The cell death-promoting gene DP5, which interacts with the BCL2 family, is induced during neuronal apoptosis following exposure to amyloid beta protein

DP5, which contains a BH3 domain, was cloned as a neuronal apoptosis-inducing gene. To confirm that DP5 interacts with members of the Bcl-2 family, 293T cells were transiently co-transfected with DP5 and Bcl-xl cDNA constructs, and immunoprecipitation was carried out. The 30-kDa Bcl-xl was co-immunoprecipitated with Myc-tagged DP5, suggesting that DP5 physically interacts with Bcl-xl in mammalian cells. Previously, we reported that DP5 is induced during neuronal apoptosis in cultured sympathetic neurons. Here, we analyzed DP5 gene expression and the specific interaction of DP5 with Bcl-xl during neuronal death induced by amyloid-beta protein (A beta). DP5 mRNA was induced 6 h after treatment with A beta in cultured rat cortical neurons. The protein encoded by DP5 mRNA showed a specific interaction with Bcl-xl. Induction of DP5 gene expression was blocked by nifedipine, an inhibitor of L-type voltage-dependent calcium channels, and dantrolene, an inhibitor of calcium release from the endoplasmic reticulum. These results suggested that the induction of DP5 mRNA occurs downstream of the increase in cytosolic calcium concentration caused by A beta. Moreover, DP5 specifically interacts with Bcl-xl during neuronal apoptosis following exposure to A beta, and its binding could impair the survival-promoting activities of Bcl-xl. Thus, the induction of DP5 mRNA and the interaction of DP5 and Bcl-xl could play significant roles in neuronal degeneration following exposure to A beta.     

Structural insights into mouse anti-apoptotic Bcl-xl reveal affinity for Beclin 1 and gossypol

This study reports the crystal structures of Bcl-xl wild type and three Bcl-xl mutants (Y101A, F105A, and R139A) with amino acid substitutions in the hydrophobic groove of the Bcl-xl BH3 domain. An additional 12 ordered residues were observed in a highly flexible loop between the α1 and α2 helices, and were recognized as an important deamidation site for the regulation of apoptosis. The autophagy-effector protein, Beclin 1, contains a novel BH3 domain (residues 101-125), which binds to the surface cleft of Bcl-xl, as confirmed by nuclear magnetic resonance (NMR) spectroscopy and analytical gel-filtration results. Gossypol, a potent inhibitor of Bcl-xl, had a K_d value of 0.9 μM. In addition, the structural and biochemical analysis of five Bcl-xl substitution mutants will provide structural insights into the design and development of anti-cancer drugs.     

Combining Paclitaxel with ABT-263 Has a Synergistic Effect on Paclitaxel Resistant Prostate Cancer Cells

We assessed the capability of paclitaxel, one of the taxanes, to induce death in two prostate cancer lines, LNCaP and PC3. Paclitaxel drove an apoptotic pathway in LNCaP, but not in PC3 cells, in response to G2/M arrest. An examination of the levels of anti-apoptotic proteins revealed that Bcl-xl was much higher in PC3 cells than in LNCaP cells and Bcl2 could be detected only in PC3 cells, not in LNCaP cells. Knocking down Bcl-xl enhanced paclitaxel-induced apoptosis in LNCaP cells, while we were unable to knock down Bcl-xl efficiently in PC3 cells. Significantly, a comparison of ABT-263, a specific inhibitor of Bcl2 and Bcl-xl, with ABT-199, a Bcl2 selective inhibitor, disclosed that only ABT-263, not ABT-199, could induce apoptosis in LNCaP and PC3 cells. The results indicate that Bcl-xl has a protective role against paclitaxel-induced apoptosis in LNCaP and PC3 cells, and its overexpression causes the paclitaxel resistance seen in PC3 cells. Interestingly, combined paclitaxel with ABT-263 to treat LNCaP and PC3 cells demonstrated synergistic apoptosis activation, indicating that ABT-263 could enhance paclitaxel-induced apoptosis in LNCaP cells and overcome Bcl-xl overexpression to trigger paclitaxel-induced apoptosis in PC3 cells. We also observed that the activation of apoptosis in LNCaP cells was more efficient than in PC3 cells in response to paclitaxel plus ABT-263 or to ABT-263 alone, suggesting that the apoptosis pathway in PC3 cells might have further differences from that in LNCaP cells even after Bcl-xl overexpression is accounted for.     

Bim directly antagonizes Bcl-xl in doxorubicin-induced prostate cancer cell apoptosis independently of p53

Doxorubicin and other anthracycline compounds exert their anti-cancer effects by causing DNA damage and initiating cell cycle arrest in cancer cells, followed by apoptosis. DNA damage generally activates a p53-mediated pathway to initiate apoptosis by increasing the level of the BH3-only protein, Puma. However, p53-mediated apoptosis in response to DNA damage has not yet been validated in prostate cancers. In the current study, we used LNCaP and PC3 prostate cancer cells, representing wild type p53 and a p53-null model, to determine if DNA damage activates p53-mediated apoptosis in prostate cancers. Our results revealed that PC3 cells were 4 to 8-fold less sensitive than LNCaP cells to doxorubicin-inuced apoptosis. We proved that the differential response of LNCaP and PC3 to doxorubicin was p53-independent by introducing wild-type or dominant negative p53 into PC3 or LNCaP cells, respectively. By comparing several apoptosis-related proteins in both cell lines, we found that Bcl-xl proteins were much more abundant in PC3 cells than in LNCaP cells. We further demonstrated that Bcl-xl protects LNCaP and PC3 cells from doxorubicin-induced apoptosis by using ABT-263, an inhibitor of Bcl-xl, as a single agent or in combination with doxorubicin to treat LNCaP or PC3 cells. Bcl-xl rather than p53, likely contributes to the differential response of LNCaP and PC3 to doxorubicin in apoptosis. Finally, co-immunoprecipitation and siRNA analysis revealed that a BH3-only protein, Bim, is involved in doxorubicin-induced apoptosis by directly counteracting Bcl-xl.     

氨基葡萄糖硫酸盐诱导凋亡中Cathepsin D与Bcl-xL的细胞内共定位研究

目的:探讨氨基葡萄糖硫酸盐(Glucosamine Sulfate,GS)诱导K562细胞凋亡中溶酶体Cathepsin D释放后与Bcl-xL的细胞内共定位关系。方法:采用5.0mmol.L-1 GS诱导K562细胞凋亡,通过HE染色和流式细胞仪检测细胞凋亡,应用免疫荧光染色结合激光共聚焦显微镜观察5.0mmol.L-1 GS诱导凋亡前后Cathepsin D与Bcl-xL的细胞内共定位关系。结果:GS诱导K562细胞72小时后出现细胞凋亡的形态改变,流式细胞仪检测表明细胞出现凋亡,诱导凋亡后的Cathepsin D与Bcl-xL的细胞内共定位信号增强。结论:GS诱导K562细胞凋亡后Cathepsin D与Bcl-xL存在细胞内共定位关系,具备一定相互作用的空间基础。     

Delayed apoptosis by neutrophils from COPD patients is associated with altered bak, bcl-xl, and mcl-1 mRNA expression

ABSTRACT: BACKGROUND: Delayed neutrophil apoptosis may be an important factor in the persistent inflammation associated with chronic obstructive pulmonary disease (COPD). Bcl-2 family proteins are important regulators of neutrophil apoptosis. We determined the mRNA levels of proapoptotic Bak and anti-aptototic Bcl-xl and Mcl-1 members of the Bcl-2 family in unstimulated peripheral blood neutrophils from patients with mild to moderate COPD and compared these to neutrophils from healthy controls. METHODS: Neutrophils were isolated from peripheral blood samples of 47 COPD patients (smokers: N = 24) and 47 healthy controls (smokers: N = 24). Percentages of apoptotic cells were determined at 4, 24, and 36 h for unstimulated neutrophils cultured in vitro. Neutrophil mRNA expression of Bak, Bcl-xl, and Mcl-1 was determined by real-time polymerase chain reaction (PCR). FEV1 (% predicted) and FVC were determined by spirometry and correlations between mRNA levels and lung function parameters were determined. RESULTS: The percentages of apoptotic cells among unstimulated neutrophils from COPD patients were significantly lower compared to cells from controls after 4, 24, and 36 h in culture; smoking history had only a minimal effect on these differences. Unstimulated neutrophils from COPD patients had significantly lower Bak mRNA expression and higher expressions of Bcl-xl and Mcl-1 mRNA than cells from healthy controls. Again, smoking history had only a minimal effect on these trends. Bak mRNA expression was significantly positively correlated with both %predicted FEV1 and the FEV1/FVC ratio, while Bcl-xl and Mcl-1 mRNA expressions were significantly negatively correlated with %predicted FEV1 and the FEV1/FVC ratio. CONCLUSIONS: The genes for pro-apoptotic Bak, and anti-apoptotic Bcl-xl and Mcl-1 may be important in regulating the delayed neutrophil apoptosis observed in COPD, which may contribute to COPD pathogenesis. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1605269445677066.     

红托竹荪多糖诱导肿瘤细胞凋亡的作用初探

探讨红托竹荪多糖对小鼠腹水瘤S180细胞凋亡的作用。分别使用不同浓度的红托竹荪多糖处理S180细胞24h,Western blot法检测Bcl-xl、Bax、caspase-9和Caspase-3等蛋白表达,使用流式细胞仪检测细胞凋亡。Western blot 法检测结果显示Bcl-xl表达量随竹荪多糖剂量增加而降低,Bax表达量则相反,每组Bax/Bcl-xl的比值增加,Caspase-9、Caspase-3表达量增加;流式细胞仪检测结果显示：0、25、50和100mg/L组细胞凋亡率分别为5.12%±0.11%、9.61%±0.61%、16.39%±0.19%和17.05%±0.13%,与对照组相比细胞凋亡率增高,差异具有统计学意义（P<0.05）,推断红托竹荪多糖具有诱导S180细胞凋亡的功能。     

Air pollution induces enhanced mitochondrial oxidative stress in cystic fibrosis airway epithelium

We studied the effects of airborne particulate matters (PM) on cystic fibrosis (CF) epithelium. We noted that PM enhanced human CF bronchial epithelial apoptosis, activated caspase-9 and PARP-1; and reduced mitochondrial membrane potential. Mitochondrial inhibitors (4,4-diisothiocyanatostilbene-2,2'disulfonic acid, rotenone and thenoyltrifluoroacetone) blocked PM-induced generation of reactive oxygen species and apoptosis. PM upregulated pro-apoptotic Bad, Bax, p53 and p21; and enhanced mitochondrial localization of Bax. The anti-apoptotic Bcl-2, Bcl-xl, Mcl-1 and Xiap remained unchanged; however, overexpression of Bcl-xl blocked PM-induced apoptosis. Accordingly, we provide the evidence that PM enhances oxidative stress and mitochondrial signaling mediated apoptosis via the modulation of Bcl family proteins in CF.     

Behavior in the forced-swimming test and expression of BDNF and Bcl-xl genes in the rat brain

A single exposure of rats to the forced-swimming stress decreased BDNF mRNA levels in the cortex and increased Bcl-xl gene expression in the hippocampus and amygdala 24 h after the stress. The animals demonstrated a depressive-like behavior and elevated blood corticosterone level. There was a significant negative correlation between BDNF mRNA level in the cortex and immobility time during swimming. Repeated exposure to swimming stress caused the elevation of the hippocampal BDNF mRNA level assessed 24 h after the second swimming session. The data suggest that stress-induced down-regulation of cortical BDNF gene expression and behavioral despair in the forced-swimming test may be interrelated. The increase in the BDNF and Bcl-xl mRNA levels may contribute to the mechanisms protecting the brain against negative effects of stress.     

Effect of Hyperoxia on Serine Phosphorylation of Apoptotic Proteins in Mitochondrial Membranes of the Cerebral Cortex of Newborn Piglets

Previous studies have shown that hyperoxia results in cerebral cortical neuronal apoptosis. Studies have also shown that phosphorylation of anti-apoptotic proteins Bcl-2 and Bcl-xl results in loss of their anti-apoptotic potential leading to alteration in mitochondrial membrane permeability and the release of apoptogenic proteins in the neuronal cell of the newborn piglets. The present study tests the hypothesis that cerebral hyperoxia will result in increased serine phosphorylation of apoptotic proteins Bcl-2, Bcl-xl, Bax, and Bad in the mitochondrial membranes of the cerebral cortex of newborn piglets. Twelve newborn piglets were divided into normoxic (Nx, n = 6) exposed to an FiO₂ of 0.21 for 1 h and hyperoxic (Hyx, n = 6) exposed to FiO₂ of 1.0 for 1 h. In the Hyx group, PaO₂ was maintained above 400 mmHg while the Nx group was kept at 80–100 mmHg. Cerebral cortical tissue was harvested and mitochondrial fractions were isolated. Mitochondrial membrane proteins were separated using 12% SDS-PAGE, and probed with anti-serine phosphorylated Bcl-2, Bcl-xl, Bax, and Bad antibodies. Protein bands were detected, analyzed by imaging densitometry and density expressed as absorbance (OD × mm²). Phosphorylated Bcl-2 (p-Bcl-2) protein density (OD × mm²) was 81.81 ± 9.24 in Nx and 158.34 ± 10.66 in Hyx (P < 0.05). Phosphorylated Bcl-xl (p-Bcl-xl) protein density was 52.98 ± 3.59 in Nx and 99.62 ± 18.22 in Hyx (P < 0.05). Phosphorylated Bax (p-Bax) protein was 161.13 ± 6.27 in Nx and 174.21 ± 15.95 in Hyx (P = NS). Phosphorylated Bad (p-Bad) protein was 166.24 ± 9.47 in Nx 155.38 ± 12.32 in Hyx (P = NS). The data show that there is a significant increase in serine phosphorylation of Bcl-2 and Bcl-xl proteins while phosphorylation of Bad and Bax proteins were not altered during hyperoxia in the mitochondrial fraction of the cerebral cortex of newborn piglets. We conclude that hyperoxia results in differential post-translational modification of anti-apoptotic proteins Bcl-2 and Bcl-xl as compared to pro-apoptotic proteins Bax and Bad in mitochondria. We speculate that phosphorylation of Bcl-2 and Bcl-xl will result in loss of their anti-apoptotic potential by preventing their dimerization with Bax leading to activation of the caspase cascade of neuronal death.     

幽门螺杆菌感染促进胃炎儿童胃粘膜细胞的增殖

本研究旨在探讨幽门螺杆菌感染对小儿慢性胃炎患者细胞增殖的影响,使用内镜检查消化不良患者的上消化道症状,使用改良的Giemsa染色检测胃粘膜活组织中幽门螺杆菌,用苏木精/曙红和改良的吉姆萨染色活组织,并通过光学显微镜研究染色后胃粘膜样品组织病理学变化,RT-PCR检测各组胃粘膜细胞中调控细胞凋亡的Bcl-2、Bcl-xl、Bax和PCNA的mRNA表达,提取胃粘膜细胞蛋白质,利用蛋白质免疫印迹分析蛋白质浓度。组织化学染色结果表明,与对照相比,患有胃炎和幽门杆菌感染后的胃炎患者胃粘膜细胞明显增加,且幽门螺杆菌感染后细胞增殖更显著(p<0.05);幽门螺杆菌感染后Bcl-2和Bcl-xl,PCNA在患者体内表达显著上调(p<0.05),而细胞促凋亡因子Bax基因在胃炎患者感染幽门螺杆菌后被显著下调(p<0.05),蛋白免疫印迹分析Bcl-2,Bcl-xl,Bax和PCNA蛋白表达趋势与基因表达一致,说明结果可靠。幽门螺杆菌感染会显著提高慢性胃炎儿童患者胃粘膜细胞的增殖。     

Effect of Hypoxia on Expression of Apoptotic Proteins in Nuclear,Mitochondrial and Cytosolic Fractions of the Cerebral Cortex of Newborn Piglets: The Role of Nuclear Ca<Superscript>++</Superscript>-influx

We have shown that hypoxia results in increased influx of nuclear Ca++ and increased expression of nuclear apoptotic proteins. The present study tests the hypothesis that hypoxia alters the distribution of pro-apoptotic proteins Bad and Bax, and the anti-apoptotic proteins Bcl-xl, and Bcl-2 in the nuclear, mitochondrial and cytosolic compartments of the cerebral cortex of newborn piglets and the administration of Clonidine, an inhibitor of high affinity nuclear Ca++ -ATPase, will prevent the hypoxia-induced increase in apoptotic proteins' expression. Studies were conducted in 19 newborn piglets, 6 normoxic (Nx), 7 hypoxic and 6 Clonidine-treated hypoxic (Hx-Clo). Tissue hypoxia was documented biochemically by measuring cerebral tissue ATP and phosphocreatine (PCr) levels. Bax and Bad protein expression increased in all the three compartments during hypoxia, while there was no significant change in the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl. In Clonidine pretreated hypoxic group, the hypoxia-induced increased expression of pro-apoptotic proteins Bad and Bax was prevented in all the three fractions. We conclude that hypoxia results in increased expression of pro-apoptotic proteins in nuclear, mitochondrial and cytosolic compartments and that the increased expression of pro-apoptotic proteins during hypoxia is nuclear Ca++ -influx-dependent. We propose that during hypoxia the increased ratio of (pro-apoptotic Bad and Bax/anti-apoptotic Bcl-xl and Bcl-2) in all the three compartments, will lead to altered mitochondrial and nuclear membrane permeability as well as caspase-9 activation in the cytosolic compartment.     

Molecular Basis for Bcl-2 Homology 3 Domain Recognition in the Bcl-2 Protein Family: IDENTIFICATION OF CONSERVED HOT SPOT INTERACTIONS*

The proteins of the Bcl-2 family are important regulators of apoptosis, or programmed cell death. These proteins regulate this fundamental biological process via the formation of heterodimers involving both pro- and anti-apoptotic family members. Disruption of the balance between anti- and pro-apoptotic Bcl-2 proteins is the cause of numerous pathologies. Bcl-xl, an anti-apoptotic protein of this family, is known to form heterodimers with multiple pro-apoptotic proteins, such as Bad, Bim, Bak, and Bid. To elucidate the molecular basis of this recognition process, we used molecular dynamics simulations coupled with the Molecular Mechanics/Poisson-Boltzmann Surface Area approach to identify the amino acids that make significant energetic contributions to the binding free energy of four complexes formed between Bcl-xl and pro-apoptotic Bcl-2 homology 3 peptides. A fifth protein-peptide complex composed of another anti-apoptotic protein, Bcl-w, in complex with the peptide from Bim was also studied. The results identified amino acids of both the anti-apoptotic proteins as well as the Bcl-2 homology 3 (BH3) domains of the pro-apoptotic proteins that make strong, recurrent interactions in the protein complexes. The calculations show that the two anti-apoptotic proteins, Bcl-xl and Bcl-w, share a similar recognition mechanism. Our results provide insight into the molecular basis for the promiscuous nature of this molecular recognition process by members of the Bcl-2 protein family. These amino acids could be targeted in the design of new mimetics that serve as scaffolds for new antitumoral molecules.     

Osmolarity at early culture stage affects development and expression of apoptosis related genes (Bax-alpha and Bcl-xl) in pre-implantation porcine NT embryos

This study examined whether high osmolarity of culture medium at the early culture stage affects development and expression of apoptosis related genes (Bax-alpha and Bcl-xl) of porcine nuclear transfer (NT) and in vitro fertilization (IVF) embryos. NT and IVF embryos were divided into three groups and the basic medium was PZM-3 (260-270 mOsmol, control group). The control group of embryos was cultured in PZM-3 for whole culture period. Other two groups of embryos were cultured in a modified PZM-3 with 0.05 M sucrose (300-320 mOsmol, sucrose group) or increased NaCl to 138 mM (300-320 mOsmol, NaCl group) for the first 2 days, and then cultured in PZM-3 for 4 days. NT embryos cultured in NaCl group showed a significantly higher developmental rate to the blastocyst stage with a decreased apoptosis rate compared to the control (P < 0.05). There was no difference in blastocyst formation and apoptosis incidence among the three culture treatments for IVF-derived embryos. Bax-alpha mRNA expression was significantly higher in the control than sucrose or NaCl group for both NT and IVF embryos (P < 0.05). Moreover, the relative abundance of Bax-alpha/Bcl-xl was higher in the control than the treatment groups. These results indicate that the higher osmolarity at the early embryonic stage of porcine NT and IVF embryos can improve the in vitro development with reduced apoptosis through regulating the Bax-alpha/Bcl-xl gene expression.     

Proscillaridin A induces apoptosis and inhibits the metastasis of osteosarcoma in vitro and in vivo

The side effects of chemotherapy, drug resistance, and tumor metastasis hinder the development of treatment for osteosarcoma, leading to poor prognosis of patients with the disease. Proscillaridin A, a kind of cardiac glycoside, has been proven to have anti-proliferative properties in many malignant tumors, but the efficacy of the drug in treating osteosarcoma is unclear. In the present study, we assessed the effects of Proscillaridin A on osteosarcoma and investigated its underlying action mechanism. The cell cytotoxicity assay showed that Proscillaridin A significantly inhibited the proliferation of 143B cells in a dose- and time-dependent manner. Also, flow cytometry and invasion assay revealed that Proscillaridin A induced apoptosis and reduced 143B cell motility. Western blotting and PCR were used to detect the expressions of Bcl-xl and MMP2 and showed that mRNA/protein expression levels decreased significantly in Proscillaridin A-treated osteosarcoma cells. Using a mouse xenograft model, we found that Proscillaridin A treatment significantly inhibited tumor growth and lung metastasis in vivo and decreased the expression levels of Bcl-xl and MMP2. No noticeable side effect was observed in the liver, kidney, and hematological functions. Conclusively, Proscillaridin A suppressed proliferation, induced apoptosis, and inhibited 143B cell metastasis in vitro and in vivo, and these effects could be mediated by downregulating the expressions of Bcl-xl and MMP2.     

Nitric oxide induces apoptosis in cutaneous T cell lymphoma (HuT-78) by downregulating constitutive NF-kappaB

Constitutive active NF-kappaB have been shown to protect cutaneous T cell lymphoma (CTCL) cells from apoptosis. In the present study, we have studied the cytotoxic potential of nitric oxide generating compound, sodium nitroprusside (SNP) on CTCL cell line, HuT-78. Treatment of cells with SNP resulted in decrease in mitochondrial membrane potential, cytochrome c release, activation of caspase-3 and poly (ADP ribose) polymerase cleavage. SNP treatment inhibited activation of NF-kappaB in a concentration dependent manner. SNP increased the expression of IkappaBalpha without affecting the phosphorylation of IkappaBalpha. Downregulation of NF-kappaB by SNP decreased p65 nuclear translocation as evident by confocal fluorescence microscopy. Further it was found that SNP treatment caused downregulation of Bcl-2 family member (Bcl-xl) in HuT-78 cells. Thus, we have provided evidence that SNP induces apoptosis in CTCL cell line, HuT-78 by downregulating constitutive NF-kappaB and thereby Bcl-xl expression.