Expression Of NF-κB Subunit P65 Research Articles

PIM1 inhibitor SMI-4a attenuated lipopolysaccharide-induced acute lung injury through suppressing macrophage inflammatory responses via modulating p65 phosphorylation

PIM kinase is involved in the cellular processes of growth, differentiation and apoptosis. However, the role of PIM1 in lipopolysaccharide (LPS)-induced acute lung injury (ALI) remains largely unknown. A trend of PIM1 in the lung tissue of LPS-induced ALI at different time points was detected. Histology, wet/dry (W/D) ratio, inflammatory cells in the bronchoalveolar lavage fluid (BALF) and survival rate analyses were performed when mice received the PIM1 inhibitor SMI-4a intratracheally 3 h before LPS administration. Cytokine production in vivo and in vitro was measured after SMI-4a pretreatment. NF-κB subunit p65 expression in nuclei and phosphorylation at Ser276 in lung tissues or cells were detected by Western blot analysis. The results showed that PIM1 mRNA and protein were upregulated in the lung tissue of LPS-induced ALI. The PIM1 inhibitor SMI-4a markedly improved the survival rate after lethal LPS administration, reduced the severity of lung edema, attenuated the histologic injuries of the lung tissue and reduced the counts of infiltrated inflammatory cells in the BALF. The PIM1 inhibitor SMI-4a suppressed the production of cytokines in LPS-treated RAW264.7 cell supernatants and BALF. Furthermore, LPS administration upregulated the levels of nuclear p65 and phosphorylated p65 (p-p65) at Ser276, whereas pretreatment with the PIM1 inhibitor SMI-4a reduced p65 upregulation in the nucleus and p-p65 at Ser276. Taken together, these data indicate that the PIM1 inhibitor SMI-4a may serve as a promising therapeutic strategy for LPS-induced ALI by suppressing macrophage production of cytokines via a reduction of p65 activities.

P16-specific DNA methylation by engineered zinc finger methyltransferase inactivates gene transcription and promotes cancer metastasis

BackgroundP16 DNA methylation is well known to be the most frequent event in cancer development. It has been reported that genetic inactivation of P16 drives cancer growth and metastasis, however, whether P16 DNA methylation is truly a driver in cancer metastasis remains unknown.ResultsA P16-specific DNA methyltransferase (P16-dnmt) expression vector is designed using a P16 promoter-specific engineered zinc finger protein fused with the catalytic domain of dnmt3a. P16-dnmt transfection significantly decreases P16 promoter activity, induces complete methylation of P16 CpG islands, and inactivates P16 transcription in the HEK293T cell line. The P16-Dnmt coding fragment is integrated into an expression controllable vector and used to induce P16-specific DNA methylation in GES-1 and BGC823 cell lines. Transwell assays show enhanced migration and invasion of these cancer cells following P16-specific DNA methylation. Such effects are not observed in the P16 mutant A549 cell line. These results are confirmed using an experimental mouse pneumonic metastasis model. Moreover, enforced overexpression of P16 in these cells reverses the migration phenotype. Increased levels of RB phosphorylation and NFκB subunit P65 expression are also seen following P16-specific methylation and might further contribute to cancer metastasis.ConclusionP16 methylation could directly inactivate gene transcription and drive cancer metastasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0819-6) contains supplementary material, which is available to authorized users.

P53 amplifies Toll-like receptor 5 response in human primary and cancer cells through interaction with multiple signal transduction pathways.

The p53 tumor suppressor regulates transcription of genes associated with diverse cellular functions including apoptosis, growth arrest, DNA repair and differentiation. Recently, we established that p53 can modulate expression of Toll-like receptor (TLR) innate immunity genes but the degree of cross-talk between p53 and TLR pathways remained unclear. Here, using gene expression profiling we characterize the global effect of p53 on the TLR5-mediated transcription in MCF7 cells. We found that combined activation of p53 and TLR5 pathways synergistically increases expression of over 200 genes, mostly associated with immunity and inflammation. The synergy was observed in several human cancer cells and primary lymphocytes. The p53-dependent amplification of transcriptional response to TLR5 activation required expression of NFκB subunit p65 and was mediated by several molecular mechanisms including increased phosphorylation of p38 MAP kinase, PI3K and STAT3 signaling. Additionally, p53 induction increased cytokine expression in response to TNFα, another activator of NFκB and MAP kinase pathways, suggesting a broad interaction between p53 and these signaling pathways. The expression of many synergistically induced genes is elevated in breast cancer patients responsive to chemotherapy. We suggest that p53's capacity to enhance immune response could be exploited to increase antitumor immunity and to improve cancer treatment.

Effect of casein glycomacropeptide on subunit p65 of nuclear transcription factor-κB in lipopolysaccharide-stimulated human colorectal tumor HT-29 cells

ObjectiveThe effect of casein glycomacropeptide (CGMP) on the expression of NF-κB subunit p65 in human colorectal cancer HT-29 cells induced by lipopolysaccharide (LPS) was investigated to explore the therapeutic efficacy of CGMP for human colorectal cancer. MethodsHT-29 cells cultured in 96-well plates were stimulated with LPS for 30min at the concentrations of 0, 0.001, 0.01, 0.1, 1 and 10μg/mL. After stimulation, the expression of p65 in HT-29 cells was evaluated by immunofluorescence method. Similarly, HT-29 cells were incubated with 0, 0.001, 0.01, 0.1, 1, 10 and 100μg/mL CGMP for 24h at the optimal LPS concentration. After treatment with CGMP under the incubation with optimal LPS concentration for 30min, the protein expression of p65 was analyzed by Western blotting. The optimum culture time was evaluated by incubating HT-29 cells with the optimal CGMP concentration for 6, 12, 24, 48 and 72h. ResultsNF-κB-p65 revealed that the highest protein expression was achieved with 1μg/mL LPS treatment. Meanwhile, CGMP could inhibit the protein expression of NF-κB-p65 in LPS-stimulated HT-29 cells, and the optimal inhibitory effect was observed at a CGMP concentration of 0.01μg/mL with 48h incubation. ConclusionCGMP can regulate NF-κB signaling pathway through inhibiting the expression of its subunit p65, which is beneficial for the further improvement of human colorectal cancer treatment.

Effects of LPS on NF-κB p65 and anti-inflammatory cytokines mRNA expression in MSC (P1096)

Abstract Abstract Objective To investigation the effects of Inflammatory simulated by LPS on mRNA expression of NF-κB p65, anti-inflammatory cytokines (IL-10, TGF-β3) and IDO in rat bone-marrow MSC. Methods The MSC cultured in vitro was divided into 6 groups: The normal group, the PDTC(100μmol/L) treatment group, the PDTC(100μmol/L)plus LPS(5µg/mL) treatment group and the 3 LPS dose (1, 5, 10μg/mL) treatment groups. After LPS supplement for 4 hr, the total RNA of the MSC was extracted and the mRNA expression of p65, IL-10, TGF-β3 and IDO were detected by RT-PCR. The gray scale was used to compare the mean levels (X±S) of PCR product within groups were determined. Results Compared with the control group, the mRNA expression of NF-κB Subunit p65 and anti-inflammatory cytokines IL-10, TGF-β3 were significant increased in the LPS treated groups (P <0.05). The correlation coefficient between p65 and IL-10, TGF-β3 were 0.844(P<0.05), 0.837(P<0.05) respectively. The correlation coefficient of p65 with IDO was -0.942 (P<0.05). Conclusion 1 LPS stimulation could induce MSC produce more anti-inflammatory cytokines through the activation of NF-κB. 2 IDO was constitutively expressed in normal cultured MSC, and its expression may be down regulated by inflammatory stimulation. This result indicates that there are different immune-modulation pathways in MSC under different microenvironments. Key words MSC; NF-κB; IL-10; TGF β3; IDO

Resveratrol reverses temozolomide resistance by downregulation of MGMT in T98G glioblastoma cells by the NF-κB-dependent pathway

Glioblastoma multiforme (GBM) is the most common intracranial tumor, with a dismal prognosis. Although temozolomide (TMZ)-based chemotherapy following neurosurgery has been proven to be effective, not all patients benefit clinically because of TMZ resistance. Given that protein expression of O(6)-methylguanine-DNA-methyltransferase (MGMT) is the most important determinant of TMZ resistance, great efforts have been made to suppress it by regulating MGMT-related transcription factors. The study presented here demonstrates that resveratrol, a natural polyphenol, is able to reverse TMZ resistance of glioblastoma T98G cells which have relatively high MGMT activity. The data showed that combination treatment with TMZ and resveratrol resulted in an enhanced antitumor potential of TMZ, decreased the 50% inhibiting concentration (IC50) of TMZ and increased the induction of apoptosis in TMZ-resistant T98G cells. Hoechst 33258 staining revealed increased apoptotic morphology, such as chromatin aggregation and nuclear and cytoplasmic condensation, in cells receiving combination treatment. Western blot analysis manifested a significant decreased intracellular content and nuclear translocation of NF-κB and increased cleavage of caspase-3 in cells exposed to combination treatment, compared to those in cells treated with TMZ alone. In addition, recombinant expression of NF-κB subunit p65 remarkably promoted nuclear translocation of NF-κB and abolished the TMZ-resistance reversal induced by combination treatment, suggesting an underlying NF-κB-dependent mechanism. Our study improved the knowledge on the mechanism of TMZ resistance and suggested a novel strategy for TMZ-based chemotherapy in glioblastoma patients.