Proapoptotic effect of propofol on T cells]

BACKGROUND: Surgery and general anesthesia induce excessive apoptosis on peripheral lymphocytes and this phenomenon significantly contributes to the postoperative lymphocytopenia. However, the role played by anesthetic agents on this event remains to be elucidated. In this study we examined whether an anesthetic compound such as propofol is able to exert, in vitro, a proapoptotic effect on T cells. METHODS: Lymphocytes were isolated from heparinized venous blood in healthy volunteers and were incubated with propofol at clinically relevant concentration (5 mg/ml) and at 10 times this concentration (50 mg/ml). The counts of cells either bearing the Fas/FasL phenotype or expressing the intracellular Bcl2 protein were determined by means of flow cytometry. Assessment of lymphocytes undergoing apoptosis was made both by staining of apoptotic nuclei with propidium iodide (PI) and by phenotypic analysis of apoptotic cells with 7-amino-actinomycin D (7-AAD). RESULTS: Treated lymphocytes exhibit a significantly enhanced expression of Fas/FssL system associated with a decrease of Bcl2 expression at both 5 and 50 mg/ml propofol concentrations (p<0.05). On the other hand, the rate of apoptotic cells was not significantly different as compared to control in the absence of propofol. CONCLUSIONS: Propofol impairs, in vitro, both Fas/FasL and Bcl2 expressions on lymphocytes at clinically relevant concentrations but fails to induce apoptotic cell death. It is suggested that the antioxidative properties of the drug could inhibit some way the mechanisms by which mitochondrial free-radicals mediating apoptosis ultimately lead to cell death execution.     

Cisplatin induces apoptosis in LLC-PK1 cells via activation of mitochondrial pathways

Cisplatin, a commonly used chemotherapeutic agent, has a major limitation because of its nephrotoxicity. Recent studies have shown that cisplatin causes apoptotic cell death in renal tubule cells, but the underlying molecular mechanisms remain to be elucidated. In this study, cisplatin was found to induce apoptosis in a dose- and duration-dependent manner in cultured proximal tubule (LLC-PK1) cells, as evidenced by DNA laddering and TdT-mediated dUTP nick end-labeling assay. Pretreatment with the specific caspase 9 inhibitor LEHD-CHO completely prevented the apoptosis, whereas the caspase 8 inhibitor IETD-fmk had no effect. Furthermore, the activity of caspase 9 was upregulated about sixfold by cisplatin in a dose-dependent manner. These results implicated the caspase 9-dependent mitochondrial apoptotic pathways. Indeed, cisplatin triggered a duration-dependent translocation of cytochrome c from the mitochondria to the cytosol, by immunofluorescence and Western blots. Cisplatin treatment also resulted in the duration-dependent activation and mitochondrial translocation of the pro-apoptotic molecule Bax, by immunofluorescence. Finally, cisplatin induced a duration-dependent onset of the mitochondrial permeability transition. Our results indicate that cisplatin induces apoptosis in LLC-PK1 cells via activation of mitochondrial signaling pathways. The sequence of events may be summarized as follows: activation of Bax induces mitochondrial permeability transition, leading to release of cytochrome c, activation of caspase 9, and entry into the execution phase of apoptosis. Inhibition of this specific pathway may provide a strategy to minimize cisplatin-induced nephrotoxicity.     

Inhibitory Effects of Propofol on Intracellular Signaling by Endothelin-1 in Aortic Smooth Muscle Cells

Background: Blood pressure decreases when propofol is administered. However, the exact mechanism underlying the vascular effects of propofol has not yet been elucidated. Endothelin produced by vascular endothelial cells is a potent vasoactive peptide that elicits prolonged contraction of vascular smooth muscle cells. The effects of propofol on endothelin-1-induced intracellular signaling in an aortic smooth muscle cell line, A10 cells, were examined.

Methods: Cultured A10 cells were pretreated with propofol for 20 min and then stimulated with endothelin-1. The effect of propofol on the endothelin-1-induced Ca2+ influx into A10 cells was evaluated by measuring intracellular45 Ca2+. The effects of propofol on the endothelin-1-induced activation of phosphatidylinositol-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D were evaluated by measuring the formation of inositol phosphates and choline, respectively. The effect of propofol on endothelin-1 binding to its receptor was determined by an [sup 125 I] endothelin-1-binding assay.

Results: Propofol inhibited the the endothelin-1-induced Ca2+ influx, but this was significant only at supuraclinical concentrations. The endothelin-1-stimulated formation of inositol phosphates was significantly suppressed by propofol. However, propofol had no effect on the formation of inositol phosphates induced by NaF, an activator of heterotrimeric guanosine triphosphate (GTP)-binding proteins. Propofol inhibited the endothelin-1-induced formation of choline. Propofol had no effect on the binding of endothelin-1 to its receptor.     

Lidocaine induces apoptosis via the mitochondrial pathway independently of death receptor signaling

BACKGROUND: Local anesthetics, especially lidocaine, can lead to persistent cauda equina syndrome after spinal anesthesia. Recently, lidocaine has been reported to trigger apoptosis, although the underlying mechanisms remain unknown. To elucidate the pathway of lidocaine-induced apoptosis, the authors used genetically modified cells with overexpression or deficiencies of key regulators of apoptosis. METHODS: Human Jurkat T-lymphoma cells overexpressing the antiapoptotic protein B-cell lymphoma 2 as well as cells deficient of caspase 9, caspase 8, or Fas-associated protein with death domain were exposed to lidocaine and compared with parental cells. The authors evaluated cell viability, mitochondrial alterations, cytochrome c release, caspase activation, and early apoptosis. Apoptosis was in addition investigated in neuroblastoma cells. RESULTS: In Jurkat cells, lidocaine reduced viability, associated with a loss of the mitochondrial membrane potential. At low concentrations (3-6 mm) of lidocaine, caspase 3 was activated and release of cytochrome c was detected, whereas at higher concentrations (10 mm), no caspase activation was found. Apoptosis by lidocaine was strongly reduced by B-cell lymphoma-2 protein overexpression or caspase-9 deficiency, whereas cells lacking the death receptor pathway components caspase 8 and Fas-associated protein with death domain were not protected and displayed similar apoptotic alterations as the parental cells. Lidocaine also induced apoptotic caspase activation in neuroblastoma cells. CONCLUSIONS: Apoptosis is triggered by concentrations of lidocaine occurring intrathecally after spinal anesthesia, whereas higher concentrations induce necrosis. The data indicate that death receptors are not involved in lidocaine-induced apoptosis. In contrast, the observation that B-cell lymphoma-2 protein overexpression or the lack of caspase 9 abolished apoptosis clearly implicates the intrinsic mitochondrial death pathway in lidocaine-induced apoptosis.     

Tacrolimus-Induced Apoptosis is Mediated by Endoplasmic Reticulum–derived Calcium-dependent Caspases-3,-12 in Jurkat Cells

Apoptotic signal pathways are delivered to caspase-3, caspase-9, or both in different cells via the death receptor pathway, mitochondrial pathway, or by the endoplasmic reticulum (ER) pathway through initiators of caspase-3, -8, -9, or -12. Tacrolimus (Tac)–induced apoptosis was characterized by nuclear fragmentation and caspase-3 activation. We examined the effect of tacrolimus on ER-derived calcium and caspase-3,-12–mediated apoptosis on Jurkat human T lymphocyte. Tac decreased the viability of Jurkat cells in a dose-dependent manner. Tac also increased continuously intracellular concentration of calcium from 24 hours to 72 hours. We did not find intracellular calcium changes on the treatment of calcium ionorpore (A23187) regardless of 1 nmol/L Tac concentration level. However, calcium adenosine triphosphatase inhibitor (thapsigargin) increased intracellular calcium accumulation and co-treating 1 nmol/L Tac further induced intracellular calcium accumulation. Interestingly, we found that 1 nmol/L Tac treatment induced activation of caspase-12 protease as well as the catalytic activity of caspase-3 but not catalytic activation of caspase-6, -8, and -9 proteases in Jurkat cells. These data advance our understanding of Tac-induced apoptosis is ER-derived calcium and caspases-3,-12– mediated apoptosis in human Jurkat cell line.     

Mechanisms underlying the inhibitory effect of propofol on the contraction of canine airway smooth muscle.

BACKGROUND: Propofol has been shown to produce relaxation of preconstricted airway smooth muscle. Although the inhibition of calcium mobilization is supposed to be the major mechanism of action, the whole picture of the mechanisms is not completely clear. METHODS: Contractile response was performed using canine tracheal rings. The effects of propofol on carbachol-induced mobilization of intracellular Ca2+ and phosphoinositide hydrolysis were measured using cultured canine tracheal smooth muscle cells by monitoring fura-2 signal and assessing the accumulation of [3H]-inositol phosphates. To detect the effect of propofol on muscarinic receptor density and affinity, [3H]N-methyl-scopolamine was used as a radioligand for receptor binding assay. RESULTS: Pretreatment with propofol shifts the concentration-response curves of carbachol-induced smooth muscle contraction to the right in a concentration-dependent manner without changing the maximal response. Propofol not only decreased the release of Ca2+ from internal stores but also inhibited the calcium influx induced by carbachol. In addition, carbachol-induced inositol phosphate accumulation was attenuated by propofol; the inhibitory pattern was similar to the contractile response. Moreover, propofol did not alter the density of muscarinic receptors. The dissociation constant value was not altered by pretreatment with 100 microM propofol but was significantly increased by 300 microM (propofol, 952+/-229 pM; control, 588+/-98 pM; P<0.05). CONCLUSIONS: Propofol attenuates the muscarinic receptor-mediated airway muscle contraction. The mechanism underlying these effects was attenuation of inositol phosphate generation and inhibition of Ca2+ mobilization through the inhibition of the receptor-coupled signal-transduction pathway.     

Homocysteine enhances apoptosis in human bone marrow stromal cells

INTRODUCTION: High plasma homocysteine (Hcy) levels have been associated with increased risk of fracture. Since Hcy has been shown to induce apoptosis in many cell types, including vascular endothelial cells, we hypothesized that Hcy would have a similar apoptotic effect on osteoblasts, leading to osteoporosis by reducing bone formation. MATERIALS AND METHODS: Using primary human bone marrow stromal cells (hBMSC) and HS-5 cell line (human bone marrow stromal cell line), we investigated the effects of Hcy on these cells by cell viability assay and analysis of cytoplasmic histone-associated DNA fragments. Caspase activity assay, Western blots, and electrophoresis mobility shift assay (EMSA) were performed to find the mechanism of apoptosis. Intracellular reactive oxygen species (ROS) were measured by spectrometry using dichlorofluorescein diacetate, and cellular total glutathione level was determined by a commercially available kit. N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) were used as tools for investigating the role of ROS and nuclear factor-kappaB (NF-kappaB), respectively. RESULTS: Hcy induced apoptosis in primary human bone marrow stromal cells and the HS-5 cell line, and this apoptotic effect was caspase-dependent. In addition, Hcy increased cytochrome c release into the cytosol, and activated caspase-9 and caspase-3, but not caspase-8, indicating that Hcy induces apoptosis via the mitochondria pathway. Hcy increased ROS, and NAC inhibited the apoptotic effect of Hcy. Western blot and EMSA showed that Hcy activated the NF-kappaB pathway. PDTC blocked Hcy-induced caspase-3 activation and apoptosis. CONCLUSION: These results suggest that Hcy induces apoptosis via the ROS-mediated mitochondrial pathway and NF-kappaB activation in hBMSCs, and that Hcy may contribute to the development of osteoporosis by reducing bone formation. Antioxidants may have a role in preventing bone loss in individuals with hyperhomocysteinemia.     

Propofol suppresses macrophage functions and modulates mitochondrial membrane potential and cellular adenosine triphosphate synthesis

BACKGROUND: Propofol is an intravenous anesthetic agent that may impair host defense system. The aim of this study was to evaluate the effects of propofol on macrophage functions and its possible mechanism. METHODS: Mouse macrophage-like Raw 264.7 cells were exposed to propofol, at 3, 30 (a clinically relevant concentration), and 300 microm. Cell viability, lactate dehydrogenase, and cell cycle were analyzed to determine the cellular toxicity of propofol to macrophages. After administration of propofol, chemotactic, phagocytic, and oxidative ability and interferon-gamma mRNA production were carried out to validate the potential effects of propofol on macrophage functions. Mitochondrial membrane potential and cellular adenosine triphosphate levels were also analyzed to evaluate the role of mitochondria in propofol-induced macrophage dysfunction. RESULTS: Exposure of macrophages to 3 and 30 microm propofol did not affect cell viability. When the administered concentration reached 300 microm, propofol would increase lactate dehydrogenase release, cause arrest of cell cycle in G1/S phase, and lead to cell death. In the 1-h-treated macrophages, propofol significantly reduced macrophage functions of chemotactic and oxidative ability in a concentration-dependent manner. However, the suppressive effects were partially or completely reversed after 6 and 24 h. Propofol could reduce phagocytic activities of macrophages in concentration- and time-dependent manners. Exposure of macrophages to lipopolysaccharide induced the mRNA of interferon-gamma, but the induction was significantly blocked by propofol. Propofol concentration-dependently decreased the membrane potential of macrophage mitochondria, but the effects were descended with time. The levels of cellular adenosine triphosphate in macrophages were also reduced by propofol. CONCLUSIONS: A clinically relevant concentration of propofol can suppress macrophage functions, possibly through inhibiting their mitochondrial membrane potential and adenosine triphosphate synthesis instead of direct cellular toxicity.     

蟾毒灵抑制胆总管癌细胞QBC-939增殖和诱导凋亡的实验研究

目的观察蟾毒灵对人胆管癌细胞QBC-939增殖和凋亡的影响,以及凋亡相关蛋白表达量的变化。方法体外培养人胆管癌QBC-939细胞,运用MTT检测蟾毒灵对胆管癌细胞增殖的影响,采用流式细胞仪检测蟾毒灵对胆管癌细胞凋亡率和线粒体膜电位的影响;并采用Western blotting检测凋亡相关蛋白Caspase3、激活型的Caspase3(Cleaved-Caspase3)和Cleaved-PARP的表达量变化。结果蟾毒灵对人胆管癌细胞QBC-939增殖具有明显抑制作用,在一定浓度下表现出时间、剂量依赖性。流式细胞仪检测显示:细胞的凋亡率明显增加;线粒体膜电位随蟾毒灵浓度增加,膜电位发生下调。Western blotting结果显示:蟾毒灵处理QBC-939细胞48h后,Caspase3蛋白被剪接活化,激活型的Caspase3(Cleaved-Caspase3)表达量升高;同时,Caspase3的作用底物多聚聚合酶PARP的降解产物(Cleaved-PARP)表达明显升高,且呈浓度依赖性。结论蟾毒灵能够明显抑制人胆管癌QBC-939细胞的增殖,并呈时间和浓度依赖性;线粒体凋亡途径在蟾毒灵的抗癌过程中发挥了作用。     

Lidocaine Induces Apoptosis via the Mitochondrial Pathway Independently of Death Receptor Signaling

Background: Local anesthetics, especially lidocaine, can lead to persistent cauda equina syndrome after spinal anesthesia. Recently, lidocaine has been reported to trigger apoptosis, although the underlying mechanisms remain unknown. To elucidate the pathway of lidocaine-induced apoptosis, the authors used genetically modified cells with overexpression or deficiencies of key regulators of apoptosis.

Methods: Human Jurkat T-lymphoma cells overexpressing the antiapoptotic protein B-cell lymphoma 2 as well as cells deficient of caspase 9, caspase 8, or Fas-associated protein with death domain were exposed to lidocaine and compared with parental cells. The authors evaluated cell viability, mitochondrial alterations, cytochrome c release, caspase activation, and early apoptosis. Apoptosis was in addition investigated in neuroblastoma cells.

Results: In Jurkat cells, lidocaine reduced viability, associated with a loss of the mitochondrial membrane potential. At low concentrations (3-6 mm) of lidocaine, caspase 3 was activated and release of cytochrome c was detected, whereas at higher concentrations (10 mm), no caspase activation was found. Apoptosis by lidocaine was strongly reduced by B-cell lymphoma-2 protein overexpression or caspase-9 deficiency, whereas cells lacking the death receptor pathway components caspase 8 and Fas-associated protein with death domain were not protected and displayed similar apoptotic alterations as the parental cells. Lidocaine also induced apoptotic caspase activation in neuroblastoma cells.     

Molecular mechanisms of Fas-mediated cell death in oligodendrocytes

Oligodendrocyte cell death is a significant component of the secondary damage following spinal cord injury (SCI) and other neurodegenerative disorders. However, the mechanisms underlying oligodendroglial apoptotic cell death and the potential relationship to Fas receptor (FasR) activation require further clarification. Here, using MO3.13, a human oligodendroglial cell line, we show clear evidence of apoptosis upon exposure to soluble Fas ligand (sFasL). Apoptosis was linked to caspase-8, -9, and -3 activity and resulted in DNA fragmentation detected by deoxynucleotide transferase dUTP nick end-labeling (TUNEL). Dissipation of mitochondrial membrane potential (DeltaPsim) was an early event and temporally coincided with mitochondrial outer membrane permeability (MOMP), demonstrated by the presence of cytochrome c and apoptosis inducing factor (AIF) in cytosolic fractions. Pretreatment with 100 microM of the caspase inhibitor zVAD-fmk prior to sFasL exposure reduced caspase activation, the dissipation of DeltaPsim, MOMP, and apoptotic cell death. These data provide clear evidence that Fas activation induces apoptosis in oligodendrocytes signaling through intrinsic and extrinsic events. Moreover, we provide evidence for the first time that AIF may play a role in caspase-independent apoptotic execution following Fas activation of oligodendrocytes. These data also add to an emerging body of evidence, which strongly implicates Fas-mediated apoptosis of oligodendrocytes as a potential mediator in the pathobiology of a variety of neurological disorders, including SCI.     

Hyperglycemia-induced apoptosis in mouse myocardium: mitochondrial cytochrome C-mediated caspase-3 activation pathway

Diabetic cardiomyopathy is related directly to hyperglycemia. Cell death such as apoptosis plays a critical role in cardiac pathogenesis. Whether hyperglycemia induces myocardial apoptosis, leading to diabetic cardiomyopathy, remains unclear. We tested the hypothesis that apoptotic cell death occurs in the diabetic myocardium through mitochondrial cytochrome c-mediated caspase-3 activation pathway. Diabetic mice produced by streptozotocin and H9c2 cardiac myoblast cells exposed to high levels of glucose were used. In the hearts of diabetic mice, apoptotic cell death occurred as detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Correspondingly, caspase-3 activation as determined by enzymatic assay and mitochondrial cytochrome c release detected by Western blotting analysis were observed. Supplementation of insulin inhibited diabetes-induced myocardial apoptosis as well as suppressed hyperglycemia. To explore whether apoptosis in diabetic hearts is related directly to hyperglycemia, we exposed cardiac myoblast H9c2 cells to high levels of glucose (22 and 33 mmol/l) in cultures. Apoptotic cell death was detected by TUNEL assay and DAPI nuclear staining. Caspase-3 activation with a concomitant mitochondrial cytochrome c release was also observed. Apoptosis or activation of caspase-3 was not observed in the cultures exposed to the same concentrations of mannitol. Inhibition of caspase-3 with a specific inhibitor, Ac-DEVD-cmk, suppressed apoptosis induced by high levels of glucose. In addition, reactive oxygen species (ROS) generation was detected in the cells exposed to high levels of glucose. These results suggest that hyperglycemia directly induces apoptotic cell death in the myocardium in vivo. Hyperglycemia-induced myocardial apoptosis is mediated, at least in part, by activation of the cytochrome c-activated caspase-3 pathway, which may be triggered by ROS derived from high levels of glucose.     

Propofol attenuates beta-adrenoreceptor-mediated signal transduction via a protein kinase C-dependent pathway in cardiomyocytes

BACKGROUND: Activation of beta adrenoreceptors by catecholamines is an important mechanism for increasing the inotropic state of the heart. The objectives of the current study were to investigate the effects of propofol on beta-adrenoreceptor-mediated increases in cardiomyocyte intracellular Ca2+ concentration ([Ca2+]i), cell shortening, L-type Ca2+ current (ICa) and cyclic adenosine monophosphate (cAMP) accumulation. The authors also investigated the site of action of propofol in the beta-adrenoreceptor signaling pathway, as well as the role of protein kinase C (PKC), and tested the hypothesis that propofol would inhibit the beta-adrenoreceptor signaling pathway via a PKC-dependent mechanism. METHODS: Freshly isolated ventricular myocytes were obtained from adult rat and guinea pig hearts. Myocyte shortening (video edge detection) and [Ca2+]i (fura-2, 340/380 ratio) were monitored simultaneously in individual cells. Conventional whole cell patch clamp analysis was used to measure the ICa in individual myocytes. cAMP production was assessed in suspensions of myocytes using an enzyme immunoassay kit. RESULTS: Propofol (0.1-10 mum) had no effect on steady state [Ca2+]i, cell shortening, ICa, or cAMP production. In contrast, propofol caused dose-dependent decreases in isoproterenol-stimulated increases in [Ca2+]i, shortening, ICa, and cAMP. Forskolin-induced increases in [Ca2+]i, shortening, and cAMP production were not altered by propofol. PKC activation with phorbol myristate acetate attenuated isoproterenol-stimulated cAMP production. Inhibition of PKC with bisindolylmaleimide (broad range inhibitor) or G? 6976 (inhibitor of Ca2+-dependent PKC isoforms) abolished propofol-induced inhibition of isoproterenol-stimulated increases in [Ca2+]i, shortening, and cAMP production. CONCLUSIONS: Clinically relevant concentrations of propofol attenuate beta-adrenergic signal transduction in cardiac myocytes via inhibition of cAMP production. The inhibitory site of action of propofol is upstream of adenylyl cyclase and involves activation of PKC alpha.     

Direct effect of zinc on mitochondrial apoptogenesis in prostate cells

BACKGROUND: Prostate epithelial cells uniquely accumulate significantly higher levels of zinc than other mammalian cells. We previously showed that the accumulation of high intracellular zinc levels in specific prostate cells results in the induction of apoptosis and the inhibition of cell growth. The apoptotic effect is due to zinc induction of mitochondrial apoptogenesis. We now report additional studies that corroborate this effect of zinc and provide insight into the mechanism of this unique effect. METHODS: The effect of exposure to physiological levels of zinc on apoptosis was determined for three human prostate cell lines (PC-3, BPH, and HPR-1). Zinc-induced apoptosis was identified by DNA fragmentation. The direct effect of zinc on isolated mitochondrial preparations from each cell line was determined. The mitochondrial release of cytochrome c was determined by Western blot. RESULTS: Exposure to zinc induced apoptosis in PC-3 and BPH cells but not in HPR-1 cells. The zinc accumulation in PC-3 (4.3 +/- 0.3) and BPH (2.8 +/- 0.4) was higher than that in HPR-1 cells (1.8 +/- 0.1). The apoptotic effect of zinc on PC-3 cells could be observed as early as 4-6 hr of zinc treatment, and this effect was not reversible. The exposure of isolated mitochondria from PC-3 and BPH cells to zinc resulted in the release of cytochrome c; but zinc had no effect on mitochondria from HPR-1 cells. CONCLUSIONS: Exposure to zinc induces apoptosis in PC-3 and BPH cells, which accumulate high intracellular levels of zinc, but not in HPR-1 cells, which do not accumulate high levels of zinc. Once initiated, the induction of apoptosis is not reversed by the removal of zinc, i.e., it is an irreversible process. The apoptogenic effect is due to a direct effect of zinc on mitochondria that results in the release of cytochrome c. The cell specificity of zinc induction of apoptogenesis is dependent on the ability of the cells to accumulate high levels of intracellular zinc and on the ability of the mitochondria to respond to the direct effect of zinc.     

异丙酚预先给药对缺氧诱导胎鼠肺泡Ⅱ型上皮细胞凋亡的影响

目的 评价异丙酚预先给经对缺氧诱导胎鼠肺泡Ⅱ型上皮细胞凋亡的影响.方法 体外分离、培养SD胎鼠的肺泡Ⅱ型上皮细胞,接种于96孔培养板,细胞浓度为1×106/L,每孔180μl,随机分为3组(n=72),正常对照组(C组)常氧培养,缺氧组(H组)和异丙酚-缺氧组(P-H组)缺氧(5%O2)培养,P-H组于缺氧前1 h加入异丙酚(终浓度为5μmol/L).分别于缺氧3、12、24或48 h时测定细胞凋亡率、缺氧诱导因子-1α(HIF-lα)mRNA、Bnip3L mRNA、HIF-lα蛋白和Bnip3L蛋白的表达水平.结果 与C组比较,H组细胞凋亡率升高,HIF-lα mRNA、Bnip3L mRNA及其蛋白表达水平上调(P＜0.05).异丙酚预先给药可抑制缺氧诱导的上述改变(P＜0.05).结论 异丙酚预先给药可抑制缺氧诱导胎鼠肺泡Ⅱ型上皮细胞凋亡,其机制与抑制HIF-1的激活,从而下调Bnip3L蛋白表达有关.     

Role of mitochondria in ciprofloxacin induced apoptosis in bladder cancer cells

PURPOSE: In our earlier series we showed that ciprofloxacin inhibits bladder tumor cell growth with concomitant S/G2M cell cycle arrest and reported an increased Bax-to-Bcl-2 ratio in cells undergoing cell death. In the current series we elucidated the molecular mechanisms by which ciprofloxacin induces apoptotic processes. MATERIALS AND METHODS: Ciprofloxacin mediated mitochondrial depolarization was detected by flow cytometry in HTB9 cells. Mitochondrial permeability transition was measured by spectrophotometry in isolated mitochondria treated with ciprofloxacin in the presence and absence of cyclosporin. The consequential decrease in mitochondrial calcium, cytochrome c release and Bax translocation to mitochondria, which resulted in the activation of caspase 3 leading to apoptotic cell death, was measured by biochemical and confocal microscopy. RESULTS: Mitochondrial depolarization was observed during ciprofloxacin induced apoptotic processes. Cyclosporin A, a known inhibitor of the mitochondrial permeability transition pore, protected cells against decreased mitochondrial potential. Also, ciprofloxacin induced an alteration of mitochondrial calcium as early as 5 minutes and this disruption of intracellular calcium homeostasis was prevented by cyclosporin. Ciprofloxacin also had a direct effect on swelling of isolated mitochondria, which was absent in the presence of cyclosporin. Mitochondrial changes were accompanied by cytochrome c release and caspase 3 activation. Our findings also showed Bcl-2 dependent subcellular redistribution of Bax to the mitochondrial membrane in ciprofloxacin treated bladder tumor cells. CONCLUSIONS: The disruption of calcium homeostasis, mitochondrial swelling and redistribution of Bax to the mitochondrial membrane are key events in the initiation of apoptotic processes in ciprofloxacin treated bladder cancer cells.     

Enhanced proapoptotic effects of tumor necrosis factor-related apoptosis-inducing ligand on temozolomide-resistant glioma cells

OBJECT: Death receptor targeting is an attractive approach in experimental treatment for tumors such as malignant gliomas, which are resistant to radiation and chemotherapy. Among the family of cytokines referred to as death li gands, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted clinical interest. The aim of this study was to assess whether TRAIL can be used as an adjuvant to temozolomide (TMZ) for apoptosis induction in malignant glioma cell lines. METHODS: Six human malignant glioma cell lines (A172, U87, U251, T98, U343, and U373) were exposed to human (h)TRAIL, TMZ, or an hTRAIL/TMZ combined treatment. Cell viability was assayed using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide and phase-contrast microscopy. Cell apoptosis was detected using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling technique and quantified using flow cytometric analysis. The apoptosis signaling cascade was studied with Western blotting. The additive effects of hTRAIL and TMZ resulted in a significant decrease in cell viability and an increased apoptotic rate. Expression of the death receptors DR5 and DR4 in two cell lines (A172 and U251) upregulated significantly when they were used in combination hTRAIL/TMZ treatment (p < 0.05 compared with baseline control), leading to activation of caspase-8 and caspase-3 (p < 0.05 compared with baseline control) and confirming an extrinsic apoptotic pathway. A cell intrinsic pathway through mitochondrial cytochrome c was not activated. CONCLUSIONS: Based on this work, one may infer that hTRAIL should be considered as an adjuvant treatment for TMZ-resistant human malignant gliomas.     

Methylglyoxal induces apoptosis through activation of p38 mitogen-activated protein kinase in rat mesangial cells

BACKGROUND: The formation of methylglyoxal (MG), a highly reactive dicarbonyl compound, is accelerated through several pathways, including the glycation reaction under diabetic conditions, presumably contributing to tissue injury in diabetes. On the other hand, apoptotic cell death of glomerular cells has been suggested to play a role in the development of glomerulosclerosis in various types of glomerular injuries. We therefore examined whether MG was capable of inducing apoptosis in rat mesangial cells to address the possible mechanism by which hyperglycemia-related products accelerated pathologic changes in diabetic glomerulosclerosis. METHODS: Rat mesangial cells were incubated with 0 to 400 micromol/L MG, followed by the detection of apoptosis by both TUNEL method and electrophoretic analysis for DNA fragmentation. In addition, we investigated intracellular mechanisms mediating MG-induced apoptosis, focusing especially on the p38 mitogen-activated protein kinase (MAPK) pathway. RESULTS: MG induced apoptosis in rat mesangial cells in a dose-dependent manner and was accompanied by the activation of p38alpha isoform. Aminoguanidine and N-acetyl-l-cysteine inhibited the MG-induced p38 MAPK activation, as well as apoptosis in rat mesangial cells, suggesting the involvement of oxidative stress in these phenomena. SB203580, a specific inhibitor of p38 MAPK also suppressed the MG-induced apoptosis in rat mesangial cells. CONCLUSIONS: These results suggest a potential role for MG in glomerular injury through p38 MAPK activation under diabetic conditions and may serve as a novel insight into the therapeutic strategies for diabetic nephropathy.     

Volatile anesthetics induce caspase-dependent, mitochondria-mediated apoptosis in human T lymphocytes in vitro

BACKGROUND: Volatile anesthetics modulate lymphocyte function during surgery, and this compromises postoperative immune competence. The current work was undertaken to examine whether volatile anesthetics induce apoptosis in human T lymphocytes and what apoptotic signaling pathway might be used. METHODS: Effects of sevoflurane, isoflurane, and desflurane were studied in primary human CD3 T lymphocytes and Jurkat T cells in vitro. Apoptosis and mitochondrial membrane potential were assessed using flow cytometry after green fluorescent protein-annexin V and DiOC6-fluorochrome staining. Activity and proteolytic processing of caspase 3 was measured by cleaving of the fluorogenic effector caspase substrate Ac-DEVD-AMC and by anti-caspase-3 Western blotting. Release of mitochondrial cytochrome c was studied after cell fractionation using anti-cytochrome c Western blotting and enzyme-linked immunosorbent assays. RESULTS: Sevoflurane and isoflurane induced apoptosis in human T lymphocytes in a dose-dependent manner. By contrast, desflurane did not exert any proapoptotic effects. The apoptotic signaling pathway used by sevoflurane involved disruption of the mitochondrial membrane potential and release of cytochrome c from mitochondria to the cytosol. In addition, the authors observed a proteolytic cleavage of the inactive p32 procaspase 3 to the active p17 fragment, increased caspase-3-like activity, and cleavage of the caspase-3 substrate poly-ADP-ribose-polymerase. Sevoflurane-induced apoptosis was blocked by the general caspase inhibitor Z-VAD.fmk. Death signaling was not mediated via the Fas/CD95 receptor pathway because neither anti-Fas/CD95 receptor antagonism nor FADD deficiency or caspase-8 deficiency were able to attenuate sevoflurane-mediated apoptosis. CONCLUSION: Sevoflurane and isoflurane induce apoptosis in T lymphocytes via increased mitochondrial membrane permeability and caspase-3 activation, but independently of death receptor signaling.     

High glucose evokes an intrinsic proapoptotic signaling pathway in mesangial cells

BACKGROUND: In response to chronic hyperglycemia, microvascular cells undergo stress and injury, which can lead to cell death. We characterized a proapoptotic signaling pathway whereby high glucose evokes an intrinsic, caspase-9-dependent mechanism of cell death in human mesangial cells. METHODS: Biochemical (caspase activity, cytochrome-c release, etc.) and morphologic (chromatin condensation and nuclear segmentation) features of apoptotic cell death were assessed in cultured human mesangial cells exposed to high glucose, a risk factor for mesangial cell injury and diabetic glomerulosclerosis. Proapoptotic signaling was also analyzed in the db/db murine model of kidney injury in diabetes. RESULTS: Incubation in high glucose caused cytotoxicity and apoptosis in mesangial cells. High glucose stimulated mitochondrial release of cytochrome-c, cleavage of procaspase-9, and caspase-9 enzyme activity, suggesting an intrinsic pathway of proapoptotic signaling. In contrast, caspase-8 was unaffected by high glucose. A cell-permeable, caspase-9-selective inhibitor blocked caspase-3 activation and prevented chromatin condensation and nuclear segmentation in cells treated with high glucose. To determine whether an intrinsic signaling pathway occurs in the diabetic kidney in vivo, apoptosis was investigated in diabetic 8- and 16-week db/db murine kidneys. Effector caspases-3 and -7 were activated in diabetic db/db kidneys but not in age-matched nondiabetic db/m controls. At 16 weeks, apoptotic cells in db/db glomeruli were identified on the basis of nuclear segmentation and DNA fragmentation. Apoptosis of glomerular cells correlated with expansion of the mesangial matrix and with worsening of albuminuria. Consistent with an intrinsic signaling pathway, caspase-9 cleavage was elevated only in db/db kidneys, whereas activation of caspase-8 and caspase-12 was undetectable. CONCLUSION: These findings support the hypothesis that hyperglycemia evokes an intrinsic pathway of proapoptotic signaling in mesangial cells. In addition, these results point to an important role for the intrinsic pathway in microvascular injury in the diabetic kidney in vivo.