冬凌草甲素通过改变Bax/Bcl-xL表达激活caspase-3诱导A375-S2细胞凋亡

目的　研究冬凌草甲素诱导人黑色素瘤A375 S2细胞凋亡的作用原理。方法　形态学观察 ,DNA凝胶电泳法及WesternBlot法。结果　冬凌草甲素能明显诱导A375 S2细胞发生凋亡 ,其作用呈明显的量效关系和时间依赖性。形态学观察可见凋亡小体的形成 ,琼脂糖凝胶电泳可见凋亡典型的DNA梯带 ;caspase 3的抑制剂能阻止caspase 3的活力升高 ;免疫印迹结果显示冬凌草甲素作用A375 S2细胞 12h改变Bax与Bcl xL蛋白的表达 ;且发现caspase 3的底物PARP蛋白在 12h时被降解。结论　冬凌草甲素 (34 3μmol·L-1)诱导A375 S2细胞凋亡 ,这种作用是通过改变了Bax/Bcl xL的表达比率 ,激活caspase 3而实现的。     

High cyclin-dependent kinase inhibitors in Bcl-2 and Bcl-xL-expressing CD34+-proliferating haematopoietic progenitors

We have previously described the isolation of primitive, slow-proliferating progenitors from normal, circulating CD34+ cells by using the fluorescent dye 5-6-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE). CFDA-SE(bright) (primitive) and CFDA-SE(dim) (differentiating) cells were isolated following cytokine stimulation on the basis of their different proliferation rates. In the present work we analysed the expression levels of a number of proteins involved with differentiation, proliferation and survival/apoptosis in CFDA-SE(bright)/CD34+/slow-proliferating cells that were previously defined as progenitors capable of differentiating into different lineages. The aim of this work was to gain a better understanding of our model system in order to define some of the important parameters that regulate differentiation in haematopoietic progenitors. GATA-1 and PU.1 RNA levels were similar in freshly isolated (d 0) CD34+ and in CFDA-SE(bright) (bright) cells, whereas they increased in CFDA-SE(dim) (dim) cells. Accordingly, Nm23 was expressed at higher levels in bright cells. Moreover, bright cells had higher p21WAF1/CIP1, p27KIP1 and p16Ink4 protein levels than dim cells. Consistently, Cdc2 and Cdk2 kinase activity was much higher in the dim than in the slower proliferating bright cells. C-myc and p53 levels were higher in bright cells than in d 0 CD34+ and dim cells, and so was Bcl-xL, which followed the trend we have previously described for Bcl-2. Thus, bright cells, despite having a higher proliferation rate than the starting d 0 CD34+ population, have strikingly elevated levels of cyclin-dependent kinase inhibitors, which are likely to also act as inhibitors of differentiation.     

Glucocorticoid Induced Leucine Zipper inhibits apoptosis of cardiomyocytes by doxorubicin

Doxorubicin (Dox) is an indispensable chemotherapeutic agent for the treatment of various forms of neoplasia such as lung, breast, ovarian, and bladder cancers. Cardiotoxicity is a major concern for patients receiving Dox therapy. Previous work from our laboratory indicated that glucocorticoids (GCs) alleviate Dox-induced apoptosis in cardiomyocytes. Here we have found Glucocorticoid-Induced Leucine Zipper (GILZ) to be a mediator of GC-induced cytoprotection. GILZ was found to be induced in cardiomyocytes by GC treatment. Knocking down of GILZ using siRNA resulted in cancelation of GC-induced cytoprotection against apoptosis by Dox treatment. Overexpressing GILZ by transfection was able to protect cells from apoptosis induced by Dox as measured by caspase activation, Annexin V binding and morphologic changes. Western blot analyses indicate that GILZ overexpression prevented cytochrome c release from mitochondria and cleavage of caspase-3. When bcl-2 family proteins were examined, we found that GILZ overexpression causes induction of the pro-survival protein Bcl-xL. Since siRNA against Bcl-xL reverses GC induced cytoprotection, Bcl-xL induction represents an important event in GILZ-induced cytoprotection. Our data suggest that GILZ functions as a cytoprotective gene in cardiomyocytes.     

Apoptosis and its pathway in X gene-transfected HepG2 cells

AIM: To investigate the effect of hepatitis B virus (HBV) X gene on apoptosis and expressions of apoptosis factors in X gene-transfected HepG2 cells. METHODS: The HBV X gene eukaryon expression vector pcDNA3-X was transiently transfected into HepG2 cells by lipid-media transfection. Untransfected HepG2 and HepG2 transfected with pcDNA3 were used as controls. Expression of HBx in HepG2 was identified by RT-PCR. MTT and TUNEL were employed to measure proliferation and apoptosis of cells in-three groups. Semi-quantified RT-PCR was used to evaluate the expression levels of Fas/FasL, Bax/Bcl-xL, and c-myc in each group. RESULTS: HBV X gene was transfected into HepG2 cells successfully. RT-PCR showed that HBx was only expressed in HepG2/pcDNA3-X cells, but not expressed in HepG2 and HepG2/pcDNA3 cells. Analyzed by MTT, cell proliferation capacity was obviously lower in HepG2/pcDNA3-X cells (0.08910±0.003164) than in HepG2(0.14410±0.004927) and HepG2/pcDNA3 cells (0.12150±0.007159) (P<0.05 and P<0.01). Analyzed by TUNEL, cell apoptosis was much more in HepG2/pcDNA3-X cells (980/2 000) than HepG2 (420/2 000), HepG2/pcDNA3 cells (520/2 000) (P<0.05 and P<0.01). Evaluated by semi-quantified RT-PCR, the expression level of Fas/FasL was significantly higher in HepG2 cells transfected with HBx than in HepG2 and HepG2/ pcDNA3 cells (P<0.05 and P<0.01). Bax/Bcl-xL expression level was also elevated in HepG2/pcDNA3-X cells (P<0.05 and P<0.01). Expression of c-myc was markedly higher in HepG2/pcDNA3-X cells than in HepG2 and HepG2/pcDNA3 cells (P<0.05 and P<0.01). CONCLUSION: HBV X gene can impair cell proliferation capacity, improve cell apoptosis, and upregulate expression of apoptosis factors. The intervention of HBV X gene on the expression of apoptosis factors may be a possible mechanism responsible for the change in cell apoptosis and proliferation.     

Na+/H+ exchanger mediates TNF-α-induced hepatocyte apoptosis via the calpain-dependent degradation of Bcl-xL

Background and Aim:  It is well known that tumor necrosis factor-α (TNF-α) induces hepatocyte apoptosis and contributes to liver diseases. However, the exact mechanisms are not well understood.
Methods:  In the present study, we reported that Na⁺/H⁺ exchanger (NHE) is involved in TNF-α-induced hepatocyte apoptosis.
Results:  TNF-α time dependently induced an increase in NHE activity in hepatocytes, but cariporide, an NHE inhibitor, blocked the TNF-α-induced increase of NHE activity in a dose-dependent manner. Increased NHE activity induced by TNF-α was associated with increased intracellular calcium (Ca²⁺_i) concentration and calpain activity. Cariporide reversed these effects induced by TNF-α. In addition, TNF-α downregulated Bcl-xL, an anti-apoptotic protein, but not mRNA levels. The inhibition of either calpain or NHE blocked the TNF-α-induced decrease of the Bcl-xL protein. TNF-α did not change the pro-apoptotic Bax and Bak protein levels. Cariporide, calcium remover 1,2-bis (2-aminophenoxy) ethane-N,N,N0,N0–tetraacetic acid, or calpain inhibitor benzyloxycarbonyl-leucyl-leucinal attenuated TNF-α-induced hepatocyte apoptosis.
Conclusion:  TNF-α via NHE results in hepatocyte apoptosis through the calcium/calpain/Bcl-xL pathway.     

Dilinoleoylphosphatidylcholine reproduces the antiapoptotic actions of polyenylphosphatidylcholine against ethanol-induced hepatocyte apoptosis

BACKGROUND: Polyenylphosphatidylcholine (PPC), a mixture of polyunsaturated phosphatidylcholines extracted from soybeans, attenuates hepatocyte apoptosis induced by ethanol feeding of rats. Our aims were to evaluate whether dilinoleoylphosphatidylcholine (DLPC), the main component of PPC, reproduces the antiapoptotic actions of PPC against alcohol-induced apoptosis and to identify the apoptotic proteins that are affected by PPC and DLPC. METHODS: Rats were fed Lieber-DeCarli liquid diets containing ethanol (35% of energy) or an isocaloric amount of carbohydrate for 4 weeks. Another group of rats were given the ethanol diet supplemented with PPC (3 g/liter) or DLPC (1.5 and 3 g/liter). Hepatocyte apoptosis was assessed by terminal transferase-mediated dUTP nick end labeling staining and by caspase 3 enzyme activity. Activity of caspases 3 and 9 was assayed by using fluorogenic peptide substrates. Cytochrome c was quantified by enzyme-linked immunosorbent assay. The protein contents of cytochrome c, procaspase 3, caspase 3, Bcl-x(L), and Bax were analyzed by Western blot and quantified by densitometry. Lobular localization of active caspase 3 was examined by immunoperoxidase staining. RESULTS: PPC and DLPC decreased ethanol-induced increases in hepatocyte apoptosis, cytosolic cytochrome c, and caspase 3 content and its activity. Caspase 3 activity correlated with the number of apoptotic hepatocytes. Active caspase 3 was present predominantly in perivenular hepatocytes, and ethanol feeding extended it to lobular hepatocytes; this ethanol effect was reduced by PPC and DLPC. Ethanol significantly decreased Bcl-x(L) in homogenate, mitochondria, and cytosol, and there was a trend for increased Bcl-x(L) in these fractions after PPC and DLPC supplementation. Microsomal Bcl-x(L) did not differ between treatment groups. Bax was detected in homogenate and cytosol, and its level was not affected by ethanol. CONCLUSIONS: DLPC, at a dose contained in PPC, reproduces the antiapoptotic actions of PPC through a reduction in cytosolic cytochrome c concentration and caspase 3 activity, possibly in association with up-regulation of Bcl-x(L) expression. Because DLPC is a pure and well defined compound, it may be more suitable than PPC for intervention against alcohol-induced apoptosis.     

Reduced expression of both Bax and Bcl-2 is independently associated with lymph node metastasis in human breast carcinomas

Imbalance between pro-apoptotic and anti-apoptotic proteins, causing altered apoptosis, may lead to tumour development and tumour progression, and reduced response to adjuvant therapy. In this study, we evaluated the expression patterns of Bcl-2, Bcl-xL, and Bax protein in 126 primary invasive breast carcinomas, and the association with other clinicopathological parameters. We used immunohistochemical methods to evaluate protein expression. Reduced expression of both Bax and Bcl-2 was associated with lymphnode metastases in univariate analyses (one-way ANOVA) as well as in multivariate analysis (binary logistic regression) (Bcl-2 p=0.003 univariate, p=0.01 multivariate, Bax p=0.05 univariate, p=0.03 multivariate). Bcl-2 overexpression showed an inverse association with cyclin A (p=0.05), while expression of Bcl-xL showed an association only with cyclin D3 (p=0.04). Bcl-xL expression also showed a highly significant association with oestrogen receptor status (p=0.009). Bcl-2 and Bcl-xL showed an association with different D-type cyclins, indicating different pathways of pathogenesis. Expression of Bcl-2 was associated with better patient survival in univariate analysis (Kaplan meyer p=0.04), but lost its prognostic value in multivariate analysis (Cox regression p=0.2).     

外周血中BAFF、BAFF-R及Bcl-xL的表达与B细胞非霍奇金淋巴瘤疾病进展的相关性研究

目的研究B细胞性非霍奇金淋巴瘤(B-NHL)患者外周血B cell-activating factor(BAFF)、其受体BAFF-R及Bcl-xL基因的表达,探讨其与B-NHL疾病进展的相关性。方法应用实时荧光定量PCR法检测36例B-NHL患者外周血单个核细胞(PBMC)BAFF、BAFF-R、Bcl-xL mRNAs的相对表达水平,以7例健康献血员作为对照,并据性别、淋巴瘤国际预后指数(IPI)、年龄、临床分期、是否耐药进行分组比较。结果 B-NHL患者PBMC BAFF、BAFF-R及Bcl-xL的mRNAs表达较正常对照组显著升高(P<0.05),Ⅲ/Ⅳ期高于Ⅰ/Ⅱ期,耐药B-NHL组高于非耐药组(P<0.05)。结论外周血单个核细胞BAFF、BAFF-R、Bcl-xL mRNAs的表达水平与B-NHL临床分期有关,在耐药B-NHL外周血中明显升高,提示BAFF/BAFF-R介导的信号通路可能参与B-NHL的临床进展及耐药机制。     

Stat3通路相关基因在散发性大肠管状腺瘤癌变中的表达及意义

目的 探讨Stat3及CyclinD1、Bcl-xL在散发性大肠管状腺瘤癌变过程中的可能作用。方法 采用免疫组化方法检测107例散发性大肠管状腺瘤异型增生及癌变组织中Stat3和CyclinD1、Bcl-xL在蛋白水平的表达情况。结果 腺瘤伴异型增生组和癌变组中的Stat3蛋白的表达明显高于正常大肠黏膜组(P=0.000),且癌变组中Stat3蛋白的表达高于腺瘤伴异型增生组(P=0.003);腺瘤伴异型增生组和癌变组中CyclinD1蛋白的表达均明显高于正常大肠黏膜组(P=0.000),且癌变组中CyclinD1蛋白的表达高于腺瘤伴异型增生组(P=0.000);大肠管状腺瘤伴异型增生组和癌变组中Bcl-xL在蛋白水平的阳性表达率均明显高于正常大肠黏膜组（P=0.000),而且癌变组中Bcl-xL的表达明显高于腺瘤伴异型增生组(P=0.000);Stat3、CyclinD1和Bcl-xL均随腺瘤异型增生程度的增高呈递增趋势。Stat3与CyclinD1、Bcl-xL 的表达存在正相关关系。结论 Stat3、CyclinD1和Bcl-xL可能在散发性大肠管状腺瘤癌变过程中起一定的作用。