RESUMO
Despite a previous report on less inflammatory responses in mice with an absence of the enhancer of zeste homologue 2 (Ezh2), a histone lysine methyltransferase of epigenetic regulation, using a lipopolysaccharide (LPS) injection model, proteomic analysis and cecal ligation and puncture (CLP), a sepsis model that more resembles human conditions was devised. As such, analysis of cellular and secreted protein (proteome and secretome) after a single LPS activation and LPS tolerance in macrophages from Ezh2 null (Ezh2flox/flox; LysM-Crecre/-) mice (Ezh2 null) and the littermate control mice (Ezh2fl/fl; LysM-Cre-/-) (Ezh2 control) compared with the unstimulated cells from each group indicated fewer activities in Ezh2 null macrophages, especially by the volcano plot analysis. Indeed, supernatant IL-1ß and expression of genes in pro-inflammatory M1 macrophage polarization (IL-1ß and iNOS), TNF-α, and NF-κB (a transcription factor) were lower in Ezh2 null macrophages compared with the control. In LPS tolerance, downregulated NF-κB compared with the control was also demonstrated in Ezh2 null cells. In CLP sepsis mice, those with CLP alone and CLP at 2 days after twice receiving LPS injection, representing sepsis and sepsis after endotoxemia, respectively, symptoms were less severe in Ezh2 null mice, as indicated by survival analysis and other biomarkers. However, the Ezh2 inhibitor improved survival only in CLP, but not LPS with CLP. In conclusion, an absence of Ezh2 in macrophages resulted in less severe sepsis, and the use of an Ezh2 inhibitor might be beneficial in sepsis.
Assuntos
Endotoxemia , Sepse , Animais , Humanos , Camundongos , Endotoxemia/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Epigênese Genética , Ligadura , Lipopolissacarídeos , Macrófagos/metabolismo , Camundongos Knockout , NF-kappa B/metabolismo , Proteômica , Punções , Sepse/genética , Sepse/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Dendritic cells (DCs) are the most potent antigen-presenting cells that have multifaceted functions in the control of immune activation and tolerance. Hyperresponsiveness and altered tolerogenicity of DCs contribute to the development and pathogenesis of system lupus erythematosus (SLE); therefore, DC-targeted therapies aimed at inducing specific immune tolerance have become of great importance for the treatment of SLE. This study developed a new nanoparticle (NP) containing a biodegradable PDMAEMA-PLGA copolymer for target-oriented delivery to DCs in situ. PDMAEMA-PLGA NPs provided sustained drug release and exhibited immunosuppressive activity in FLT3L and GM-CSF-derived bone marrow in conventional DCs (BM-cDCs). PDMAEMA-PLGA NPs improved dexamethasone capability to convert wild-type and Fcgr2b-/- BM-cDCs from an immunogenic to tolerogenic state, and BM-cDCs treated with dexamethasone-incorporated PDMAEMA-PLGA NPs (Dex-NPs) efficiently mediated regulatory T cell (Treg) expansion in vitro. Dex-NP therapy potentially alleviated lupus disease in Fcgr2b-/- mice by mediating Foxp3+ Treg expansion in an antigen-specific manner. Our findings substantiate the superior efficacy of DC-targeted therapy using the PDMAEMA-PLGA NP delivery system and provide further support for clinical development as a potential therapy for SLE. Furthermore, PDMAEMA-PLGA NP may be a versatile platform for DC-targeted therapy to induce antigen-specific immune tolerance to unwanted immune responses that occur in autoimmune disease, allergy, and transplant rejection.
Assuntos
Lúpus Eritematoso Sistêmico , Nanopartículas , Camundongos , Animais , Antígenos , Tolerância Imunológica , Lúpus Eritematoso Sistêmico/terapia , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Dexametasona/farmacologia , Células Dendríticas , Receptores de IgG/genéticaRESUMO
BACKGROUND: Abnormally high expression of the mammalian de novo lipogenesis (DNL) pathway in various cancer cells promotes cell over-proliferation and resistance to apoptosis. Inhibition of key enzymes in the DNL pathway, namely, ATP citrate lyase, acetyl-CoA carboxylase, and fatty acid synthase (FASN) can increase apoptosis without cytotoxicity to non-cancerous cells, leading to the search for and presentation of novel selective and powerful targets for cancer therapy. Previous studies reported that epistructured catechins, epigallocatechin gallate (EGCG) and epicatechin (EC) exhibit different mechanisms regarding a strong inducer of apoptosis in various cancer cell lines. Thus, the current study investigated the growth inhibitory effect of EGCG and EC, on the enzyme expression and activity of the DNL pathway, which leads to the prominent activity of carnitine palmitoyl transferase-1 (CPT-1) mediating apoptosis in HepG2 cells. METHODS: The cytotoxicity on HepG2 cells of EGCG and EC was determined by MTT assay. Cell death caused by apoptosis, the dissipation of mitochondrial membrane potential (MMP), and cell cycle arrest were then detected by flow cytometry. We further investigated the decrease of fatty acid levels associated with DNL retardation, followed by evaluation of DNL protein expression. Then, the negative inhibitory effect of depleted fatty acid synthesis on malonyl-CoA synthesis followed by regulating of CPT-1 activity was investigated. Thereafter, we inspected the enhanced reactive oxygen species (ROS) generation, which is recognized as one of the causes of apoptosis in HepG2 cells. RESULTS: We found that EGCG and EC decreased cancer cell viability by increasing apoptosis as well as causing cell cycle arrest in HepG2 cells. Apoptosis was associated with MMP dissipation. Herein, EGCG and EC inhibited the expression of FASN enzymes contributing to decreasing fatty acid levels. Notably, this decrease consequently showed a suppressing effect on the CPT-1 activity. We suggest that epistructured catechin-induced apoptosis targets CPT-1 activity suppression mediated through diminishing the DNL pathway in HepG2 cells. In addition, increased ROS production was found after treatment with EGCG and EC, indicating oxidative stress mechanism-induced apoptosis. The strong apoptotic effect of EGCG and EC was specifically absent in primary human hepatocytes. CONCLUSION: Our supportive evidence confirms potential alternative cancer treatments by EGCG and EC that selectively target the DNL pathway.
RESUMO
The de novo lipogenesis (DNL) pathway has been identified as a regulator of cancer progression and aggressiveness. Downregulation of key lipogenesis enzymes has been shown to activate apoptosis in cancerous cells. Epigallocatechin gallate (EGCG) inhibits cancer cell proliferation without causing cytotoxicity in healthy cells. The present study aimed to investigate the effects of EGCG on the promotion of apoptosis associated with the DNL pathway inhibition in cancer cells, both in vitro and in vivo. We observed that two colorectal cancer cell lines (HCT116 and HT-29) had a higher cytotoxic response to EGCG treatment than hepatocellular carcinoma cells, including HepG2 and HuH-7. EGCG treatment decreased cell viability and increased mitochondrial damage-triggered apoptosis in both HCT116 and HT-29 cancer cells. Additionally, we treated mice transplanted with HCT116 cells with 30 or 50 mg·kg-1 EGCG for 7 days to evaluate the apoptotic effects of EGCG treatment in a xenograft mouse model of cancer. We observed a decrease in intracellular fatty acid levels, which suggested that EGCG-induced apoptosis was associated with a decrease in fatty acid levels in cancer. Suppression of ATP synthesis by EGCG indicated that cell death induction in cancer cells could be mediated by shared components of the DNL and energy metabolism pathways. In addition, EGCG-induced apoptosis suppressed the expression of the phosphorylation protein kinase B and extracellular signal-regulated kinase 1/2 signaling proteins in tumors from xenografted mice. Cytotoxic effects in unaffected organs and tissues of the mouse xenograft model were absent upon EGCG treatment.