急性白血病细胞中JAK/STAT5和PI3K/AKT信号通路对eIF4B的协同调控作用

人类急性白血病(Acute leukemia,AL)是一类造血干细胞异常的克隆性恶性疾病。在临床上,急性白血病由于发病急、病程短等原因使其非常难以治愈。已有研究表明,慢性白血病的发生与真核转译起始因子4B(Eukaryotic initiation factor 4B,eIF4B)的活化密切相关,但是其在急性白血病发生中的作用尚不明确。为了探究eIF4B在急性白血病发生中的作用及其机理,利用PI3K抑制剂LY294002、AKT抑制剂AKTi以及Pim抑制剂SMI-4A特异性地分别阻断JAK/STAT5/Pim和PI3K/AKT/mTOR信号通路,检测这两条信号通路下游共同靶标分子eIF4B的磷酸化水平。研究发现,阻断一条信号通路可明显降低eIF4B的磷酸化水平,而同时阻断两条信号通路能够更为显著地降低eIF4B活性并以一种协同作用的方式诱导细胞发生凋亡。进一步通过检测细胞凋亡和裸鼠致瘤实验,发现干扰eIF4B表达抑制了急性白血病细胞的存活及其在裸鼠体内的肿瘤形成。此外,敲低eIF4B可显著降低抗凋亡蛋白Bcl-2和Bcl-XL的蛋白表达水平。综上所述,在急性白血病细胞中eIF4B的活...     

Bone marrow cells are differentiated into MDSCs by BCC-Ex through down-regulating the expression of CXCR4 and activating STAT3 signalling pathway

Studies showed that the increase of myeloid-derived suppressor cells (MDSCs) in tumour microenvironment is closely related to the resistant treatment and poor prognosis of metastatic breast cancer. However, the effect of tumour-derived exosomes on MDSCs and its mechanism are not clear. Here, we reported that breast cancer cells (4T1)-secreted exosomes (BCC-Ex) were able to differentiate bone marrow cells into MDSCs and significantly inhibited the proliferation of T lymphocytes to provide an immunosuppressive microenvironment for cancer cells in vivo and in vitro. The number of MDSCs in bone marrow and spleen of 4T1 tumour-bearing mice and BCC-Ex infused mice was significantly higher than that of normal mice, whereas the number of T lymphocytes in spleen was significantly decreased. In addition, BCC-Ex markedly promoted the differentiation of MDSCs from bone marrow cells or bone marrow cells derived macrophages, seen as the increased expressions of MDSCs-related functional proteins Arginase-1 (Arg-1) and inducible nitric oxide synthase (iNOS). Furthermore, BCC-Ex significantly down-regulated the expressions of chemokine receptor CXCR4 and markedly up-regulated the levels of inflammatory cytokines IL-6 and IL-10 in bone marrow cells and macrophages and remarkably inhibited the division and proliferation of T cells. Importantly, CXCR4 agonist, CXCL12, could reverse the function of BCC-Ex, indicating that BCC-Ex-induced MDSCs might be dependent on the down-regulation of CXCR4. Western blot showed that BCC-Ex significantly promoted the phosphorylation of STAT3 in bone marrow cells, resulting in the inhibitions of the proliferation and apoptosis of bone marrow cells, and the aggravation of the differentiation of bone marrow cells into MDSCs.     

The human perforin gene is a direct target of STAT4 activated by IL-12 in NK cells

IL-12 activates STAT4 by inducing tyrosine phosphorylation, homo-dimerization, and nuclear translocation in NK cells and thereby stimulates proliferation and activation of these cells. The pore-forming protein perforin is a key effector protein for NK cell- and cytotoxic T lymphocyte-mediated cytolysis. Here we demonstrate that IL-12 induces the expression of the perforin gene in human NK cell line, NKL. Electrophoretic mobility shift assays using a probe containing two putative STAT-binding sequences located at -1085 and -1059 in the human perforin gene showed that STAT4 or STAT5 activated by IL-12 or IL-2, respectively, in NKL cells binds this region. Further analyses using various probes with or without mutated STAT-binding sequences showed that, although either of the two tandem STAT-binding sequences binds STAT4 weakly, the presence of both is required for significant binding of activated STAT4 and for formation of the STAT4-DNA-binding complex with lower electrophoretic mobility. Furthermore, mutation of either of the tandem STAT-binding sequences abolished the IL-12-induced activation of the perforin gene promoter in reporter gene assays. These results indicate that the IL-12-induced expression of the perforin gene in NK cells is directly regulated by STAT4, which binds, most likely as a homo-tetramer, to the tandem STAT-binding sequences in the perforin gene promoter.     

Anti-apoptotic effects of SERPIN B3 and B4 via STAT6 activation in macrophages after infection with Toxoplasma gondii

Toxoplasma gondii penetrates all kinds of nucleated eukaryotic cells but modulates host cells differently for its intracellular survival. In a previous study, we found out that serine protease inhibitors B3 and B4 (SERPIN B3/B4 because of their very high homology) were significantly induced in THP-1-derived macrophages infected with T. gondii through activation of STAT6. In this study, to evaluate the effects of the induced SERPIN B3/B4 on the apoptosis of T. gondii-infected THP-1 cells, we designed and tested various small interfering (si-) RNAs of SERPIN B3 or B4 in staurosporine-induced apoptosis of THP-1 cells. Anti-apoptotic characteristics of THP-1 cells after infection with T. gondii disappeared when SERPIN B3/B4 were knock-downed with gene specific si-RNAs transfected into THP-1 cells as detected by the cleaved caspase 3, poly-ADP ribose polymerase and DNA fragmentation. This anti-apoptotic effect was confirmed in SERPIN B3/B4 overexpressed HeLa cells. We also investigated whether inhibition of STAT6 affects the function of SERPIN B3/B4, and vice versa. Inhibition of SERPIN B3/B4 did not influence STAT6 expression but SERPIN B3/B4 expression was inhibited by STAT6 si-RNA transfection, which confirmed that SERPIN B3/B4 was induced under the control of STAT6 activation. These results suggest that T. gondii induces SERPIN B3/B4 expression via STAT6 activation to inhibit the apoptosis of infected THP-1 cells for longer survival of the intracellular parasites themselves.     

Tannic acid inhibits EGFR/STAT1/3 and enhances p38/STAT1 signalling axis in breast cancer cells

Tannic acid (TA), a naturally occurring polyphenol, is a potent anti‐oxidant with anti‐proliferative effects on multiple cancers. However, its ability to modulate gene‐specific expression of tumour suppressor genes and oncogenes has not been assessed. This work investigates the mechanism of TA to regulate canonical and non‐canonical STAT pathways to impose the gene‐specific induction of G1‐arrest and apoptosis. Regardless of the p53 status and membrane receptors, TA induced G1‐arrest and apoptosis in breast cancer cells. Tannic acid distinctly modulated both canonical and non‐canonical STAT pathways, each with a specific role in TA‐induced anti‐cancer effects. Tannic acid enhanced STAT1 ser727 phosphorylation via upstream serine kinase p38. This STAT1 ser727 phosphorylation enhanced the DNA‐binding activity of STAT1 and in turn enhanced expression of p21^Waf1/Cip1. However, TA binds to EGF‐R and inhibits the tyrosine phosphorylation of both STAT1 and STAT3. This inhibition leads to the inhibition of STAT3/BCL‐2 DNA‐binding activity. As a result, the expression and mitochondrial localization of BCl‐2 are declined. This altered expression and localization of mitochondrial anti‐pore factors resulted in the release of cytochrome c and the activation of intrinsic apoptosis cascade involving caspases. Taken together, our results suggest that TA modulates EGF‐R/Jak2/STAT1/3 and P38/STAT1/p21^Waf1/Cip1 pathways and induce G1‐arrest and intrinsic apoptosis in breast carcinomas.     

GABPalpha regulates Oct-3/4 expression in mouse embryonic stem cells

There is a dire need for novel therapeutics to treat the virulent malarial parasite, Plasmodium falciparum. Recently, the X-ray crystal structure of enoyl-acyl carrier protein reductase (ENR) in complex with triclosan has been determined and provides an opportunity for the rational design of novel inhibitors targeting the active site of ENR. Here, we report the discovery of several compounds by virtual screening and their experimental validation as high potency PfENR inhibitors.     

The Pros1/Tyro3 axis protects against periodontitis by modulating STAT/SOCS signalling

Periodontitis, an oral inflammatory disease caused by periodontal pathogen infection, is the most prevalent chronic inflammatory disease and a major burden on healthcare. The TAM receptor tyrosine kinases (Tyro3, Axl and Mertk) and their ligands (Gas6 and Pros1) play a pivotal role in the resolution of inflammation and have been associated with chronic inflammatory and autoimmune diseases. In this study, we evaluated the effects of exogenous Pros1 in in vitro and in vivo models of periodontitis. We detected higher Pros1 but lower Tyro3 levels in inflamed gingival specimens of periodontitis patients compared with healthy controls. Moreover, Pros1 was mostly localized in the gingival epithelium of all specimens. In cultured human gingival epithelial cells (hGECs), Porphyromonas gingivalis LPS (p.g‐LPS) stimulation down‐regulated Pros1 and Tyro3. Exogenous Pros1 inhibited p.g‐LPS–induced production of TNF‐α, IL‐6, IL‐1β, MMP9/2 and RANKL in a Tyro3‐dependent manner as revealed by PCR, Western blot analysis, ELISA and gelatin zymography. Pros1 also restored Tyro3 expression down‐regulated by p.g‐LPS in hGECs. In rats treated with ligature and p.g‐LPS, administration of Pros1 attenuated periodontitis‐associated gingival inflammation and alveolar bone loss. Our mechanistic studies implicated SOCS1/3 and STAT1/3 as mediators of the in vitro and in vivo anti‐inflammatory effects of Pros1. Collectively, the findings from this work supported Pros1 as a novel anti‐inflammatory therapy for periodontitis.     

Scutellarin inhibits proliferation and invasion of hepatocellular carcinoma cells via down‐regulation of JAK2/STAT3 pathway

The prognosis of hepatocellular carcinoma (HCC) is poor because of high incidence of recurrence and metastasis. JAK/STAT signalling pathway regulates cell proliferation, apoptosis, differentiation and migration and epithelial‐mesenchymal transition (EMT) is also considered to contribute to invasion and metastasis of epithelial malignant tumours. Scutellarin is an active component found in many traditional Chinese herbs and has been regularly used in anti‐inflammatory and antitumour medicine. This study aimed to identify the effect of scutellarin and its possible mechanism of action in HCC cells. Proliferation, colony‐forming, apoptosis and cell migration assays were used to examine the effect of scutellarin on HCC cells. Quantitative real‐time PCR and Western blotting were performed to study the molecular mechanisms of action of scutellarin. Light and electron microscopy and immunofluorescence analysis were performed to study the effect of scutellarin on cellular mechanics. We show that scutellarin potentially suppresses invasiveness of HepG2 and MHCC97‐H cells in vitro by remodelling their cytoskeleton. The molecular mechanism behind it might be the inhibition of the EMT process, which could be attributed to the down‐regulation of the JAK2/STAT3 pathway. These findings may provide new clinical ideas for the treatment of liver cancer.     

Development of a STAT3 reporter prostate cancer cell line for high throughput screening of STAT3 activators and inhibitors

STAT3 is constitutively activated in several cancers, including prostate cancer, and is therefore, a potential target for cancer therapy. DU-145 prostate cancer cells were stably co-transfected with STAT3 reporter and puromycin resistant plasmids to create a stable STAT3 reporter cell line that can be used for high throughput screening of STAT3 modulators. The applicability of this cell line was tested with two known activators and inhibitors of STAT3. As expected, EGF and IL-6 increased STAT3 reporter activity and enhanced the nuclear localization of phosphorylated STAT3 (pSTAT3); whereas Cucurbitacin I and AG490 decreased STAT3 reporter activity dose and time-dependently and reduced the localization of pSTAT3 in the nuclei of prostate cancer cells. Given the importance of STAT3 in cancer initiation and progression, the development of a stable STAT3 reporter cell line in prostate cancer cells provides a rapid, sensitive, and cost effective method for the screening of potential STAT3 modulators.