Fibrate prevents cisplatin-induced proximal tubule cell death

BACKGROUND: In previous studies we have shown that cisplatin inhibits peroxisome proliferator-activated receptor-alpha (PPAR-alpha) activity and consequently fatty acid oxidation, and these events precede proximal tubule cell death. In addition the use of fibrate class of PPAR-alpha ligands ameliorate renal function by preventing both inhibition of fatty acid oxidation and proximal tubule cell death. METHODS: LLC-PK1 cells were treated with cisplatin and apoptosis was established by the presence of nuclear fragmentation and by cell cycle analysis. Proximal tubular cells treated with cisplatin and bezafibrate were subjected to sub cellular fractionation and the presence of Bax, Bcl-2, cytochrome c, and active caspase-3 in the cytosolic and mitochondrial membrane fractions was determined by Western blot analysis. PPAR-alpha activity was measured by determining luciferase activity after transfection of LLC-PK1 cells with TK-Luc 3x PPAR response elements (PPRE), and the accumulation of nonesterified free fatty acids was measured in lysates obtained from cells treated with cisplatin and bezafibrate. RESULTS: Incubation of LLC-PK1 cells with 25 micromol/L cisplatin for 18 hours induced 41.5% apoptosis measured by cell cycle analysis. Cisplatin-induced apoptosis was significantly suppressed by bezafibrate, a fibrate class of PPAR-alpha ligand. Bezafibrate treatment of LLC-PK1 cells prevented cisplatin-induced translocation of proapoptotic Bax from the cytosol to the mitochondrial fraction, and increased the expression of antiapoptotic molecule Bcl-2. Cisplatin-induced inhibition of PPAR-alpha activity was accompanied by increased accumulation of nonesterified free fatty acids. Pretreatment with bezafibrate prevented both the inhibition of PPAR-alpha activity and the accumulation of nonesterified free fatty acids induced by cisplatin. Finally, bezafibrate prevented cisplatin-induced release of cytochrome c from the mitochondria to the cytosol, and the cleavage of procaspase-3 to active caspase-3. CONCLUSION: Bezafibrate treatment inhibits cisplatin-mediated tubular injury by preventing the activation of various cellular mechanisms that lead to proximal tubule cell death. These findings support our previous observations where the use of fibrates represents a novel strategy to ameliorate proximal tubule cell death in cisplatin-induced acute renal failure.     

周期素依赖激酶2在非小细胞肺癌中的表达及其临床意义

目的 观察细胞周期因子周期素依赖激酶2(CDK2)在非小细胞肺癌(NSCLC)中的表达，探讨其临床意义。方法 采用免疫组织化学链霉素亲生物蛋白—过氧化酶(S—P)法，观察66例临床资料完整的NSCLC组织和12例正常肺组织中CDK2的表达，并对其与临床预后的关系进行相关分析。结果 在66例NSCLC患者中，CDK2在细胞核呈阳性免疫反应，其阳性表达率为84．85％。CDK2表达对年龄、性别、组织学类型、pTNM分期和淋巴结转移均无显著的影响(P＞O．05)，而对肿瘤的大小、病理分级有显著影响(P＜O．05)。CDK2表达阳性者与阴性者比较生存时间明显缩短(P＝O．O14)，CDK2阳性患者估计增加相对风险为2．5。结论 CDK2在NSCLC组织中细胞核呈阳性免疫反应，其过度表达与NSCLC患者临床病理特征和预后不良有关。     

Growth factor-mediated phosphorylation of proapoptotic BAD reduces tubule cell death in vitro and in vivo

BACKGROUND: Exogenous growth factors administered during unilateral ureteral obstruction (UUO) in neonatal rats significantly reduce apoptosis and tubular atrophy. Because the mechanism underlying these salutary effects is largely unknown, we investigated signaling pathways potentially activated by growth factors to determine their roles in therapeutic action. METHODS: Mechanical strain was applied to confluent cultures of immortalized rat proximal tubule cells to simulate obstruction-induced stretch injury in vivo. Growth factors, inhibitory antibodies or pharmacological inhibitors were added to cultures that were subsequently processed for TUNEL analysis or immunoblots to identify signaling pathways that could be modulating cell survival. For in vivo studies, kidneys harvested from rats +/- UUO +/- epidermal growth factor (EGF) were fixed or frozen for immunohistochemistry or immunoblot analysis. RESULTS: Treatment with EGF or insulin-like growth factor-1 (IGF-1) during stretch decreased apoptosis by 50% (P < 0.001). Neutralizing antibodies (Abs) directed against either growth factor or its receptor blocked the reduction in apoptosis. Stretch decreased BAD phosphorylation by approximately 50% (P < 0.001) relative to unstretched cells and each growth factor restored phosphorylation to basal levels. Kinase-specific inhibitors that blocked growth factor-mediated BAD phosphorylation promoted apoptosis in vitro. BAD phosphorylation decreased by approximately 50% (P < 0.001) in the tubules of obstructed hydronephrotic rat kidneys and administration of EGF restored BAD phosphorylation to basal levels. CONCLUSIONS: Signaling pathways converging at BAD phosphorylation are key to growth factor-mediated attenuation of stretch-induced apoptosis in vitro and in vivo.     

人肠癌RKO细胞周期依赖性激抑制因子基因启动子区甲基化状态的研究

目的 探讨人结肠癌RKO细胞周期依赖性激酶抑制因子(CKIs)家族启动子区CpG岛甲基化状态及其甲基化可逆性特征.方法 应用特异性DNA甲基转移酶(DNMTs)抑制剂5-Aza-2]-deoxycytidine(5-Aza-CdR)处理肠癌细胞,甲基特异性聚合酶链反应(Methylation-Specific PCR,MSP)、T-A克隆及DNA测序法分析RKO细胞CKIs家族抑癌基因p15ink4b、p16ink4a/CDKN2、p21/cip、p27/kip启动子CpG岛甲基化状态.结果 未经5-Aza-CdR作用的肠癌RKO细胞,其p15ink4b、p16ink4a/CDKN2基因组DNA胞嘧啶(C)保持不变,这提示在其单链DNA中胞嘧啶呈甲基化状态;而经5-Aza-CdR作用的肠癌RKO细胞,其p15ink4b、p16ink4a/CDKN2、p21/cip和p27/kip基因组DNA胞嘧啶均已变为胸腺嘧啶,表明在DNA单链中胞嘧啶已呈去甲基化状态.结论 肠癌RKO细胞周期INK4家族(p15ink4b和p16ink4a/CDKN2)基因的启动子区处于异常的高甲基化状态,而KIP/CIP家族(p21/cip和p27/kip)基因未处于高甲基化状态;5-Aza-CdR能较好地逆转肿瘤细胞INK4家族基因DNA高甲基化状态.     

死亡相关蛋白激酶1与细胞死亡

背景 细胞死亡是多种疾病的伴随过程,死亡相关蛋白激酶1（death-associated protein kinase 1,DAPK1）是一类与细胞死亡有关的酶,最初人们主要研究它在肿瘤抑制方面的作用,随后人们发现它在细胞死亡方面也有重要作用. 目的 DAPK1与细胞死亡密切相关,是近年来的研究热点.了解其研究现状以及未来发展趋势是非常有意义的. 内容 从DAPK1的结构、活性调节等方面人手,着重分析DAPK1与多种作用物的相互作用过程及其与疾病的关系. 趋向 随着人们对DAPK1研究的日益深入,DAPK1将成为疾病治疗的一个新靶点.     

The map kinase ERK regulates renal activity of cyclin-dependent kinase 2 in experimental glomerulonephritis.

BACKGROUND: In vitro, the extracellular signal-regulated kinase (ERK) is an intracellular convergence point of multiple stimuli, which affect the cell cycle. However, the role of ERK in cell cycle regulation in vivo is unknown. METHODS: To address this issue, ERK activity was blocked both in vitro in mesangial cells (MC) and in vivo in experimental glomerulonephritis (GN) by a pharmacological inhibitor (U0126) of the ERK-activating kinase. RESULTS: In stimulated MC, inhibition of ERK reduced cyclin-dependent kinase 2 (CDK2) phosphorylation, CDK2 activity and cyclin E/A expression, whereas downregulation of CDK inhibitor p27(Kip1) expression was inhibited. In vivo, U0126 was given to rats in the acute phase of anti-Thy 1.1 GN. We previously showed that glomerular cell proliferation was reduced by 67% upon treatment with the inhibitor compared to nephritic controls. Now, we detected a significant increase in renal CDK2-activity/phosphorylation in the nephritic controls, that was significantly and dose-dependently reduced by ERK inhibition. CDK2 activation was accompanied by an increase in renal expression of cyclins E/A and the enhanced binding of these cyclins to CDK2 in the nephritic controls. These changes were blunted by U0126 treatment. Finally, we noted an increased expression and CDK2-binding of p27(KIP1) protein in the nephritic controls which was decreased in U0126 treated rats. CONCLUSIONS: Our observations provide the first evidence that ERK is an intracellular regulator of renal CDK2 activity in vivo in a glomerulonephritis model.     

9-硝基喜树碱脂质体对肝癌细胞株HepG2的抑制作用及机制

目的 观察9-硝基喜树碱脂质体对HepG2肝癌细胞的抑制作用及其机制.方法 9-硝基喜树碱(9NC)及其脂质体(9NC-LP)处理HepG2细胞后,MTT法测定细胞活性,流式细胞术检测细胞周期及凋亡相关蛋白表达.建立HepG2细胞裸鼠皮下移植瘤模型,随机分为空白对照组,空白脂质体组,DMSO组,9NC低、高剂量组,9NC-LP低、高剂量组,每组10只.尾静脉给药后持续监测各组裸鼠肿瘤体积、体重变化.给药后28 d处死裸鼠,切取肿瘤提取总蛋白后Western Blot检测蛋白表达变化.结果 在体外实验,9NC-LP对肿瘤细胞生长呈时间和剂量依赖性抑制.药物处理后S期和G2/M期细胞明显增多,浓度大于0.1 btmol/L时24 h后细胞完全阻滞于S期;0.1μmol/L 孵育72 h后,超过95%HepG2细胞阻滞于G2/M期.在体内实验,与空白对照组比较,各剂量的9NC及9NC-LP均引起肿瘤体积下降(P<0.05)和裸鼠体重减轻(P<0.05).空白脂质体组与空白对照组、DMSO组比较差异无显著性(P>0.05).肿瘤生长最高抑制率9NC-LP(2.5 mg/kg)组、9NC-LP(1.5 mg/kg)组、9NC(1.5 mg/kg)组,分别为87.02%、51.57%和35.47%.9NC (2.5 mg/kg)组用药14 d过半动物死亡.结论 9NC及9NC-LP可以通过调控细胞周期和诱导凋亡有效抑制肝癌细胞生长.与原料药相比9-硝基喜树碱脂质体在体内对肝癌细胞的抑制增强,副作用减弱.9-硝基喜树碱脂质体是一种极具前景的纳米靶向药物.

Abstract：

Objective To observe the inhibitory effect and mechanism of 9-nitrocamptothecin liposomes on HepG2 liver carcinoma cells. Methods HepG2 cells were incubated with 9-nitrocampto-thecin(9NC) or with 9-nitrocamptothecin liposomes(9NC-LP) for 24 h, 48 h and 72 h. Cell viability was then measured by the MTT assay. Cell cycle and apoptosis were evaluated by flow cytometry.Western Blot was used to determine the expression of cell cycle and apoptosis related proteins. HepG2tumor-bearing mouse models were then established. The HepG2 tumor-bearing mice were randomly divided into control group, free liposomes group, DMSO group, 9NC low dose group, 9NC high dose group, 9NC-LP low dose group and 9NC-LP high dose group. There were 10 mice in each group.Drugs were administered by tail vein and tumor volume and body weight were observed 28 days after administration. Then animals were sacrificed and the expression of proteins from tumor homogenates was analyzed by Western blotting. Results In vitro, HepG2 cell viability was apparently inhibited by 9NC and 9NC-LP, and the inhibitory effect increased in a time-dependent and dose-dependent manner.Both S and G2/M phase arrests were observed after incubation with drugs. HepG2 cells were completely arrested in S phase with 9NC concentration over than 0.1 μmol/L after incubation for 24 h,while more than 95% of cells arrested in G2/M phase when 9NC concentration was 0.1 μmol/L after incubation for 72 h. In vivo, compared with the control group, the average tumor volume was reduced in both the 9NC and 9NC-LP group (P<0.05) , and the average animal body weight also decreased in both the 9NC and 9NC-LP group (P<0.05). There was no significant difference among the control group, free liposomes group, and DMSO group. The lights inhibition rates of tumor growth in the 9NC-LP(2.5 mg/kg),9NC-LP(1.5 mg/kg),and 9NC(1.5 mg/kg)groups were 87.02%, 51.57%and 35.47%, respectively. In the 9NC-LP(2.5 mg/kg)group, >50% of animals died 14 days after drug administration. Conclusion 9NC and 9NC-LP can inhibit HepG2 cell growth via cell cycle arrest and apoptosis induction. 9NC-LP has a more potent anti-tumor effect and fewer side effects in vivo,which means 9NC-LP is a promising compound for cancer therapy via intravenous administration.     

Double-stranded RNA-dependent protein kinase is involved in 2-methoxyestradiol-mediated cell death of osteosarcoma cells.

We studied the involvement of interferon-regulated, PKR on 2-ME-mediated actions in human osteosarcoma cells. Our results show that PKR is activated by 2-ME treatment and is necessary for 2-ME-mediated induction of osteosarcoma cell death. INTRODUCTION: Osteosarcoma is the most common primary bone tumor and most frequently develops during adolescence. 2-Methoxyestradiol (2-ME), a metabolite of 17beta-estradiol, induces interferon gene expression and apoptosis in human osteosarcoma cells. In this report, we studied the role of interferon-regulated double-stranded (ds)RNA-dependent protein kinase (PKR) protein on 2-ME-mediated cell death in human osteosarcoma cells. MATERIALS AND METHODS: Western blot analyses were used to measure PKR protein and phosphorylation levels. Cell survival and apoptosis assays were measured using trypan blue exclusion and Hoechst dye methods, respectively. A transient transfection protocol was used to express the dominant negative PKR mutants. RESULTS AND CONCLUSIONS: PKR was increased in 2-ME-treated MG63 cells, whereas 17beta-estradiol, 4-hydroxyestradiol, and 16alpha-hydroxyestradiol, which do not induce cell death, had no effect on PKR protein levels. Also, 2-ME treatment induced PKR kinase activity as indicated by increased autophosphorylation and phosphorylation of the endogenous substrate, eukaryotic initiation factor (eIF)-2alpha. dsRNA poly (I).poly (C), an activator of PKR protein, increased cell death when osteosarcoma cells were treated with a submaximal concentration of 2-ME. In contrast, a serine-threonine kinase inhibitor SB203580 and a specific PKR inhibitor 2-aminopurine (2-AP) blocked the 2-ME-induced cell death in MG63 cells. A dominant negative PKR mutant protein conferred resistance to 2-ME-induced cell death to MG63 osteosarcoma and 2-ME-mediated PKR regulation did not require interferon gene expression. PKR protein is activated in cell free extracts by 2-ME treatment, resulting in autophosphorylation and in the phosphorylation of the substrate eIF-2alpha. We conclude from these results that PKR is regulated by 2-ME independently of interferon and is essential for 2-ME-mediated cell death in MG63 osteosarcoma cells.     

IL-6 protects pancreatic islet beta cells from pro-inflammatory cytokines-induced cell death and functional impairment in vitro and in vivo

Protection of pancreatic islet beta cells from pro-inflammatory cytokines-induced cell death and functional impairment is a key issue in developing therapeutic interventions of type 1 diabetes mellitus including islet transplantation. The effects of IL-6 on the protection of beta cells in vitro and in vivo were examined. Freshly isolated islets or MIN6 beta cells, when pre-incubated with IL-6, showed significantly higher viabilities measured by MTT assay and FACS analysis of PI stained cells against pro-apoptotic signaling delivered by IL-1beta, TNF-alpha and IFN-gamma. Insulin secretory function was also significantly protected in static culture with glucose and KCl stimulation. In vivo assessment using marginal mass syngeneic islet transplantation in mouse model revealed IL-6 conferred significantly better blood glucose control and graft survival rate over 50 days. Conclusively, IL-6 protects pancreatic islets or beta-cells from inflammatory cytokines-induced cell death and functional impairment both in vitro and in vivo. This strategy could be exploited in the clinical setting to maintain functional islet mass.     

Overexpression of parathyroid hormone-related protein inhibits pancreatic beta-cell death in vivo and in vitro

Pancreatic beta-cell survival is critical in the setting of diabetes as well as in islet transplantation. Transgenic mice overexpressing parathyroid hormone-related protein (PTHrP) targeted to beta-cells using the rat insulin II promoter (RIP) display hyperinsulinemia, hypoglycemia, and islet hyperplasia, without a concomitant increase in beta-cell proliferation rate or enlargement of individual beta-cell size. Thus, the mechanism for increased beta-cell mass is unknown. In this study, we demonstrated that beta-cells of transgenic mice are resistant to the cytotoxic effects of streptozotocin (STZ) in vivo, as documented by a sixfold reduction in the rate of STZ-induced beta-cell death in RIP-PTHrP mice relative to their normal siblings. The reduced cell death in transgenic mice is due neither to their increased islet mass nor to a decrease in their sensing of STZ, but rather results from PTHrP-induced resistance to beta-cell death. This is also demonstrated in vitro by markedly reduced cell death rates observed in beta-cells of transgenic mice compared with normal mice when cultured in the absence of serum and glucose or in the presence of STZ. Finally, we demonstrated that NH(2)-terminal PTHrP inhibits beta-cell death. These findings support the concept that PTHrP overexpression increases islet mass in transgenic mice through inhibition of beta-cell death.     

Bisphosphonates induce breast cancer cell death in vitro.

Breast cancer frequently spreads to bone and is almost always associated with osteolysis. This tumor-induced osteolysis is caused by increased osteoclastic bone resorption. Bisphosphonates are used successfully to inhibit bone resorption in tumor bone disease and may prevent development of new osteolytic lesions. The classical view is that bisphosphonates only act on bone cells. We investigated their effects on breast cancer cells using three human cell lines, namely, MCF-7, T47D, and MDA.MB.231, and we tested four structurally different bisphosphonates: clodronate, pamidronate, ibandronate, and zoledronate. We performed time course studies for each bisphosphonate at various concentrations and found that all four compounds induced a nonreversible growth inhibition in both MCF-7 and T47D cell lines in a time- and dose-dependent manner. The MDA.MB.231 cell line was less responsive. Bisphosphonates induced apoptosis in MCF-7 and cell necrosis in T47D cells. The inhibition of MCF-7 cell proliferation could be reverted almost completely by the benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone (z-VAD-fmk) inhibitor of caspases, suggesting that the apoptotic process observed in the MCF-7 cell line is mediated, at least partly, by the caspase system. Caspase activity was little changed by bisphosphonates in T47D cells and the inhibitor of caspase did not modify bisphosphonates effects. In summary, we found that bisphosphonates inhibit breast cancer cell growth by inducing cell death in vitro. Such effects could contribute to the beneficial role of bisphosphonates in the treatment and the prevention of tumor-induced osteolysis.     

普伐他汀对肝癌HepG2细胞增殖的抑制作用

目的探讨HMG-CoA还原酶抑制剂(Statins)在体内外抑制肝细胞癌增殖和侵犯的作用及其机制。方法以不同浓度的普伐他汀(Pr)作用于培养的HepG2细胞,用噻唑蓝比色试验检测细胞的增殖活性,并观察细胞的形态学变化。建立HepG2细胞裸小鼠皮下移植瘤模型,腹腔注射浓度为5 g／L的Pr,每只每日10 ml／kg体重。观察肿瘤的生长情况,实验结束时切取肿瘤称重,检测血中AFP水平。肿瘤标本石蜡切片,免疫组织化学检测CyclinD1和FⅧRA的表达。结果 Pr作用96 h后明显抑制了HepG2细胞的生长,细胞出现明显的形态学改变。Pr也抑制裸小鼠皮下移植型肝癌的生长,Pr组和对照组瘤重分别是(0．246±0．129)g和(0．376±0．102)g(P< 0．05),抑瘤率34．6％。两组的AFP表达差异有统计学意义[(2．657±1．465)μg／L比(4．206± 1．204)μg／L,P<0．05]。Cydin D1在肿瘤间质的成纤维细胞表达,Pr组的积分吸光度(OPTID)为 219．40±24．00,对照组为451．54±47．97(P<0．05)。两组的微血管密度(MVD)分别为27．43± 6．83和41．22±6．26(P<0．05)。结论 Pr能抑制肝癌细胞的增殖,其机制主要是抑制肿瘤的血管形成和肿瘤基质中的成纤维细胞的增生。     

Mouse molar cell proliferation kinetics in vivo and in vitro

Patterns of cell proliferation kinetics have been examined in mouse first mandibular molars growing in vivo and in vitro. In vitro teeth were cultured on semi-solid medium or on Millipore filters. These conditions led respectively to normal and abnormal cusp formation. Similar cell-compartment specific mitotic activities existed in vivo and in vitro. Intradental control mechanisms exist. In vitro however the duration of cell cycle was lengthened, leading to lower rate of decrease of mitotic indices. The implications of these findings are discussed in the context of tooth morphogenesis and cytodifferentiation.     

The cyclin-dependent kinase inhibitor p57Kip2 regulates cell cycle exit, differentiation, and migration of embryonic cerebral cortical precursors

Mounting evidence indicates cyclin-dependent kinase (CDK) inhibitors (CKIs) of the Cip/Kip family, including p57(Kip2) and p27(Kip1), control not only cell cycle exit but also corticogenesis. Nevertheless, distinct activities of p57(Kip2) remain poorly defined. Using in vivo and culture approaches, we show p57(Kip2) overexpression at E14.5-15.5 elicits precursor cell cycle exit, promotes transition from proliferation to neuronal differentiation, and enhances process outgrowth, while opposite effects occur in p57(Kip2)-deficient precursors. Studies at later ages indicate p57(Kip2) overexpression also induces precocious glial differentiation, suggesting stage-dependent effects. In embryonic cortex, p57(Kip2) overexpression advances cell radial migration and alters postnatal laminar positioning. While both CKIs induce differentiation, p57(Kip2) was twice as effective as p27(Kip1) in inducing neuronal differentiation and was not permissive to astrogliogenic effects of ciliary neurotrophic factor, suggesting that the CKIs differentially modulate cell fate decisions. At molecular levels, although highly conserved N-terminal regions of both CKIs elicit cycle withdrawal and differentiation, the C-terminal region of p57(Kip2) alone inhibits in vivo migration. Furthermore, p57(Kip2) effects on neurogenesis and gliogenesis require the N-terminal cyclin/CDK binding/inhibitory domains, while previous p27(Kip1) studies report cell cycle-independent functions. These observations suggest p57(Kip2) coordinates multiple stages of corticogenesis and exhibits distinct and common activities compared with related family member p27(Kip1).