灵芝多糖对AD模型大鼠海马组织Caspase-3和FasL表达的影响

目的研究灵芝多糖（GLP）对阿尔茨海默病（AD）大鼠海马组织半胱氨酰天冬氨酸特异性蛋白静3（caspase-3）和FasL基因以及空间学习记忆能力的影响。方法双侧海马内一次性注射淀粉样多肽25-35片段（Aβ25—35）制作大鼠AD模型,治疗组24h后腹腔注射灵芝多糖水溶液,1周后进行Morris水迷宫检测大鼠空间学习记忆能力变化。采用免疫组织化学法和逆转录-聚合酶链式反应法（RT-PCR）分别检测大鼠海马组织caspase-3蛋白的表达量和FasL基因的表达,以及灵芝多糖对上述各指标的影响。结果灵芝多糖能明显改善AD模型大鼠低下的空间学习记忆能力。显著降低模型大鼠海马组织caspase-3和FasL的表达,而且灵芝多糖能明显改善模型大鼠脑组织海马CAI区神经元的退行性变化。结论灵芝多糖能降低海马组织内FasL和caspase-3表达量,改善海马CAI区神经元的退行性变化。对老年性痴呆大鼠学习记忆能力可能有增强和提高作用。     

内耳免疫反应诱导Fas和FasL表达与凋亡的关系

目的研究内耳免疫反应过程中是否存在细胞凋亡,以及细胞凋亡是否与Fas和FasL信号转导有关.方法选用雌性白色豚鼠16只,随机分为实验组和对照组各8只,以钥孔虫戚血蓝蛋白(keyhole limpet hemocyanin,KLH)全身免疫后,实验组以相同抗原进行内耳免疫,对照组内耳注射等量的磷酸盐缓冲生理盐水(phosphate buffered saline,PBS),在内耳免疫5d后处死动物,取内耳免疫侧耳蜗做石蜡切片.通过脱氧核糖核苷酸末端转移酶介导的缺口末端标记技术(terminal-deoxynucleotidyl transferase mediated nick end labeling,TUNEL)检测内耳凋亡细胞,免疫组化检测内耳Fas和FasL的表达.结果实验组豚鼠内耳Corti器毛细胞,血管纹的缘细胞和螺旋神经节细胞存在TUNEL染色阳性细胞,而对照组动物切片仅在支持细胞、血管纹和螺旋神经节细胞中发现极少数TUNEL染色阳性细胞.免疫组化染色实验组Corti器、螺旋神经节细胞、血管纹和螺旋韧带Fas和FasL蛋白表达阳性,而对照组只有螺旋神经节细胞和血管纹有较弱的Fas蛋白表达,FasL蛋白表达阴性.结论内耳免疫反应可诱导细胞凋亡的发生,Fas-FasL途径是参与此过程重要的信号转导途径之一.     

Advanced glycation end-product (AGE) induces apoptosis in human retinal ARPE-19 cells via promoting mitochondrial dysfunction and activating the Fas-FasL signaling

Advanced glycation end-products (AGEs) are extremely accumulated in the retinal vascular and epithelial cells of diabetes mellitus (DM) patients, particularly with diabetic retinopathy (DR). To elucidate the pathogenesis of the AGE-induced toxicity to retinal epithelial cells, we investigated the role of Fas–Fas ligand (FasL) signaling and mitochondrial dysfunction in the AGE-induced apoptosis. Results demonstrated that the AGE-BSA- induced apoptosis of retinal ARPE-19 cells. And the AGE-BSA treatment caused mitochondrial dysfunction, via deregulating the B-cell lymphoma 2 (Bcl-2) signaling. Moreover, the Fas/FasL and its downstreamer Caspase 8 were promoted by the AGE-BSA treatment, and the exogenous α-Fas exacerbated the activation of Caspase 3/8. On the other side, the siRNA-mediated knockdown of Fas/FasL inhibited the AGE-BSA-induced apoptosis. Taken together, we confirmed the activation of Fas–FasL signaling and of mitochondrial dysfunction in the AGE-BSA-promoted apoptosis in retinal ARPE-19 cells, implying the important role of Fas–FasL signaling in the DR in DM.     

p38 inhibitor SB203580 sensitizes the resveratrol-induced apoptosis in human lung adenocarcinoma (A549) cells

Based on our recent findings that resveratrol, a natural plant polyphenol found in red grape skins as well as other food products, induces apoptosis via a caspase-independent intrinsic pathway in human lung adenocarcinoma cells, this study is designed to explore whether SB203580, a p38 inhibitor, potentiates the resveratrol-induced apoptosis of human lung adenocarcinoma (A549) cells. We found that pretreatment with SB203580 enhanced the resveratrol-induced apoptosis by accelerating the intrinsic apoptotic pathway including Bax activation, loss of mitochondrial membrane potential, and activation of both caspase-9 and -3. Although treatment with resveratrol alone did not induce caspase-8 activation, cotreatment with both SB203580 and resveratrol not only enhanced FasL cleavage but also activated caspase-8, indicating that the extrinsic apoptotic pathway may be involved in the synergistic effect. Collectively, we for the first time demonstrate that SB203580 synergistically enhances the resveratrol-induced apoptosis by accelerating Bax-mediated intrinsic pathway and initiating extrinsic pathway, suggesting a possible alternative therapeutic strategy for human lung cancer.     

A let-7/Fas double-negative feedback loop regulates human colon carcinoma cells sensitivity to Fas-related apoptosis

Interferon-γ (IFN-γ) is considered essential for the regulation of anti-tumor reactions as it sensitizes Fas-related apoptosis in HT29 cells, but the mechanism is unclear. In the current study, our data demonstrated that IFN-γ stimulation and Fas activation suppressed Dicer processing and let-7 microRNA biogenesis, while let-7 microRNA strongly inhibited Fas expression by directly targeting Fas mRNA. Accordingly, our results indicate that Fas and let-7 microRNAs form a double-negative feedback loop in IFN-γ and Fas induced apoptosis in colon carcinoma cell line HT29, which may be an important synergistic mechanism in anti-tumor immune response. We also found that a let-7 microRNA inhibitor increased Fas expression and sensitized cells to Fas-related apoptosis, which may have future implications in colon carcinoma therapy.     

Down-regulation of Fas-mediated apoptosis by plasma transglutaminase factor XIII that catalyzes fetal-specific cross-link of the Fas molecule

The Fas antigen, also designated as APO-1 or CD95, is a member of the tumor necrosis factor receptor superfamily and can mediate apoptotic cell death in various cells. We report here that blood coagulation factor XIII (plasma transglutaminase, fibrin stabilizing factor) inhibits apoptosis induced by a cytotoxic anti-Fas monoclonal antibody in Jurkat cells. When cells were treated with the antibody in fetal calf serum-containing media, higher-molecular-weight (180 K) polypeptides containing Fas molecule were detected by immunoblotting. Under conditions where the transglutaminase activity was eliminated or suppressed, the cross-link of Fas was not observed, and concurrently cell death was hastened. Moreover, an antibody against factor XIII strongly accelerated the Fas-mediated apoptosis. Furthermore, addition of partially purified factor XIII neutralized the apoptosis-promoting effect of anti-factor XIII antibody, indicating that this enzyme is involved in cross-link of Fas and down-regulates Fas-mediated apoptotic cell death. Significantly, the cross-link of Fas was seen only in fetal calf serum but not in newly-born calf serum, 1-year-old calf serum or adult bovine serum. These data suggest that plasma transglutaminase factor XIII may play a key role in fetal development of vertebrates via cross-link of Fas antigen.     

FasL regulatory B-cells during Mycobacterium tuberculosis infection and TB disease

Tuberculosis (TB) disease remains a major health crisis. Infection with Mycobacterium tuberculosis (M.tb) cause a range of diseases ranging from latent infection to active TB disease. This active state of the disease is characterised by the formation of granulomas (a physical barrier in the lung), a structure thought to protect the host by controlling the infection through preventing the growth of the bacilli. Subsequently, the surviving bacteria become inactive and in most cases, TB reactivation is prevented by the immune response of the host. B-cells perform numerous immunological functions beyond antibody production to positively regulate the response to pathogenic assault. A subgroup of B-cells with regulatory functions express death-inducing ligands, such as Fas ligand (FasL). Expression and interaction of the Fas receptor-ligand promotes the induction of apoptosis and the induction of T-cell tolerance. Here, we focus on the significance of B-cells by addressing their FasL phenotype and regulatory functions during TB, with reference to disease in humans, non-human primates and mice.     

Selective induction of cyclooxygenase-2 plays a role in lysophosphatidic acid regulated Fas ligand cell surface presentation

Previous studies found that lysophosphatidic acid (LPA) upregulated Fas ligand (FasL) presentation on the ovarian cancer cell surface and lead to apoptosis of activated lymphocytes. In this report, we investigated the role of selective induction of cyclooxygenase-2 (Cox-2) in FasL cell surface presentation stimulated by LPA. Ovarian cancer cells pretreated with general aspirin derivative acetylsalicylic acid and specific Cox-2 inhibitor (NS-398) before stimulation with LPA, FasL cell surface presentation was significantly blocked, so was the apoptosis of activated lymphocytes mediated by increasing FasL on the ovarian cancer cell surface. Using the specific inhibitors PD98059, AG1478 or dominant-negative epidermal-growth-factor receptor (EGFR-DN) plasmid, we found that the activation of ERK1/2 played a role in Cox-2 induction, and the transactivation of EGFR worked as an upstream signaling pathway in ERK1/2 phosphorylation. This study first revealed the selective induction of Cox-2 by LPA led to FasL presentation on ovarian cancer cell surface and provide cancer cell immune privilege, and might provide important information of Cox-2 in cancer progression and Cox-2 inhibitors' application in cancer chemoprevention.     

Disruption of IGF-1R signaling increases TRAIL-induced apoptosis: A new potential therapy for the treatment of melanoma

Resistance of cancer cells to apoptosis is dependent on a balance of multiple genetic and epigenetic mechanisms, which up-regulate efficacy of the surviving growth factor-receptor signaling pathways and suppress death-receptor signaling pathways. The Insulin-like Growth Factor-1 Receptor (IGF-1R) signaling pathway is highly active in metastatic melanoma cells by mediating downstream activation of PI3K-AKT and MAPK pathways and controlling general cell survival and proliferation. In the present study, we used human melanoma lines with established genotypes that represented different phases of cancer development: radial-growth-phase WM35, vertical-growth-phase WM793, metastatic LU1205 and WM9 [1]. All these lines have normal NRAS. WM35, WM793, LU1205 and WM9 cells have mutated BRAF (V600E). WM35 and WM9 cells express normal PTEN, while in WM793 cells PTEN expression is down-regulated; finally, in LU1205 cells PTEN is inactivated by mutation. Cyclolignan picropodophyllin (PPP), a specific inhibitor of IGF-1R kinase activity, strongly down-regulated the basal levels of AKT activity in WM9 and in WM793 cells, modestly does so in LU1205, but has no effect on AKT activity in the early stage WM35 cells that are deficient in IGF-1R. In addition, PPP partially down-regulated the basal levels of active ERK1/2 in all lines used, highlighting the role of an alternative, non-BRAF pathway in MAPK activation. The final result of PPP treatment was an induction of apoptosis in WM793, WM9 and LU1205 melanoma cells. On the other hand, dose-dependent inhibition of IGF-1R kinase activity by PPP at a relatively narrow dose range (near 500 nM) has different effects on melanoma cells versus normal cells, inducing apoptosis in cancer cells and G2/M arrest of fibroblasts. To further enhance the pro-apoptotic effects of PPP on melanoma cells, we used a combined treatment of TNF-Related Apoptosis-Inducing Ligand (TRAIL) and PPP. This combination substantially increased death by apoptosis for WM793 and WM9 cells, but did so only modestly for LU1205 cells with very high basal activity of AKT. The ultimate goal of this direction of research is the discovery of a new treatment method for highly resistant human metastatic melanomas. Our findings provide the rationale for further preclinical evaluation of this novel treatment.