Apoptotic properties of polysaccharide isolated from fruiting bodies of medicinal mushroom Fomes fomentarius in human lung carcinoma cell line

Mushrooms are known to complement chemotherapy and radiation therapy by countering the side effects of cancer. Recently, there has been great interest in isolation of novel bioactive compounds from mushrooms due to their numerous health beneficial effects. Chemically water-extractable polysaccharide (MFKF-AP1β), with a molecular weight of 12 kDa, was isolated from fruiting bodies of mushroom Fomes fomentarius. In this research, we investigated the anti-tumor effects of MFKF-AP1β on human lung carcinoma A549 cells. Results showed that MFKF-AP1β markedly inhibited A549 cell growth in a dose-dependent manner based on the amount of lactate dehydrogenase (LDH) released and morphological alterations. In addition, MFKF-AP1β induced cellular apoptosis by causing single-stranded DNA breakage, as evidenced by apoptosis assay. Furthermore, MFKF-AP1β (25–100 μg/ml) significantly induced single-stranded DNA breakage in A549 cells, as shown by comet assay. Taken together, our results demonstrate that MFKF-AP1β has strong anti-tumor effects mediated through induction of apoptosis. Therefore, MFKF-AP1β could be useful in lung chemotherapy.     

IAP家族抑制衰老的骨髓间充质干细胞的凋亡

目的:探讨衰老的大鼠骨髓间充质干细胞凋亡抗性是否增加且是否通过凋亡抑制蛋白(IAP)家族介导。方法:用DMEM/F12中加入10%胎牛血清(FBS)的完全培养基培养大鼠骨髓间充质干细胞(BMSC)至第三代,使用D-半乳糖(D-gal)诱导成衰老的BMSC并用β-半乳糖苷酶染色做衰老鉴定。用肿瘤坏死因子α(TNF-α)和环己酰亚胺(CHX)共同处理正常对照组与衰老组的BMSC,通过贴壁细胞计数统计其生存率,并通过蛋白印迹法检测两组细胞中Cleaved PARP、Cleaved Caspase3蛋白、IAP家族成员c-IAP1、c-IAP2、XIAP以及衰老相关标志蛋白的水平。再用IAP抑制剂AT-406处理衰老的细胞,CCK-8检测处理组以及未处理组细胞的细胞活力。最后用凋亡抑制剂z-VAD-fmk和AT-406共同处理正常对照组与衰老组细胞,观察z-VAD-fmk能否减少IAP蛋白被抑制后所引起的细胞死亡。结果:与正常对照组相比,衰老的BMSC中由TNF-α和CHX共同诱导引起的细胞死亡数明显减少,凋亡标志物Cleaved PARP和Cleaved Caspase3蛋白的水平降低,而IAP家族成员c-IAP1、c-IAP2和XIAP的蛋白水平升高。用AT-406处理衰老BMSC后细胞的死亡数明显高于未处理的衰老BMSC组,通过z-VAD-fmk抑制凋亡通路后,AT-406处理的衰老细胞的死亡数减少。结论:衰老BMSC通过升高IAP家族蛋白水平从而提高对凋亡的抵抗能力。     

Prevention of TGF-β-induced apoptosis by interlukin-4 through Akt activation and p70S6K survival signaling pathways

In this study, we demonstrate that interleukin-4 (IL-4) protects human hepatocellular carcinoma (HCC) cell line Hep3B from apoptosis induced by transforming growth factor-β (TGF-β). Further investigation of IL-4-transduced signaling pathways revealed that both insulin response substrate 1 and 2 (IRS-1/-2) and extracellular signal-regulated kinase (ERK) pathways were activated after IL-4 stimulation. The IRS-1/-2 activation was accompanied by the activation of phosphotidylinositol-3-kinase (PI3K), leading to Akt and p70 ribosomal protein S6 kinase (p70S6K). Interestingly, a protein kinase C (PKC) inhibitor, Gö6976, inhibited the phosphorylation of Akt, suggesting that the Akt activation was PKC-dependent. Using specific inhibitors for PI3K or ERK, we demonstrated that the PI3K pathway, but not the ERK pathway, was required for protection. The constitutively active form of PI3K almost completely rescued TGF-β-induced apoptosis, further supporting the importance of the PI3K pathway in the protective effect of IL-4. Furthermore, a dominant negative Akt and/or Gö6976 only partially blocked the anti-apoptotic effect of IL-4. Similarly, rapamycin, which interrupted the activation of p70S6K, also only partially blocked the protective effect of IL-4. However, in the presence of both rapamycin and dominant negative Akt with or without Gö6976, IL-4 almost completely lost the anti-apoptotic effect, suggesting that both Akt and p70S6K pathways were required for the protective effect of IL-4 against TGF-β-induced apoptosis.     

单纯疱疹病毒2型潜伏相关转录体ORF2的表达及其抗凋亡作用

本文探讨了单纯疱疹病毒2型 (HSV-2)潜伏相关转录体 (LAT)的开放读码框2 (ORF2)在细胞中的表达, 及其对5-氟尿嘧啶 (5-FU)诱导的非洲绿猴肾细胞 (Vero)凋亡的影响。通过将重组质粒pEGPF-ORF2转染Vero细胞, 绿色荧光蛋白检测转染效率, RT-PCR验证目的基因的表达, 5-FU 诱导细胞凋亡, 通过荧光显微镜观察凋亡小体, Gimesa染色检测细胞核形态, MTT法检测细胞的存活率, DNA ladder片段分析, 结果表明, 转染后绿色荧光蛋白表达效率很高, RT-PCR验证有目的基因的转录。凋亡诱导后的细胞形态正常, MTT法分析活性率与正常无差异, 而显著高于空质粒组, DNA ladder未见凋亡条带。由此我们认为HSV-2 LAT ORF2 基因在Vero细胞中得到了高效表达, 并且具有抗5-FU诱导的凋亡作用。     

Carbon monoxide protects PC12 cells from peroxynitrite-induced apoptotic death by preventing the depolarization of mitochondrial transmembrane potential

Heme oxygenase-1 (HO-1), the rate-limiting enzyme in catalyzing heme degradation into biliverdin, free iron, and carbon monoxide (CO), serves as a protective enzyme against oxidative and nitrosative stresses. In the present study, we investigated the cytoprotective effects of HO-1 upregulation and its product CO against the peroxynitrite-induced PC12 cell death. PC12 cells treated with 3-morphoinosydonimine (SIN-1), a generator of peroxynitrite (ONOO-), underwent apoptotic cell death as evidenced by dissipation of mitochondrial transmembrane potential (DeltaPsim), release of mitochondrial cytochrome c into cytoplasm, cleavage of poly(ADP-ribose)polymerase and fragmentation of internucleosomal DNA. Pretreatment of PC12 cells with a low non-toxic concentration of SIN-1 (0.5 mM) induced HO-1 expression and abrogated the cell death caused by subsequent challenge with high dose SIN-1 (2.5 mM). Furthermore, pretreatment of PC12 cells with SnCl2, a potent inducer of HO-1 expression, increased endogenous production of CO (HO activity) and rescued the PC12 cells from peroxynitrite-induced apoptosis. The cytoprotective effect of SnCl2 was abolished when the HO activity was inhibited by zinc protoporphyrin IX (ZnPP IX). PC12 cells treated directly with the CO-releasing molecule, tricarbonyldichlororuthenium (II) dimer ([Ru(CO)3Cl2]2) became tolerant to the depolarization of DeltaPsim and apoptosis induced by high dose peroxynitrite. Taken together, these data demonstrate that the adaptive protection against peroxynitrite-induced apoptotic death in PC12 cells is mediated by CO formed as a consequence of HO-1 induction.     

斜纹夜蛾核多角体病毒p49基因在大肠杆菌中的表达及其在昆虫细胞中的表达时相分析

利用PCR方法从斜纹夜蛾核多角体病毒（SpltMNPV）基因组中扩增获得了细胞凋亡抑制基因〖STBX〗p49〖STBZ〗的完整ORF并将其克隆于pMD18T载体,其序列分析结果与文献报道一致。将基因重组于硫氧还蛋白融合表达载体pThioHis C,在大肠杆菌BL21(DE3)中获得了稳定表达,表达的P49融合蛋白占菌体总蛋白30%左右,主要以包涵体形式存在。分离纯化重组表达的SpltMNPV P49蛋白作为抗原,免疫家兔制备得到效价高于1∶10000的抗重组P49蛋白多克隆抗体。应用制备的抗体对受SpltMNPV感染的Sl细胞中P49蛋白的表达时相进行分析,结果显示P49蛋白在细胞感染后3h内便可检测到,并在整个感染期间维持着低水平表达。     

Inhibition of cell death by a novel 16.2 kD heat shock protein predominantly via Hsp90 mediated lipid rafts stabilization and Akt activation pathway

AlphaB-crystallin homology, heat stress induction and chaperone activity suggested that a previously encloned gene product is a novel small heat shock protein (Hsp16.2). Suppression of Hsp16.2 by siRNA sensitized cells to hydrogen peroxide or taxol induced cell-death. Over-expressing of Hsp16.2 protected cells against stress stimuli by inhibiting cytochrome c release from the mitochondria, nuclear translocation of AIF and endonuclease G, and caspase 3 activation. Recombinant Hsp16.2 protected mitochondrial membrane potential against calcium induced collapse in vitro indicating that Hsp16.2 stabilizes mitochondrial membrane systems. Hsp16.2 formed self-aggregates and bound to Hsp90. Inhibition of Hsp90 by geldanamycin diminished the cytoprotective effect of Hsp16.2 indicating that this effect was Hsp90-mediated. Hsp16.2 over-expression increased lipid rafts formation as demonstrated by increased cell surface labeling with fluorescent cholera toxin B, and increased Akt phosphorylation. The inhibition of PI-3-kinase—Akt pathway by LY-294002 or wortmannin significantly decreased the protective effect of the Hsp16.2. These data indicate that the over-expression of Hsp16.2 inhibits cell death via the stabilization of mitochondrial membrane system, activation of Hsp90, stabilization of lipid rafts and by the activation of PI-3-kinase—Akt cytoprotective pathway.     

Autotaxin (lysoPLD/NPP2) protects fibroblasts from apoptosis through its enzymatic product, lysophosphatidic acid, utilizing albumin-bound substrate

Autotaxin (ATX) was originally identified as a potent tumor cell motility-stimulating factor that displays multiple enzymatic activities including ATPase, Type I nucleotide pyrophosphatase/phosphodiesterase, and lysophospholipase D, depending on its substrates. We demonstrate herein that ATX is a key regulator of extracellular lysophosphatidic acid (LPA) that can act as survival factor, in addition to its mitogenic activity in mouse fibroblasts. Introduction of atx gene into NIH3T3 cells resulted in resistance to conditional apoptosis induced by serum-deprivation, and exogenous ATX protein prevented cells from death by starvation. Flow cytometric analysis showed that co-treatment of ATX with lysophosphatidylcholine as substrate rescued NIH3T3 cells from cellular apoptosis, and this survival activity of ATX was also demonstrated by caspase-3 degradation and PARP cleavage resulting from the enzymatic activity of extracellular ATX. Furthermore, the effect of ATX in preventing apoptosis appears to be mediated through the G-protein-coupled receptor pathway followed by the activation of phosphoinositide 3-kinase and Akt pathway leading to enhanced cell survival. These findings provide novel insights into understanding the functions of ATX as a key regulator of bioactive phospholipids and suggest interventions to correct dysfunction in conditions of tumor cell growth and metastasis.     

Evidence for a second messenger function of dUTP during Bax mediated apoptosis of yeast and mammalian cells

The identification of novel anti-apoptotic sequences has lead to new insights into the mechanisms involved in regulating different forms of programmed cell death. For example, the anti-apoptotic function of free radical scavenging proteins supports the pro-apoptotic function of Reactive Oxygen Species (ROS). Using yeast as a model of eukaryotic mitochondrial apoptosis, we show that a cDNA corresponding to the mitochondrial variant of the human DUT gene (DUT-M) encoding the deoxyuridine triphosphatase (dUTPase) enzyme can prevent apoptosis in yeast in response to internal (Bax expression) and to exogenous (H₂O₂ and cadmium) stresses. Of interest, cell death was not prevented under culture conditions modeling chronological aging, suggesting that DUT-M only protects dividing cells. The anti-apoptotic function of DUT-M was confirmed by demonstrating that an increase in dUTPase protein levels is sufficient to confer increased resistance to H₂O₂ in cultured C2C12 mouse skeletal myoblasts. Given that the function of dUTPase is to decrease the levels of dUTP, our results strongly support an emerging role for dUTP as a pro-apoptotic second messenger in the same vein as ROS and ceramide.