity and mortality in males in the Western world. In the present study, LNCaP, which is an androgen receptor-positive and androgen-responsive prostate cancer cell line derived from lymph node metastasis, and DU145, which is an androgen receptor-negative prostate cancer cell line derived from brain metastasis, were investigated. TNF? treatment decreased p105 and p50 expression and R1881 treatment slightly decreased p105 expression but increased p50 expression with or without TNF? induction. As an aggressive prostate cancer cell line, DU145 transfected with six transmembrane protein of prostate (STAMP)1 or STAMP2 was also exposed to TNF?. Western blotting indicated that transfection with either STAMP gene caused a significant increase in NF?B expression following TNF? induction. In addition, following the treatment of LNCaP cells with TNF?, reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed with a panel of apoptosis-related gene primers. The apoptosis-related genes p53, p73, caspase 7 and caspase 9 showed statistically significant increases in expression levels while the expression levels of MDM2 and STAMP1 decreased following TNF? induction. Furthermore, LNCaP cells were transfected with a small interfering NF?B (siNF?B) construct for 1 and 4 days and induced with TNF? for the final 24 h. RT-qPCR amplifications were performed with apoptosis-related gene primers, including p53, caspases and STAMPs. However, no changes in the level of STAMP2 were observed between cells in the presence or absence of TNF? induction or between those transfected or not transfected with siNF?B; however, the level of STAMP1 was significantly decreased by TNF? induction, and significantly increased with siNF?B transfection. Silencing of the survival gene NF?B caused anti-apoptotic STAMP1 expression to increase, which repressed p53, together with MDM2. NF?B silencing had varying effects on a panel of cancer regulatory genes. Therefore, the effective inhibition of NF?B may be critical in providing a targeted pathway for prostate cancer prevention.

SUBMITTER: Gonen-Korkmaz C

PROVIDER: S-EPMC4218634 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Analysis of tumor necrosis factor α-induced and nuclear factor κB-silenced LNCaP prostate cancer cells by RT-qPCR.

Gonen-Korkmaz Ceren C Sevin Gulnur G Gokce Goksel G Arun Mehmet Zuhuri MZ Yildirim Gokce G Reel Buket B Kaymak Aysegul A Ogut Deniz D

Experimental and therapeutic medicine 20141017 6

Prostate cancer is the second leading cause of morbidity and mortality in males in the Western world. In the present study, LNCaP, which is an androgen receptor-positive and androgen-responsive prostate cancer cell line derived from lymph node metastasis, and DU145, which is an androgen receptor-negative prostate cancer cell line derived from brain metastasis, were investigated. TNFα treatment decreased p105 and p50 expression and R1881 treatment slightly decreased p105 expression but increased ...[more]

PMID: 25371717

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Project description:BackgroundThe humoral system is activated and various cytokines are released due to infections in tissues and traumatic damage. Nuclear factor-kappa B dimers are encoded by nuclear factor-kappa B genes and regulate transcription of several crucial proteins of inflammation such as tumour necrosis factor-alpha.AimsTo investigate the possible effect of polymorphisms on tumour necrosis factor-alpha serum levels with clinical and prognostic parameters of sepsis by determining the nuclear factor-kappa B-1-94 ins/del ATTG and tumour necrosis factor-alpha (-308 G/A) gene polymorphisms and tumour necrosis factor-alpha serum levels.Study designCase-control study.MethodsSeventy-two patients with sepsis and 104 healthy controls were included in the study. In order to determine the polymorphisms of nuclear factor-kappa B-1-94 ins/del ATTG and tumour necrosis factor-alpha (-308 G/A), polymerase chain reaction-restriction fragment length polymorphism analysis was performed and serum tumour necrosis factor-alpha levels were determined using an enzyme-linked immunosorbent assay.ResultsWe observed no significant differences in tumour necrosis factor-alpha serum levels between the study groups. In the patient group, an increase in the tumour necrosis factor-alpha serum levels in patients carrying the tumour necrosis factor-alpha (-308 G/A) A allele compared to those without the A allele was found to be statistically significant. Additionally, an increase in the tumour necrosis factor-alpha serum levels in patients carrying tumour necrosis factor-alpha (-308 G/A) AA genotype compared with patients carrying the AG or GG genotypes was statistically significant. No significant differences were found in these 2 polymorphisms between the patient and control groups (p>0.05).ConclusionOur results showed the AA genotype and the A allele of the tumour necrosis factor-alpha (-308 G/A) polymorphism may be used as a predictor of elevated tumour necrosis factor-alpha levels in patients with sepsis.

Project description:BackgroundTumor necrosis factor α-induced protein 1 (TNFAIP1) is frequently downregulated in cancer cell lines and promotes cancer cell apoptosis. However, its role, clinical significance and molecular mechanisms in hepatocellular carcinoma (HCC) are unknown.MethodsThe expression of TNFAIP1 in HCC tumor tissues and cell lines was measured by Western blot and immunohistochemistry. The effects of TNFAIP1 on HCC proliferation, apoptosis, metastasis, angiogenesis and tumor formation were evaluated by Cell Counting Kit-8 (CCK8), Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL), transwell, tube formation assay in vitro and nude mice experiments in vivo. The interaction between TNFAIP1 and CSNK2B was validated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), Co-immunoprecipitation and Western blot. The mechanism of how TNFAIP1 regulated nuclear factor-kappaB (NF-κB) pathway was analyzed by dual-luciferase reporter, immunofluorescence, quantitative Real-time polymerase chain reaction (RT-qPCR) and Western blot.FindingsThe TNFAIP1 expression is significantly decreased in HCC tissues and cell lines, and negatively correlated with the increased HCC histological grade. Overexpression of TNFAIP1 inhibits HCC cell proliferation, metastasis, angiogenesis and promotes cancer cell apoptosis both in vitro and in vivo, whereas the knockdown of TNFAIP1 in HCC cell displays opposite effects. Mechanistically, TNFAIP1 interacts with CSNK2B and promotes its ubiquitin-mediated degradation with Cul3, causing attenuation of CSNK2B-dependent NF-κB trans-activation in HCC cell. Moreover, the enforced expression of CSNK2B counteracts the inhibitory effects of TNFAIP1 on HCC cell proliferation, migration, and angiogenesis in vitro and in vivo.InterpretationOur results support that TNFAIP1 can act as a tumor suppressor of HCC by modulating TNFAIP1/CSNK2B/NF-κB pathway, implying that TNFAIP1 may represent a potential marker and a promising therapeutic target for HCC.

Project description:BackgroundCirculating lipopolysaccharide (LPS) concentrations are often elevated in patients with sepsis or with various endogenous diseases that are associated with metabolic endotoxemia. Involuntary loss of skeletal muscle, termed muscle wasting, is commonly observed in these conditions, suggesting that circulating LPS might play an essential role in its development. Although impairment of muscle regeneration is an important determinant of skeletal muscle wasting, it is unclear whether LPS affects this process and, if so, by what mechanism. Here, we used the C2C12 myoblast cell line to investigate the effects of LPS on myogenesis.MethodsC2C12 myoblasts were grown to 80% confluence and induced to differentiate in the absence or presence of LPS (0.1 or 1 μg/mL); TAK-242 (1 μM), a specific inhibitor of Toll-like receptor 4 (TLR4) signaling; and a tumor necrosis factor (TNF)-α neutralizing antibody (5 μg/mL). Expression of a skeletal muscle differentiation marker (myosin heavy chain II), two essential myogenic regulatory factors (myogenin and MyoD), and a muscle negative regulatory factor (myostatin) was analyzed by western blotting. Nuclear factor-κB (NF-κB) DNA-binding activity was measured using an enzyme-linked immunosorbent assay.ResultsLPS dose-dependently and significantly decreased the formation of multinucleated myotubes and the expression of myosin heavy chain II, myogenin, and MyoD, and increased NF-κB DNA-binding activity and myostatin expression. The inhibitory effect of LPS on myogenic differentiation was reversible, suggesting that it was not caused by nonspecific toxicity. Both TAK-242 and anti-TNF-α reduced the LPS-induced increase in NF-κB DNA-binding activity, downregulation of myogenic regulatory factors, and upregulation of myostatin, thereby partially rescuing the impairment of myogenesis.ConclusionsOur data suggest that LPS inhibits myogenic differentiation via a TLR4-NF-κB-dependent pathway and an autocrine/paracrine TNF-α-induced pathway. These pathways may be involved in the development of muscle wasting caused by sepsis or metabolic endotoxemia.

Project description:Background: Tumor necrosis factor α-induced protein 1 (TNFAIP1) is frequently downregulated in cancer cell lines and promotes cancer cell apoptosis. However, its role, clinical significance and molecular mechanisms in hepatocellular carcinoma (HCC) are unknown. Methods: The expression of TNFAIP1 in HCC tumor tissues and cell lines was measured by Western blot and immunohistochemistry. The effects of TNFAIP1 on HCC proliferation, apoptosis, metastasis, angiogenesis and tumor formation were evaluated by Cell Counting Kit-8 (CCK8), Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL), transwell, tube formation assay in vitro and nude mice experiments in vivo. The interaction between TNFAIP1 and CSNK2B was validated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), Co-immunoprecipitation and Western blot. The mechanism of how TNFAIP1 regulated nuclear factor-kappaB (NF-κB) pathway was analyzed by dual-luciferase reporter, immunofluorescence, quantitative Real-time polymerase chain reaction (RT-qPCR) and Western blot. Findings: The TNFAIP1 expression is significantly decreased in HCC tissues and cell lines, and negatively correlated with the increased HCC histological grade. Overexpression of TNFAIP1 inhibits HCC cell proliferation, metastasis, angiogenesis and promotes cancer cell apoptosis both in vitro and in vivo, whereas the knockdown of TNFAIP1 in HCC cell displays opposite effects. Mechanistically, TNFAIP1 interacts with CSNK2B and promotes its ubiquitin-mediated degradation with Cul3, causing attenuation of CSNK2B-dependent NF-κB trans-activation in HCC cell. Moreover, the enforced expression of CSNK2B counteracts the inhibitory effects of TNFAIP1 on HCC cell proliferation, migration, and angiogenesis in vitro and in vivo. Interpretation: Our results support that TNFAIP1 can act as a tumor suppressor of HCC by modulating TNFAIP1/CSNK2B/NF-κB pathway, implying that TNFAIP1 may represent a potential marker and a promising therapeutic target for HCC. Fund: This work was supported in part by the financial support from the China Natural Science Foundation (No. 81972642, No. 81601122 and No. 81770389), Hunan Natural Science Foundation (No. 2017JJ3205), Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (No. 20134486), and the Scientific Research Fund of Hunan Provincial Education Department (17B162).

Dataset Information

Analysis of tumor necrosis factor ?-induced and nuclear factor ?B-silenced LNCaP prostate cancer cells by RT-qPCR.

Publications

Analysis of tumor necrosis factor α-induced and nuclear factor κB-silenced LNCaP prostate cancer cells by RT-qPCR.

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