肝复康经JNK信号通路在肝星状细胞凋亡中的调节机制

目的探讨中药肝复康经JNK(c-Jun N-terminal kinase,JNK)信号通路在肝星状细胞凋亡中的调节机制。方法将SD大鼠随机分为5组:正常对照组(C),模型组(M),肝复康高(HT)、中(MT)、低(LT)剂量治疗组,每组给予相应的处理;HSC-T6细胞株随机分为3组:正常对照组、乙醛组及肝复康治疗组。HE染色法观察肝组织形态结构变化;RT-PCR法测定MKK4、MKK7、JNK1、JNK2、COX-2、Survivin、Caspase-3、α-SMA、collagenⅠ和collagenⅢ的基因表达水平;免疫组织化学法和免疫蛋白印记法观察COX-2在肝组织和肝星状细胞中的表达。结果正常组中COX-2几乎不表达。与正常组相比,模型组中COX-2表达显著增加(P<0.05)。与模型组相比,肝复康治疗组各组中COX-2表达均明显降低,其中,中剂量组最明显(P<0.05)。与正常组相比,模型组中MKK4、MKK7、JNK1、JNK2、COX-2、Survivin的基因表达水平均显著上调(P<0.05)。与模型组相比,肝复康治疗组各基因表达水平均明显减弱,中剂量组最明显(P<0.05),其中Caspase-3基因表达呈相反趋势。COX-2和Survivin的表达呈正相关,和Caspase-3的表达呈负相关。检测α-SMA、collagenⅠ与collagenⅢ基因表达,与模型组相比,中剂量治疗组表达水平明显下调(P<0.05)。结论肝复康可通过抑制JNK信号通路的传导诱导肝星状细胞凋亡,发挥抗纤维化作用。     

Oleanolic acid induces apoptosis of MKN28 cells via AKT and JNK signaling pathways

Context: Oleanolic acid (OA) belongs to the triterpenoid compound group existing widely in food, medicinal herbs and other plants. Its effects on gastric cancer cells and the mechanisms involved have not been investigated.

Objective: This study aimed to substantiate whether OA induces apoptosis of gastric cancer cell line (MKN28) and to elucidate the molecular mechanism involved.

Materials and methods: Cell viability was assessed by MTT assay within the range of 0–160?μg/mL. The effects of OA (5, 10 and 20?μg/mL) on apoptosis of MKN28 cells were evaluated by flow cytometry, DNA fragmentation and mitochondrial membrane potential assays. Western blot and FQRT-PCR assays were used to investigate the mechanism of cell apoptosis induced by OA (5 and 10?μg/mL).

Results: OA evidently inhibited cell viability with IC₅₀ of 44.8 and 15.9?μg/mL at 12 and 24?h, respectively. Furthermore, OA increased JNK phosphorylation, decreased AKT phosphorylation, but did not affect p38 and ERK phosphorylation in MKN28 cells. In contrast, OA also significantly enhanced the mRNA expression levels of caspase 3, caspase 9 and Apaf-1 in MKN28 cells.

Conclusion: OA induces apoptosis of MKN28 cells via the mitochondrial pathway regulated by AKT and JNK signaling pathways.     

人参皂甙Rg1对大鼠关节软骨细胞凋亡的拮抗作用

目的 探讨人参皂甙Rg1对软骨细胞凋亡的影响.方法 正常大鼠关节软骨细胞分为三组:A、B组用白细胞介素1β(IL-1β)10 ng/ml作用24 h,建立软骨细胞凋亡模型;B组事先加入10 μg/ml的人参皂甙Rg1保护2h;C组作为空白对照.TUNEL法检测人参皂甙Rg1对软骨细胞凋亡的影响;Western blot检测人参皂甙Rg1对凋亡软骨细胞基质金属蛋白酶13(MMP-13)和基质金属蛋白酶组织抑制剂1(TIMP-1)蛋白表达的影响.结果 A、B和C组细胞凋亡率分别为(37.30±4.05)％、(10.30±1.08)％和(2.20±1.93)％,B组胞凋亡率低于A组(P＜0.05).与A组比较,B组软骨细胞TIMP-1的表达增加,MMP-13的表达降低(P＜0.05).结论 人参皂甙Rg1具有拮抗大鼠关节软骨细胞凋亡的作用.     

Cell apoptosis induced by a synthetic carbazole compound LCY-2-CHO is mediated through activation of caspase and mitochondrial pathways

The mechanisms involved in the apoptotic effect of LCY-2-CHO [9-(2-chlorobenzyl)-9H-carbazole-3-carbaldehyde], a synthetic carbazole derivative identified as an anti-inflammatory compound, were studied. Cell cycle analysis by propidium iodide staining in human THP-1 monocytic leukemia cells showed the ability of LCY-2-CHO to increase cell population in sub-G1 stage with time- and concentration-dependent manners. LCY-2-CHO-mediated cell death was also demonstrated by DNA laddering and was not related to the release of lactate dehydrogenase. Apoptosis in THP-1 cells induced by LCY-2-CHO was accompanied by the Bid cleavage, collapse of mitochondrial transmembrane potential, the release of cytochrome c and the activation of caspase-3. The apoptotic effect of LCY-2-CHO was diminished by the presence of zVEID-fmk (caspase-6 inhibitor), zIETD-fmk (caspase-8 inhibitor), and zVAD-fmk (non-selective caspase inhibitor), but was not altered by several antioxidants, and cathepsin inhibitor. The Bid cleavage and loss of mitochondrial transmembrane potential, but not the cytochrome c release, were reversed by zIETD-fmk. Comparing the cell selectivity of LCY-2-CHO, we found T-cell acute lymphoblastic CEM leukemia cells were sensitive to 1 microM LCY-2-CHO, acute myeloid leukemia HL-60 cells underwent apoptosis at 10 microM, while adherent cancer cells, such as PC3, HT29 and MCF-7, were resistant to 30 microM LCY-2-CHO within 24-h incubation. Taken together in the present study, we demonstrated LCY-2-CHO might be apoptotic for malignant hematopoietic cells but not anchorage-dependent cells. This action is mediated by an intrinsic caspase-dependent apoptotic event involving mitochondria.     

An inhibitor of p38 and JNK MAP kinases prevents activation of caspase and apoptosis of cultured cerebellar granule neurons

Both p38 mitogen-activated protein kinase (p38) and c-Jun N-terminal kinase (JNK) are known to play important roles in neuronal apoptosis. However, the relationship between these kinases and caspases, another key mediator of apoptosis, is unclear. In the present study, we investigated the possible effects of SB203580 [(4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-i mid azole], an inhibitor of p38, on caspase activation and apoptosis of cultured rat cerebellar granule neurons. In granule neurons, SB203580 prevented apoptosis that was induced by lowering the concentration of KCl in the culture medium for 24 hr. SB203580 also prevented augmentation of caspase-3-like protease activity at 8 hr after the low KCl treatment. The IC50 values of SB203580 for both events were between 3 microM and 10 microM. Expression and phosphorylation of c-Jun, potently induced by low KCl treatment, were prevented by SB203580 at 10 microM. Z-Asp-CH2-DCB, a caspase inhibitor with anti-apoptotic activity, did not inhibit the induction and phosphorylation of c-Jun. Granule neurons displayed high levels of p38 and JNK activities. SB203580 inhibited not only p38 but also JNK activities extracted from granule neurons. These results suggest that activation of c-Jun by p38 and/or JNK mediates the activation of caspase in the low KCl-induced apoptosis in cerebellar granule neurons.     

Effect of alkyltins on rabbit articular and growth-plate chondrocytes in monolayer culture

The effect of four different alkyltins (trimethyltin, triethyltin, dibutyltin, and dioctyltin) on the metabolism of rabbit articular and growth-plate chondrocytes was investigated using a monolayer cell-culture system. In most instances the compounds tested exhibited a general cytotoxic effect on these cells, inhibiting the synthesis of both DNA and sulfated proteoglycans. The effect of these compounds on proteoglycan synthesis was both quantitative and qualitative, as demonstrated by CsCl isopycnic density gradient centrifugation and gel exclusion chromatographic techniques. However, certain tin compounds tested, at specific concentrations, exerted a stimulatory effect on chondrocyte proliferation. Regarding DNA synthesis, growth-plate chondrocytes were more sensitive to the effect of the triethyltin, dibutyltin, and dioctyltin than were articular chondrocytes. The data are discussed in relation to the possible effects of the alkyltins on skeletal growth and development as well as the mechanism of action of the alkyltins at the molecular level.     

The effects of razoxane (ICRF 159) on the production of collagenase and inhibitor (TIMP) by stimulated rabbit articular chondrocytes

Monolayer cultures of rabbit chondrocytes were stimulated to produce collagenase with conditioned medium from human peripheral blood mononuclear cells (MCM), and the ability of Razoxane to modulate the production of collagenase and specific tissue inhibitor of metalloproteinases (TIMP) was studied. Collagenase production was inhibited and TIMP increased by Razoxane, in a dose-dependent manner, when cells were treated daily for 3 days. Over this period the effect of Razoxane was progressive; 50 micrograms/ml or less had no effect at day 1 but 50 micrograms/ml was effective by day 3. The effectiveness of Razoxane was inversely related to the degree of MCM stimulation and the confluency of the culture. On removal of the drug, chondrocytes stimulated with MCM recovered their ability to produce collagenase, and TIMP production returned to near normal. The results suggest that the ability of Razoxane to reduce collagenase and increase TIMP production may correlate with its effectiveness in treating psoriatic arthritis.     

双氢青蒿素诱导肿瘤细胞凋亡的信号通路研究进展

双氢青蒿素作为青蒿素的重要衍生物,是我国自行研制出的抗疟新药。近些年来,人们对双氢青蒿素进行了多方面的研究,在抗肿瘤研究方面有许多新进展,发现其诱导肿瘤细胞凋亡的机制与多条信号通路有关,例如PI3K/Akt、MAPK、STAT3、Wnt/β-catenin、NF-κB等信号通路。通过对作用机制的深入研究和阐述有望发现双氢青蒿素的新适应症,使其在临床上的应用更加广泛。     

Ofloxacin induces apoptosis in microencapsulated juvenile rabbit chondrocytes by caspase-8-dependent mitochondrial pathway

Quinolones (QNs)-induced arthropathy is an important toxic effect in immature animals leading to restriction of their therapeutic use in pediatrics. However, the exact mechanism still remains unclear. Recently, we have demonstrated that ofloxacin, a typical QN, induces apoptosis of alginate microencapsulated juvenile rabbit joint chondrocytes by disturbing the beta 1 integrin functions and inactivating the ERK/MAPK signaling pathway. In this study, we extend our initial observations to further elucidate the mechanism(s) of ofloxacin-induced apoptosis by utilizing specific caspase inhibitors. Pretreatment with both caspase-9-specific inhibitor zLEHD-fmk and caspase-8 inhibitor zIETD-fmk attenuated ofloxacin-induced apoptosis and activation of caspase-3 of chondrocyte in a concentration-dependent manner, as determined by fluorescent dye staining, enzyme activity assay and immunoblotting. Furthermore, the activation of caspase-9, -8 and -3 stimulated by ofloxacin was significantly inhibited in the presence of zIETD-fmk while pretreatment with zLEHD-fmk only blocked the activation of caspase-9 and -3. Ofloxacin also stimulated a concentration-dependent translocation of cytochrome c from mitochondria into the cytosol and a decrease of mitochondrial transmembrane potential, which was completely inhibited by zIETD-fmk. In addition, ofloxacin was found to increase the level of Bax, tBid, p53 in a concentration- and time-dependent manner. Taken together, The current results indicate that the caspase-8-dependent mitochondrial pathway is primarily involved in the ofloxacin-induced apoptosis of microencapsulated juvenile rabbit joint chondrocytes.     

The JNK,ERK and p53 pathways play distinct roles in apoptosis mediated by the antitumor agents vinblastine,doxorubicin, and etoposide

Assessment of specific apoptosis and survival pathways implicated in anticancer drug action is important for understanding drug mechanisms and modes of resistance in order to improve the benefits of chemotherapy. In order to better examine the role of mitogen-activated protein kinases, including JNK and ERK, as well as the tumor suppressor p53, in the response of tumor cells to chemotherapy, we compared the effects on these pathways of three structurally and functionally distinct antitumor agents. Drug concentrations equal to 50 times the concentration required to reduce cell proliferation by 50% were used. Vinblastine, doxorubicin, or etoposide (VP-16) induced apoptotic cell death in KB-3 carcinoma cells, with similar kinetic profiles of PARP cleavage, caspase 3 activation, and mitochondrial cytochrome c release. All three drugs strongly activated JNK, but only vinblastine induced c-Jun phosphorylation and AP-1 activation. Inhibition of JNK by SP600125 protected cells from drug-induced cytotoxicity. Vinblastine caused inactivation of ERK whereas ERK was unaffected in cells exposed to doxorubicin or VP-16. Inhibition of ERK signaling by the MEK inhibitor, U0126, potentiated the cytotoxic effects of vinblastine and doxorubicin, but not that of VP-16. Vinblastine induced p53 downregulation, and chemical inhibition of p53 potentiated vinblastine-induced cell death, suggesting a protective effect of p53. In contrast, doxorubicin and VP-16 induced p53, and inhibition of p53 decreased drug-induced cell death, suggesting a pro-apoptotic role for p53. These results highlight the differential roles played by several key signal transduction pathways in the mechanisms of action of key antitumor agents, and suggest ways to specifically potentiate their effects in a context-dependent manner. In addition, the novel finding that JNK activation can occur without c-Jun phosphorylation or AP-1 activation has important implications for our understanding of JNK function.     

Colchicine-induced apoptosis in human normal liver L-02 cells by mitochondrial mediated pathways

Colchicine is an alkaloid that has been widely used to treat gout. It also has a curative effect on cancer. Although many studies have shown that its effect on cell apoptosis was mediated by the activation of caspase-3, the pathways involved in the process remained obscure. Here we show some evidence regarding the missing information using human normal liver cells L-02 in our study. The effect of colchicine on apoptosis in L-02 cells and the apoptosis-associated signaling pathways were determined using different tests including cell viability assay, Annexin V and propidium idodide binding, PI staining, Hoechst 33342 staining, mitochondrial membrane potential assay, caspase activity assay and Western blot analysis. We found that colchicine-induced a dose-dependent drop of cell viability in L-02 cells; early apoptosis happened when cells were treated with 0.1 μM of colchicine. The colchicine-induced loss of mitochondrial membrane potential, activation of caspase-3 and 9, up-regulation of Bax and down-regulation of Bcl-2 showed an evidence for the colchicine activity on apoptosis, at least, by acting via the intrinsic apoptotic pathway.     

Protection against TNF-induced liver parenchymal cell apoptosis during endotoxemia by a novel caspase inhibitor in mice

Excessive apoptotic cell death is implicated in a growing number of acute and chronic disease states. Caspases are critical for the intracellular signaling pathway leading to apoptosis. The aim of this investigation was to evaluate the efficacy and the mechanism of action of the novel caspase inhibitor CV1013 in a well-characterized model of TNF-induced apoptosis. Administration of 700 mg/kg galactosamine/100 microg/kg endotoxin (Gal/ET) induced hepatocellular apoptosis in C3Heb/FeJ mice as indicated by increased caspase-3 activity (706% above controls) and enhanced DNA fragmentation (3400% above controls) at 6 h. In addition, apoptosis was aggravated by the neutrophil-induced injury at 7 h (ALT activities: 4220 +/- 960 U/L and 48 +/- 4% necrosis). All animals died 8-12 h after Gal/ET treatment from shock and liver failure. A dose of 10 or 1 mg/kg of CV1013 administered three times (3, 4.5, and 5.5 h after Gal/ET) effectively prevented caspase-3 activation and parenchymal cell apoptosis at 6 h as well as the subsequent neutrophil-induced aggravation of the injury at 7 h after Gal/ET treatment. Animals treated with 10 mg/kg CV1013 survived for 24 h without liver injury. CV1013 reduced the processing of caspase-3 and caspase-8. This suggests that CV1013 may have inhibited the small amount of active caspase-8 generated at the receptor level. Because of the multiple amplification loops used to activate the entire caspase cascade, blocking the initial intracellular signal by CV1013 was highly effective in preventing apoptotic cell death. CV1013 has therapeutic potential for disease states with excessive apoptosis.     

Enhanced susceptibility to kindling by chlordimeform may be mediated by a local anesthetic action

The formamidine pesticides amitraz and chlordimeform have recently been shown to be potent proconvulsants (Gilbert 1988). Two main neuroactive properties have been identified as mediators of formamidine neurotoxicity, -2 adrenergic agonism and local anesthetic actions. These two proposed mechanisms of formamidine action were contrasted using electrical kindling of the amygdala. Male rats were administered 0, 10 and 40 mg/kg of the local anesthetic lidocaine, 0, 0.01 and 0.10 mg/kg of the -2 adrenergic agonist clonidine or 0, 10 and 30 mg/kg chlordimeform, IP, once per day. After each injection, kindling stimulation was delivered through chronically-implanted electrodes. The high dosage of chlordimeform and both dosages of lidocaine enhanced the rate of kindling development ( sessions to stage 5 seizures=8.6±1.16, 10.15±1.04 and 8.5±0.95, respectively) relative to controls ( =14.59±1.36). Afterdischarge (AD) durations were increased over the first seven sessions by both treatments, but the total cumulative AD did not differ from controls. Clonidine, by contrast, delayed kindling development ( =27.57±1.97) and shortened the mean AD duration over the first seven sessions. These data provide support for a local anesthetic action of chlordimeform and stand in contrast to several recent demonstrations of -2 activity of formamidines as a primary contributor to formamidine toxicity.Although the research described in this article has been supported by the United States Environmental Protection Agency (through contract 68-02-4450 to NSI-Environmental Sciences), it has not been subjected to Agency review and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred. Mention of trade names or commerical products does not constitute endorsement or recommendation for use     

Dual intracellular signaling pathways mediated by the human cannabinoid CB1 receptor.

It has long been established that the cannabinoid CB1 receptor transduces signals through a pertussis toxin-sensitive Gi/Go inhibitory pathway. Although there have been reports that the cannabinoid CB1 receptor can also mediate an increase in cyclic AMP levels, in most cases the presence of an adenylyl cyclase costimulant or the use of very high amounts of agonist was necessary. Here, we present evidence for dual coupling of the cannabinoid CB receptor to the classical pathway and to a pertussis toxin-insensitive adenylyl cyclase stimulatory pathway initiated with low quantities of agonist in the absence of any costimulant. Treatment of Chinese hamster ovary (CHO) cells expressing the cannabinoid CB1 receptor with the cannabinoid CP 55,940, {(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hyd roxypropyl) cyclohexan-1-ol} resulted in cyclic AMP accumulation in a dose-response manner, an accumulation blocked by the cannabinoid CB1 receptor-specific antagonist SR 141716A, {N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide hydrochloride}. In CHO cells coexpressing the cannabinoid CB1 receptor and a cyclic AMP response element (CRE)-luciferase reporter gene system, CP 55,940 induced luciferase expression by a pathway blocked by the protein kinase A inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide hydrochloride (H-89). Under the same conditions the peripheral cannabinoid CB2 receptor proved to be incapable of inducing cAMP accumulation or luciferase activity. This incapacity allowed us to study the luciferase activation mediated by CB /CB2 chimeric constructs, from which we determined that the first and second internal loop regions of the cannabinoid CB1 receptor were involved in transducing the pathway leading to luciferase gene expression.     

Mig-2 attenuates cisplatin-induced apoptosis of human glioma cells in vitro through AKT/JNK and AKT/p38 signaling pathways

Aim:

Mig-2 (also known as Kindlin-2 and FERMT2) is an important regulator of integrin activation and cell-extracellular matrix adhesion, and involved in carcinogenesis and tumor progression. The aim of this study was to investigate the role of mig-2 in cisplatin-induced apoptosis of human glioma cells in vitro.

Methods:

The expression of mig-2 was modulated in human glioma H4, HS 683 and U-87 MG cells by transfection with a plasmid carrying mig-2 or mig-2 siRNA. Cisplatin-induced apoptosis was detected using Annexin V/PI staining and flow cytometry, as well as MTS analyses. The expression of apoptosis-related or signaling proteins was examined using Western blotting analysis. H4 cells were transfected with plasmids carrying mig-2 mutants to determine the functional domain of mig-2.

Results:

In the 3 glioma cell lines tested, overexpression of mig-2 significantly attenuated cisplatin-induced apoptosis, whereas knock-down of mig-2 potentiated the apoptosis. The mechanisms of action of mig-2 were further addressed in H4 cells: overexpression of mig-2 markedly reduced cleaved caspase-9, caspase-8, caspase-3 and PARP, as well as p-JNK and p-p38, and increased p-AKT in cisplatin-treated H4 cells, whereas mig-2 siRNA reversely changed these apoptosis-related and signaling proteins. Furthermore, pretreatment with JNK inhibitor SP600125 and p38 inhibitor SB203580, or with AKT inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 abolished the effects of mig-2 on cisplaxtin-induced apoptosis. In H4 cells, GFP-mig-2 F3 plasmid that contained only the F3 subdomain showed the same efficiency in attenuating cisplatin-induced apoptosis, as the mig-2 wild-type vector did, whereas GFP-mig-2 (1-541) plasmid that lacked the F3 subdomain was inactive.

Conclusion:

Mig-2 significantly attenuates the antitumor action of cisplatin against human glioma cells in vitro through AKT/JNK and AKT/p38 signaling pathways. The F3 subdomain of mig-2 is necessary and sufficient for this effect.     

Norcantharidin induces apoptosis in HeLa cells through caspase, MAPK, and mitochondrial pathways

AIM: To investigate the mechanism of norcantharidin (NCTD)-induced HeLa cell apoptosis. METHODS: HeLa cell growth inhibition was measured by MTT method. Apoptosis was detected by Hoechst 33258 staining and agarose gel electrophoresis. Caspase activities were assayed using caspase apoptosis detection kit. Western blot analysis was used to evaluate the level of ICAD, ERK/p-ERK, JNK/p-JNK, and Bcl-XL/Bax expression. RESULTS: Norcantharidin inhibited HeLa cell growth in a time- and dose-dependent manner. HeLa cells treated with norcantharidin showed typical characteristics of apoptosis including the morphological changes and DNA fragmentation. Caspase family inhibitor (z-VAD-fmk), caspase-8, -9 inhibitor (z-IETD-fmk, Ac-LEHD-CHO, respectively) and caspase-3 inhibitor (z-DEVD-fmk) partially prevent norcantharidin-induced apoptosis, but initiator caspase-1 inhibitor (Ac-YVAD-fmk) did not. The activities of caspase-3, -8, and -9 were up-regulated after norcantharidin treatment. Furthermore, NCTD-induced activat     

薯蓣苷元诱导人黑素瘤细胞A375-S2凋亡依赖半胱天冬酶和MAPK途径

目的　研究薯蓣苷元诱导人黑素瘤细胞A375 S2凋亡的机制。方法　MTT法测定细胞生长抑制率及半胱天冬酶和有丝分裂原活化的蛋白激酶(MAPK)抑制剂对薯蓣苷元诱导细胞凋亡的影响。电镜观察细胞形态变化。流式细胞仪检测细胞凋亡及周期分布。用半胱天冬酶活力检测试剂盒测定半胱天冬酶活力。结果　薯蓣苷元抑制A375 S2细胞的生长呈时间 剂量依赖性。经薯蓣苷元处理后的A375 S2细胞表现出典型的凋亡特征 :细胞表面微绒毛消失、胞质空泡化、染色质浓集、边聚。流式细胞仪检测表明薯蓣苷元导致DNA片段化和细胞周期阻滞在G0 /G1期。半胱天冬酶家族抑制剂 (z VAD fmk )和p38MAPK抑制剂 (SB2 0 35 80 )可部分抑制薯蓣苷元诱导的A375 S2细胞凋亡。结论薯蓣苷元可诱导A375 S2细胞凋亡。其诱导凋亡作用与半胱天冬酶及MAPK的活化相关。