Critical roles for JNK, c-Jun, and Fas/FasL-Signaling in vitamin E analog-induced apoptosis in human prostate cancer cells

BACKGROUND: Alpha-tocopherol ether-linked acetic acid (alpha-TEA), an analog of vitamin E (RRR-alpha-tocopherol), is a potent pro-apoptotic agent for human cancer cells in vivo and in vitro. METHODS: alpha-TEA-induced apoptosis was investigated in LNCaP and PC-3 human prostate cancer cells. Apoptosis was measured by DAPI-staining and FACS analyses of the sub-G1 fraction. Signaling molecules involved in apoptosis were measured by Western immunoblot analyses with or without prior immunoprecipitation, FACS analyses of cell surface membrane expression, RT-PCR analyses of mRNA levels, and chromatin immunoprecipitation. Functional significance was determined using siRNAs, dominant negative mutant, chemical inhibitor, or neutralizing antibody. RESULTS: Alpha-TEA treatment increased Fas and Fas ligand mRNA and protein levels; as well as, levels of cell surface membrane Fas in both cell lines. Blockage of Fas signaling attenuated alpha-TEA-induced apoptosis. alpha-TEA treatment also produced prolonged, elevated levels of activated (phosphorylated) c-Jun N-terminal kinase (JNK) and its substrate c-Jun, both of which were demonstrated to be necessary for alpha-TEA-induced apoptosis. Chromatin immunoprecipitation results showed binding of c-Jun to the promoters of both Fas and FasL in alpha-TEA treated cells. Investigations of alpha-TEA-triggered apoptosis showed dual signaling from Fas with essential roles for both FADD and Daxx with FADD initiating the classical pathway mediated by caspase-8 activation and Daxx initiating an alternate pathway involving activation of JNK, c-Jun, and increased levels of Fas and FasL. CONCLUSIONS: Collectively, data support critical roles for JNK, c-Jun, and dual signaling from Fas/FasL via FADD and Daxx in alpha-TEA-induced apoptosis of human prostate cancer cells.     

Inflammatory and apoptotic signaling after spinal cord injury

Central nervous system (CNS) destruction in spinal cord injury (SCI) is caused by a complex series of cellular and molecular events. Recent studies have concentrated on signaling by receptors in the tumor necrosis factor receptor (TNFR) superfamily that mediate diverse biological outcomes ranging from inflammation to apoptosis. From the perspective of basic science research, understanding how receptor signaling mediates these divergent responses is critical in clarifying events underlying irreversible cell injury in clinically relevant models of SCI. From a clinical perspective, this work also provides novel targets for the development of therapeutic agents that have the potential to protect the spinal cord from irreversible damage and promote functional recovery. In this review, we discuss how the formation of alternate signaling complexes and receptor membrane localization after SCI can influence life and death decisions of cells stimulated through two members of the TNFR superfamily, Fas/CD95 and TNFR1.     

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.     

Caspase activation in neuronal and glial apoptosis following spinal cord injury in mice

The involvement of caspases in apoptosis after spinal cord injury (SCI) was investigated in adult mouse spinal cord after contusion. Sections of spinal cord were processed for staining 7 days after SCI with the fluorescent dye Hoechst 33342, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL), and immunostaining with an antibody (CM1) recognizing activated caspase-3. Caspase-3- and caspase-8-like enzyme activities were measured colorimetrically at 8 hours to 7 days after SCI using the specific substrates Asp-Glu-Val-Asp-p-nitroanilide and Ile-Glu-Thr-Asp-p-nitroanilide, respectively. Hoechst 33342 staining showed small, bright areas in fragmented nuclei. Double labeling with TUNEL plus immunostaining with cell type-specific markers identified TUNEL-positive neurons stained by anti-neuronal nuclear protein/neurons antibody, and TUNEL-positive oligodendrocytes stained by anti-cyclic nucleotide 3'-phosphohydrolase antibody. Double labeling with CM1 and cell-type specific markers similarly identified CM1-positive neurons and oligodendrocytes. Caspase-8-like enzyme activity was increased significantly on days 3 and 7 (p < 0.01), whereas caspase-3-like activity increased on day 7 (p < 0.01). Intraventricular injection of a nonspecific tetrapeptide caspase inhibitor or a specific tetrapeptide inhibitor of caspase-3 just after SCI reduced enzyme activity at 7 days. Apoptotic cells were identified with TUNEL staining in both neurons and oligodendrocytes in mice after SCI, which also showed activated caspase-3. Increased caspase-3- and caspase-8-like activity was detected in the injured spinal cord on days 3 and 7. Caspase protease activities may be involved in delayed neuronal and glial apoptosis after SCI.     

甲基强的松龙对大鼠脊髓损伤后神经细胞凋亡的影响及其机理

目的研究甲基强的松龙(MP)对大鼠横断性脊髓损伤(SCI)后神经细胞凋亡的影响及其作用机理.方法60只成年Wistar大鼠随机分成正常对照组、脊髓损伤组和MP治疗组,每组20只,MP治疗组在横断T10脊髓组织后30min经尾静脉给予MP治疗,损伤组和对照组未予任何治疗.MP治疗组和脊髓损伤组大鼠于脊髓损伤后8h、24h、3d和1周取材,采用透射电镜、TUNEL染色观察细胞凋亡情况,采用免疫组织化学染色观察Fas、半胱氨酸蛋白酶(caspase)-8和caspase-3在SCI前后的变化情况,采用改良Tarlov评分方法观察大鼠后肢运动功能.结果SCI后24h大鼠后肢运动功能逐渐恢复,MP治疗组大鼠的后肢运动功能恢复优于损伤组,7d后尤其明显.TUNEL染色和电镜检查证实SCI后8h即有凋亡细胞出现,其中既有神经元也有胶质细胞3d凋亡细胞数达到高峰;7d仍有凋亡细胞存在,但已明显减少;各时间点治疗组凋亡细胞数量明显少于损伤组(P＜0.05).治疗组和损伤组在SCI后8h可以检测到少量的Fas和caspase-8阳性神经元及胶质细胞,Fas和caspase-8的表达在SCI后3d达到高峰,7d表达下降,各时间点治疗组的Fas和caspase-8灰度值明显大于损伤组,二者有显著性差异(P＜0.05).治疗组和损伤组caspase-3的表达在SCI后8h均逐渐增加(与对照组相比),7d达到高峰,治疗组灰度值大于损伤组,但二者无显著性差异.结论MP能抑制大鼠脊髓横断性脊髓损伤后的神经细胞凋亡,但不能推迟细胞凋亡出现的高峰时间;MP抑制细胞凋亡的途径可能通过非特异性抑制Fas和caspase-8的表达来实现.     

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.     

早期手术减压对损伤脊髓caspase-3表达的影响

目的探讨大鼠脊髓损伤后不同减压时间对大鼠脊髓细胞caspase-3表达的影响。方法将动物分为:大鼠脊髓挫伤即刻手术减压组(A组),大鼠脊髓挫伤2小时手术减压组(B组),大鼠脊髓挫伤8小时手术减压组(C组),大鼠脊髓挫伤24小时手术减压组(D组)。手术后1、3、7、14、28天对脊髓损伤区进行细胞凋亡caspase-3蛋白表达的测定(免疫组化法),采用计算机图像分析技术,进行定量分析。并用行为学和电生理检查观察大鼠功能恢复情况。结果A、B、C、D四组中均发现凋亡caspase-3蛋白阳性表达细胞,图象分析发现,各组凋亡细胞caspase-3免疫反应阳性细胞表达顺序为D>C>B>A,与大鼠后肢功能恢复有平行的变化趋势。结论大鼠脊髓损伤早期手术减压能抑制脊髓损伤后的细胞凋亡,促进大鼠后肢功能恢复。     

黄连素在脊髓损伤中对线粒体的保护作用及机制

目的探讨黄连素对脊髓损伤(SCI)后线粒体氧化损伤的作用和可能机制。方法将36只C57小鼠随机分为假手术组、SCI组(伤后立即腹腔注射10 mg/kg生理盐水)和黄连素组(SCI后立即腹腔注射10 mg/kg黄连素),每组12只。使用PSI-IH脊髓打击器建立小鼠SCI模型,于损伤后24 h处死小鼠,取脊髓组织。使用全自动酶标仪检测各组小鼠脊髓组织线粒体内丙二醛(MDA)、还原型谷胱甘肽(GSH)和超氧化物歧化酶(SOD)的变化;用蛋白质印迹法检测脊髓组织caspase-3、cleaved caspase-3的表达及细胞质内和线粒体内细胞色素C(Cyt C)的表达;用免疫荧光双标染色法检测脊髓组织中神经细胞凋亡情况。结果与假手术组相比,SCI组小鼠脊髓组织线粒体内MDA水平升高,GSH、SOD水平降低;细胞质内Cyt C和脊髓组织中caspase-3、cleaved caspase-3表达水平增高,线粒体内Cyt C表达水平降低;脊髓组织中神经元凋亡比例增高;差异均有统计学意义(P 0.05)。与SCI组相比,黄连素组小鼠脊髓组织线粒体内MDA水平降低,SOD和GSH水平增高;细胞质内Cyt C和脊髓组织中caspase-3、cleaved caspase-3表达水平降低,线粒体内Cyt C表达水平增高;脊髓组织中神经细胞凋亡比例减少;差异均有统计学意义(P 0.05)。结论黄连素可减轻SCI小鼠脊髓组织中神经细胞凋亡,这可能与其抑制线粒体氧化损伤、减少Cyt C释放、降低凋亡蛋白表达有关。     

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.     

Intracellular mechanisms of cyclosporin A-induced tubular cell apoptosis

Tubular cell apoptosis contributes to the pathogenesis of renal injury. However, the intracellular pathways that are active in tubular epithelium are poorly understood. The lethal pathways activated by cyclosporin A (CsA), a nephrotoxin that induces caspase-dependent apoptosis in tubular epithelium, were explored. Fas expression, caspase activation, and mitochondrial injury were assessed by Western blot, flow cytometry, and microscopy in cultured murine tubular epithelial cells exposed to CsA. The influence of FasL antagonists, Bax antisense oligodeoxynucleotides, and caspase inhibitors on cell survival was explored. Tubular cells constitutively express FasL. CsA increased the expression of Fas. However, Fas had no role in CsA-induced apoptosis, as CsA did not sensitize to FasL-induced apoptosis, caspase-8 activity was not increased, and neither blocking anti-FasL antibodies nor caspase-8 inhibition prevented CsA-induced apoptosis. Apoptosis induced by CsA is associated with the translocation of Bax to the mitochondria and Bax antisense oligodeoxynucleotides protected from CsA-induced apoptosis. CsA promoted a caspase-independent release of cytochrome c and Smac/Diablo from mitochondria. CsA also led to a caspase-dependent loss of mitochondrial membrane potential. Caspase-2, caspase-3, and caspase-9 were activated, and specific caspase inhibitor prevented apoptosis and increased long-term survival. Evidence for endoplasmic reticulum stress, such as induction of GADD153, was also uncovered. However, endoplasmic reticulum-specific caspase-12 was not activated. CsA induces changes in several apoptotic pathways. However, the main lethal apoptotic pathway in CsA-exposed tubular epithelial cells involves mitochondrial injury.     

Tamoxifen induces permanent growth arrest through selective induction of apoptosis in growth plate chondrocytes in cultured rat metatarsal bones

Estrogen affects skeletal growth and promotes growth plate fusion in humans. High doses of estrogen have been used to limit growth in girls with predicted extreme tall stature; a treatment which has been associated with severe side effects. Selective estrogen receptor modulators (SERMs) could potentially be used as an alternative treatment. We chose to study the effects of Tamoxifen (Tam), a first generation SERM that has been used in the treatment of pubertal gynecomastia or McCune-Albright syndrome. Cultured fetal rat metatarsal bones were used to study the effects of Tam on longitudinal bone growth. In sectioned bones, chondrocyte apoptosis and proliferation were analyzed by TUNEL assay and BrdU incorporation, respectively. We also used a human chondrocytic cell line, HSC-2/8, to study the effects of Tam on apoptosis (FACS analysis and Cell Death detection ELISA) and caspase activation (caspase substrate cleavage and Western immunoblotting). Tam caused a dose-dependent growth retardation of cultured metatarsal bones. No catch-up growth was observed after Tam was removed from the culture medium. Detailed analysis of sectioned growth plate cartilage revealed increased apoptosis of chondrocytes within the resting and hypertrophic zones. HCS-2/8 cells also underwent apoptosis upon Tam treatment. Tam-induced apoptosis was caspase-dependent and completely abrogated by either caspase-8 or -9 inhibitors. A substrate assay revealed that caspase-8 is first activated followed by caspase-9 and -3. Finally, FasL secretion was stimulated by Tam and blocking of either FasL or Fas decreased Tam-induced apoptosis in chondrocytes. We here describe a novel mechanism of tamoxifen-induced apoptosis in chondrocytes, involving the activation of caspases and the FasL/Fas pathway, which diminishes the potential for bone growth.     

Role of E-selectin in cell apoptosis induced by allogeneic blood perfusion in isolated mouse lung

BACKGROUND: In a model of mouse isolated lung, we have recently demonstrated that E-selectin is involved in the activation of endothelial cells induced by allogeneic blood perfusion. In the present study, we explored the signaling pathway of apoptosis induced by E-selectin triggering. METHODS: Lungs were perfused for 3 hours with fresh blood in the absence or presence of an anti-E-selectin monoclonal antibody, or a protein kinase C (PKC), protein tyrosine phosphatase (PTP), or protein tyrosine kinase (PTK) inhibitor. The number of apoptotic cells in lung sections was determined by a TUNEL method. mRNAs for Fas, FasL and caspase-8, and for Bad, Bax, Bcl-w, Bcl-xL and caspase-9, for the FasL and the mitochondrial cytochrome-c pathways of apoptosis, respectively, and mRNA for the effector caspase-3 were quantified in lung tissues by RT-PCR. PTP and Src-PTK activities were also measured. RESULTS: After 3 hours of allogeneic perfusion, we observed a significant increase in: 1) the number of apoptotic cells in lung sections, 2) mRNA levels of FasL, Bcl-xL, caspase-8 and caspase-3, and 3) PTP activity (P < 0.05 compared with isogeneic perfusion). Surprisingly, mRNA levels of the proapoptotic genes Bad and Bax were significantly decreased (P < 0.05). PTK activity and caspase-9 mRNA level were not affected. Blocking anti-E-selectin mAbs and inhibitors for PKC, PTP, and PTK resulted in a significant reduction of apoptosis. CONCLUSIONS: In our model, the engagement of E-selectin induced by endothelial cell allogeneic activation appeared to be a prerequisite for lung apoptosis, which involved FasL and increase of PTP activity. Blockade of apoptosis with selective inhibitors may be a promising approach to the treatment/prevention of lung graft injury.     

转化生长因子-β1对脊髓损伤后神经细胞凋亡的影响

 目的 探讨转化生长因子(Transforming growth factor-β1,TGF-ββ1)对大鼠脊髓损伤后神 经细胞凋亡及神经动能恢复的影响。方法 采用改良 Allen爷s撞击法制作脊髓损伤大鼠模型, 根据干预 方式不同随机分为对照组、模型组、TGF-β1治疗组(渗透微泵持续蛛网膜下腔泵入 1μg/h TGF-β1)、 TGF-β1抗体组(渗透微泵持续蛛网膜下腔泵入 1μg/h TGF-β1中和抗体), 按设定的不同时间点随机处死各组的实验动物, 取材。应用 TUNEL法、RT-PCR、Western blot和免疫组织化学染色分别检测不同组 别在神经细胞凋亡, 脊髓组织细胞中 TGF-β1和 Fas的 mRNA及蛋白质表达差异。通过 BBB运动功能 评分法评估大鼠后肢运动功能恢复情况。结果 脊髓损伤后 TGF-β1和 Fas表达时间依赖性增加, Fas 表达 24h达高峰, TGF-β1表达 7d达高峰。脊髓损伤后神经细胞凋亡增加, 其中 8h神经元凋亡明显, 7 d神经胶质细胞凋亡明显。局部应用 TGF-β1, 可显著下调损伤脊髓组织 Fas表达, 减少 8h和 7d时神 经元凋亡及神经胶质细胞凋亡数量, BBB运动功能评分结果显示, TGF-β1治疗组大鼠后肢运动功能明显改善(P约0.01)。结论 脊髓损伤部位应用 TGF-β1可抑制 Fas mRNA及蛋白质的表达, 减少脊髓损伤 部位神经细胞的凋亡, 有利于促进脊髓神经功能的恢复。     

Mechanism of chronic obstructive uropathy: increased expression of apoptosis-promoting molecules

BACKGROUND: We have demonstrated that renal tubular and interstitial cells undergo pronounced apoptosis during the course of chronic obstructive uropathy (COU). Apoptosis is a complex cellular process consisting of multiple steps, each of which is mediated by families of related molecules. These families may include receptor/ligand molecules such as Fas, Fas ligand, tumor necrosis factor receptor-1 (TNFR-1), and TNF-related apoptosis inducing ligand (TRAIL); signal transduction adapter molecules such as Fas-associated death domain (FADD), TNFR-1 associated death domain (TRADD), receptor-interacting protein (RIP), Fas-associated factor (FAF), and Fas-associated phosphatase (FAP); or effector molecules such as caspases. However, the mechanism of tubular cell apoptosis, as well as the pathogenetic relevance of these apoptosis-related molecules in COU, remains poorly understood. METHODS: Kidneys were harvested from sham-operated control mice and mice with COU created by left ureter ligation sacrificed in groups of three at days 4, 15, 30, and 45. To detect apoptotic tubular and interstitial cells, in situ end labeling of fragmented DNA was performed. To detect the expression of apoptosis-related molecules, ribonuclease protection assay was used with specific antisense RNA probes for Fas, Fas ligand, TNFR-1, TRAIL, FADD, TRADD, RIP, FAF, FAP, and caspase-8. Immunostaining for Fas, Fas ligand, TRAIL, TRADD, RIP, and caspase-8 was also performed. To assess the role of these molecules in COU-associated renal cell apoptosis, the frequencies of apoptotic tubular and interstitial cells were separately quantitated for each experimental time point, and their patterns of variation were correlated with those of apoptosis-related molecules. RESULTS: The obstructed kidneys displayed increased apoptosis of both tubular and interstitial cells. Tubular cell apoptosis appeared at day 4 after ureter ligation, peaked (fivefold of control) at day 15, and decreased gradually until the end of the experiment. In contrast, interstitial cell apoptosis sustained a progressive increase throughout the experiment. Apoptosis was minimal at all experimental time points for control and contralateral kidneys. Compared with control and contralateral kidneys, the ligated kidneys displayed a dynamic expression of mRNAs for many apoptosis-related molecules, which included an up to threefold increase for Fas, Fas ligand, TNF-R1, TRAIL, TRADD, RIP, and caspase-8, and an up to twofold increase for FADD and FAP, but there was little change for FAF. These mRNAs increased between days 4 and 15, decreased until day 30, but then increased again until day 45. The rise and fall of mRNAs between days 4 and 30 paralleled a similar fluctuation in tubular cell apoptosis in that period. The subsequent increase of mRNAs was correlated with a continuous rise of interstitial cell apoptosis. We demonstrated a positive immunostaining for Fas and Fas ligand in the tubular cells at early time points as well as in interstitial inflammatory cells at later time points. Although increased expression of TRAIL, TRADD, RIP, and caspase-8 was noted in tubular cells, there was no staining for these molecules in interstitial cells. CONCLUSION: The current study documents a dynamic expression of several molecules that are known to mediate the most crucial steps of apoptosis. It implicates these molecules in COU-associated renal cell apoptosis and in the pathogenesis of this condition. It also lays the foundation for interventional studies, including genetic engineering, to evaluate the molecular control of apoptosis associated with COU.     

c-Jun NH2-terminal kinase-dependent Fas activation contributes to etoposide-induced apoptosis in p53-mutated prostate cancer cells

BACKGROUND: The death receptor, Fas, has recently been demonstrated to contribute the chemotherapeutic agents-induced apoptosis, however, the detail mechanisms have yet to be fully understood, especially in prostate cancer cells. METHODS: PC-3 and DU145 stably transfected with dominant negative form of Fas-associated death domain (FADD) or specific kinase of c-Jun NH2-terminal kinase (JNK) (mitogen-activated protein kinase kinase, MKK7) were selected in the presence of hygromycin B (Hyg B). Cell viability was examined by (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphonyl)-2H-tetrazolium, inner salt (MTS) assay or flowcytometric analysis using green fluorescent protein (GFP). Apoptosis was examined by DNA ladder, Western blotting analysis of cleaved caspases, or morphological analysis. The expression of Fas and JNK activation were investigated by Western blotting/flowcytometric analysis and in vitro kinase assay, respectively. RESULTS: Stimulation with etoposide significantly up-regulated Fas, and the death-inducing signaling complex (DISC) was formed in PC-3 and DU145. Stable transfection with dominant-negative FADD inhibited etoposide-induced apoptosis. In addition, stable transfection with dominant-negative MKK7, by which JNK activation was inhibited, canceled both the up-regulation of Fas and the formation of DISC by etoposide. Re-introduction of wild type p53 into PC-3 and DU145 completely suppressed these inhibitory effects. CONCLUSIONS: These results suggest that, in p53-mutated prostate cancer, JNK-initiated Fas-mediated apoptotic signals may play an important role in chemosensitivity.     

Overexpressed exogenous IL-4 And IL-10 paradoxically regulate allogenic T-cell and cardiac myocytes apoptosis through FAS/FASL pathway

BACKGROUND: The authors' previous study has shown that liposome-mediated ex vivo intracoronary interleukin (IL)-4 and IL-10 combined gene therapy suppressed the allo-immune responses and prolonged the cardiac allograft survival by 15 folds. However, the mechanism for promoting long-term allograft survival remains unknown. METHODS: This study tested the hypothesis that this combined cytokine gene targeting may promote alloreactive T-cell apoptosis or prevent apoptosis of cardiac allograft myocytes through Fas/Fas ligand (FasL) pathway. A rabbit functional cervical heterotopic heart transplantation model was used, and plasmid human recombinant IL-4 and IL-10 gene complexed with cationic liposome (GAP/DLRIE) was delivered into cardiac allografts by intracoronary infusion ex vivo. RESULTS: This liposome-mediated IL-4 and IL-10 combined gene therapy significantly increased apoptotic T cells detected by TUNEL staining. The caspase-8 or caspase-3 expressing T cells were also significantly increased. The Fas+ apoptotic T cells dominated in the population of apoptotic CD4+ T cells, but FasL+ CD4+ T-cell population was less effected in the combined gene therapy group. The effect of combined gene therapy on the infiltrative Fas+ CD8+ T-cell population is much less than that on Fas+ CD4+ cells, and there was almost no effect on the FasL+ CD8+ T-cell population. Furthermore, localized IL-4 and IL-10 combined gene therapy protected cardiac allograft myocytes by down-regulating its FasL expression, but not Fas. CONCLUSIONS: These results suggest that this combined gene targeting strategy which induced localized overexpression of exogenous IL-4 and IL-10 may promote alloreactive T-cell apoptosis and prevent myocytes apoptosis through Fas/FasL cell surface interaction, therefore inducing cardiac allograft tolerance.     

Curcumin modulates TLR4/NF-κB inflammatory signaling pathway following traumatic spinal cord injury in rats

Abstract

Background

Curcumin, a polyphenolic compound extracted from the plant turmeric, has protective effects on spinal cord injury (SCI) through attenuation of inflammatory response. This study was designed to detect whether curcumin modulates toll-like receptor 4 (TLR4) and the nuclear factor-kappa B (NF-κB) inflammatory signaling pathway in the injured rat spinal cord following SCI.

Methods

Adult male Sprague–Dawley rats were subjected to laminectomy at T8–T9 and compression with a vascular clip. There were three groups: (a) sham group; (b) SCI group; and (g) SCI + curcumin group. We measured TLR4 gene and protein expression by real-time polymerase chain reaction and western blot analysis; NF-κB activity by electrophoretic mobility shift assay, inflammatory cytokines tumor necrosis factor-α, interleukin-1β, and interleukin-6 levels by enzyme-linked immunosorbent assay, hindlimb locomotion function by Basso, Beattie, and Bresnahan rating, spinal cord edema by wet/dry weight method, and apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis.

Results

The results showed that SCI induced the up-regulation of TLR4, NF-κB, and inflammatory cytokines in the injured rat spinal cord. Treatment with curcumin following SCI markedly down-regulated the levels of these agents related to the TLR4/NF-κB inflammatory signaling pathway. Administration of curcumin also significantly ameliorated SCI induced hind limb locomotion deficits, spinal cord edema, and apoptosis.

Conclusions

Post-SCI curcumin administration attenuates the TLR4/NF-κB inflammatory signaling pathway in the injured spinal cord, and this may be a mechanism whereby curcumin improves the outcome following SCI.     

Pathways of caspase-mediated apoptosis in autosomal-dominant polycystic kidney disease (ADPKD)

BACKGROUND: We have recently demonstrated an increase in apoptosis in Han:SPRD rat kidneys with autosomal-dominant polycystic kidney disease (ADPKD). Caspase-3 and caspase-7 are major mediators of apoptosis. There are two pathways of caspase-3 and caspase-7-mediated apoptosis: (1) the "extrinsic" pathway involving the death receptor Fas, Fas ligand (FasL), and caspase-8 and (2) the "mitochondrial" or "intrinsic" pathway involving Bcl-2 proteins, caspase-2, cytochrome c release, and caspase-9. The aim of the present study was to investigate the pathways of apoptosis in 3-week-old Han:SPRD rats with ADPKD. METHODS: Fluorescent substrates were used to measure caspase activity. mRNA and protein was determined by ribonuclease protection assays and immunoblotting, respectively. The effect of caspase inhibitors on caspase activity in polycystic kidneys was determined. RESULTS: Caspase-3 and caspase-7 activity was more than 100% increased in homozygous (Cy/Cy) compared to heterozygous (Cy/+) and normal littermate control (+/+) kidneys. Ribonuclease protection assays demonstrated no difference in caspase-3 mRNA. On immunoblotting, there was an increase in the proform of caspase-3 and caspase-7 in Cy/Cy compared to +/+ and Cy/+ kidneys. Caspase-8 and caspase-9 activity was more than 100% increased in Cy/Cy compared to Cy/+ and +/+ kidneys. On immunoblotting, there was an increase of the proform of both caspase-8 and caspase-9 in Cy/Cy kidneys. There was also an increase in cytochrome c release into the cytosol and an increase in caspase-2 protein and activity in Cy/Cy kidneys. On ribonuclease protection assay there was no difference in FasL mRNA between +/+, Cy/+, and Cy/Cy kidneys. Short-term treatment of Cy/Cy rats with the caspase inhibitor IDN-8050 resulted in inhibition of caspase-3 and caspase-7 activity in the kidney. CONCLUSION: In Cy/Cy kidneys with ADPKD, there was an increase of the proform of caspase-9, an increase in cytochrome c release into the cytosol, and an increase in caspase-2 protein and activity demonstrating involvement of the intrinsic pathway. There was an increase in the proform of caspase-8 demonstrating involvement of the extrinsic pathway. No differences in FasL mRNA were seen suggesting that the extrinsic pathway is independent of the death receptor ligand, FasL.