hSulf1 Sulfatase promotes apoptosis of hepatocellular cancer cells by decreasing heparin-binding growth factor signaling

: The heparin-binding growth factors fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) are potent mitogens for hepatocellular carcinomas (HCCs). Heparin-binding growth factor signaling is regulated by sulfation of cell-surface heparan sulfate proteoglycans (HSPGs). We hypothesized that hSulf1, a recently described sulfatase, regulates growth signaling in HCCs. :Expression of hSulf1 in human HCC tumors was determined by real-time PCR. Down-regulation of hSulf1 expression was investigated by analyzing loss of heterozygosity (LOH) at the hSulf1 locus and the effect of the DNA methylation inhibitor 5-aza-deoxycytidine on hSulf1 expression. The subcellular location of hSulf1 and sulfation state of cell-surface HSPGs were assessed by immunocytochemistry. FGF and HGF signaling was examined by phospho-specific immunoblot analysis. Cell growth was measured by trypan blue exclusion, and the MTT assay and apoptosis were quantitated by fluorescence microscopy. :hSulf1 expression was decreased in 29% of HCCs and 82% of HCC cell lines. There was LOH at the hSulf1 locus in 42% of HCCs. Treatment with 5-aza-deoxycytidine reactivated hSulf1 expression in hSulf1-negative cell lines. Low hSulf1-expressing cells showed increased sulfation of cell-surface HSPGs, enhanced FGF and HGF-mediated signaling, and increased HCC cell growth. Conversely, forced expression of hSulf1 decreased sulfation of cell-surface HSPGs and abrogated growth signaling. HCC cells with high-level hSulf1 expression were sensitive to staurosporine- or cisplatin-induced apoptosis, whereas low expressing cells were resistant. Transfection of hSulf1 into hSulf1-negative cells restored staurosporine and cisplatin sensitivity. :Down-regulation of hSulf1 contributes to hepatocarcinogenesis by enhancing heparin-binding growth factor signaling and resistance to apoptosis.     

PI3K抑制剂LY294002对人晶状体上皮细胞E2F-1、cyclinE蛋白表达的影响

目的探讨PI3K抑制剂LY294002对人晶状体上皮细胞（LECs）增殖及E2F-1、细胞周期素E（cyclinE）蛋白表达的影响。方法人LECs常规培养,分为实验组和对照组,实验组加入PI3K抑制剂LY294002（25μmol/L）,对照组加入同体积DMSO,两组分别培养36 h后,MTT比色法测定细胞增殖状况,Western blot法检测细胞中E2F-1及cyclinE蛋白表达。结果实验组细胞增殖受到明显抑制,与对照组比较差异有统计学意义（P〈0.05）,人LECs中E2F-1及cyclinE蛋白表达降低（P〈0.05）。结论 PI3K抑制剂LY294002可抑制人LECs的增殖,可能与下调E2F-1及cyclinE蛋白的表达水平相关。     

左西孟旦对缺氧复氧诱导的H9c2细胞凋亡、PTEN和PI3K/Akt信号通路的影响

[目的]研究左西孟旦对缺氧复氧(H/R)条件下H9c2细胞凋亡的影响及相关机制。[方法]体外培养H9c2细胞,将细胞分为空白对照组、H/R组、左西孟旦低剂量组、左西孟旦中剂量组、左西孟旦高剂量组。采用四甲基偶氮噻唑蓝法检测H9c2细胞增殖;流式细胞仪检测H9c2细胞凋亡;超氧化物歧化酶(SOD)试剂盒、丙二醛(MDA)试剂盒分别检测SOD活性及MDA含量;荧光探针法检测活性氧(ROS)水平;Western blot检测增殖细胞核抗原(PCNA)、B淋巴细胞瘤2蛋白(Bcl-2)、Bcl-2相关X蛋白(Bax)、磷酸酶及张力蛋白同源物(PTEN)及磷脂酰肌醇3激酶(PI3K)/丝氨酸苏氨酸蛋白激酶(Akt)信号通路蛋白表达。[结果]与空白对照组比较,H/R组H9c2细胞增殖抑制率、凋亡率、MDA含量、ROS水平、PTEN蛋白表达均显著升高(P<0.05),PCNA、Bcl-2、p-PI3K/PI3K、p-Akt/Akt均显著降低,Bax显著升高(P<0.05)。与H/R组比较,左西孟旦低剂量组、左西孟旦中剂量组、左西孟旦高剂量组H9c2细胞增殖抑制率、凋亡率、MDA含量、RO...     

洛伐他汀经PI3K/Akt和ERK1/2信号通路抑制大鼠骨髓间充质干细胞凋亡

目的 体外以缺氧无血清条件模拟心肌梗死后的心脏缺血微环境,研究洛伐他汀能否抑制缺氧无血清引起的骨髓间充质干细胞(MSC)凋亡并探讨其机制.方法 以Hocchst33342染色荧光显微镜观察法及Annexin V/PI流式细胞术检测洛伐他汀的抗凋亡作用,并进一步采用Westernblot方法 检测洛伐他汀对线粒体凋亡途径的抑制作用以及对磷脂酰肌醇3激酶(PI3K)/丝氨酸苏氨酸激酶(Akt)途径和丝裂原活化的蛋白激酶(MAPK)的激酶(MEK)/细胞内信号调节蛋白激酶(ERK1/2)途径的激活作用.结果 0.01～1 μmol/L浓度范围的洛伐他汀能够有效地抑制缺氧无血清引起的MSC凋亡.洛伐他汀抑制线粒体凋亡途径,洛伐他汀抑制细胞色素C释放,降低天冬氨酸特异性半胱氨酸蛋白酶-3(caspase-3)活化,从而保护线粒体功能.洛伐他汀的抗凋亡效应以及其抑制细胞色素C释放的作用均可被PI3K抑制剂LY294002和MEK抑制剂U0126阻断.洛伐他汀能够激活PI3K/Akt和MEK/ERK1/2两条细胞存活信号通路,分别导致Akt和GSK-3β及ERK1/2磷酸化.结论 洛伐他汀能够抑制线粒体凋亡途径,并激活PI3K/Akt和MEK/ERK1/2细胞存活通路,最终发挥抗缺氧无血清引起的MSC凋亡.该研究为提高移植干细胞的存活率提供了一种可能有效的干预措施.     

Inhibition of Frizzled-2 by small interfering RNA protects rat hepatic BRL-3A cells against cytotoxicity and apoptosis induced by Hypoxia/Reoxygenation

Frizzled-2 plays an important role in maintaining normal hepatic cell functionality. This study aimed to investigate the role of inhibition of Frizzled-2 in protecting rat liver BRL-3A cells from Hypoxia/Reoxygenation (H/R). In vitro H/R hepatic cell model was established by culturing BRL-3A cells under H/R condition. Frizzled-2 siRNA was transfected into BRL-3A cells to inhibit Frizzled-2 signaling. Wnt5a and Frizzled-2 were significantly increased in BRL-3A cells upon H/R treatment. H/R treatment induced cell cytotoxicity, the early apoptosis rate and the intracellular Ca²⁺ level in BRL-3A cells while silencing frizzled-2 gene decreased the H/R induced cell cytotoxicity, apoptosis and intracellular Ca²⁺ level. In vivo mice study further showed the up-regulation of Frizzled-2/Wnt 5 pathway and cleaved Caspase-3 expression in liver tissues under ischemia and reperfusion injury (IRI). In summary, inhibition of Frizzled-2 by its siRNA may protects BRL-3A cells by attenuating the H/R induced cell cytotoxicity and apoptosis.     

1,25-二羟基维生素D3和维生素K2联合诱导HL-6O细胞分化及凋亡的研究

目的研究1,25-二羟基维生素D3[1,25(OH)2D3,简称D3]与维生素K2(VK2)联合应用对HL-60细胞分化及凋亡的影响.方法通过四唑氮蓝(MTT)比色,细胞形态,流式细胞仪(FCM)测定细胞周期、凋亡率及CD14的表达,观察D3、VK2对HL-60细胞的影响.结果D3与VK2都能抑制HL-60细胞增殖,并且联合使用抑制作用显著.10-8mol/L D3与10 μmol/L VK2联合处理HL-60细胞72 h后CD14的表达率为63.15%,且G0/G1期细胞显著增多,与单独一种比较差异有统计学意义(P＜0.05).10 μmol/L、20 μmol/L VK2作用于HL-60细胞72 h凋亡率分别为11.31%、20.36%,与对照组比较差异有统计学意义(P＜0.01);而10 μmol/LVK2与10-8 mol/L D3联用72 h细胞的凋亡率为5.41%,与对照组比较差异无统计学意义(P＞0.05).结论D3与VK2联合使用可以使D3诱导分化作用加强,而VK2诱导凋亡作用受到抑制.     

The phosphatidylinositol 3‐kinases (PI3K) inhibitor GS‐1101 synergistically potentiates histone deacetylase inhibitor‐induced proliferation inhibition and apoptosis through the inactivation of PI3K and extracellular signal‐regulated kinase pathways

Previously, we showed that inhibition of the protein kinase C β (PKCβ)/AKT pathway augments engagement of the histone deacetylase inhibitor (HDI)‐induced apoptosis in lymphoma cells. In the present study, we investigated the cytotoxicity and mechanisms of cell death induced by the delta isoform‐specific phosphatidylinositide 3‐kinase (PI3K) inhibitor, GS‐1101, in combination with the HDI, panobinostat (LBH589) and suberoylanilide hydroxamic acid (SAHA). Lymphoma cell lines, primary non‐Hodgkin Lymphoma (NHL) and chronic lymphocytic leukaemia (CLL) cells were simultaneously treated with the HDI, LBH589 and GS‐1101. An interaction of the LBH589/GS‐1101 combination was formally examined by using various concentrations of LBH589 and GS‐1101. Combined treatment resulted in a synergistic inhibition of proliferation and showed synergistic effect on apoptotic induction in all tested cell lines and primary NHL and CLL cells. This study indicates that interference with PI3K signalling dramatically increases HDI‐mediated apoptosis in malignant haematopoietic cells, possibly through both AKT‐dependent or AKT‐ independent mechanisms. Moreover, the increase in HDI‐related apoptosis observed in PI3K inhibitor‐treated cells appears to be related to the disruption of the extracellular signal‐regulated kinase (ERK) signalling pathway. This study provides a strong rational for testing the combination of PI3K inhibitors and HDI in the clinic.     

Specific apoptosis induction by the dual PI3K/mTor inhibitor NVP-BEZ235 in HER2 amplified and PIK3CA mutant breast cancer cells

NVP-BEZ235 is a dual PI3K/mTOR inhibitor currently in phase I clinical trials. We profiled this compound against a panel of breast tumor cell lines to identify the patient populations that would benefit from such treatment. In this setting, NVP-BEZ235 selectively induced cell death in cell lines presenting either HER2 amplification and/or PIK3CA mutation, but not in cell lines with PTEN loss of function or KRAS mutations, for which resistance could be attributed, in part to ERK pathway activity. An in depth analysis of death markers revealed that the cell death observed upon NVP-BEZ235 treatment could be recapitulated with other PI3K inhibitors and that this event is linked to active PARP cleavage indicative of an apoptotic process. Moreover, the effect seemed to be partly independent of the caspase-9 executioner and mitochondrial activated caspases, suggesting an alternate route for apoptosis induction by PI3K inhibitors. Overall, this study will provide guidance for patient stratification for forthcoming breast cancer phase II trials for NVP-BEZ235.