锤头状核酶在HepG2细胞中抑制adr亚型的乙型肝炎病毒

针对HBV基因组设计了三种锤头状核酶－－RS3、RC2和RC1。将裸露的和用tRNA包埋的核酶（RtS3、RtC2和RtC1）基因插入RNA修剪质粒pRG523中,然后转变为真核表达载体,使用同样克隆技术,构建由不同长度（2、4、8、12个）RS3或12个RtS3连成的鸟枪型真核表达载体,将它们分别与带有HBV基因组的质粒共转染人肝癌细胞系HepG2,用G418筛选抗性细胞。分析不同种类的核酶在G418抗性细胞中的HBV抑制活性,包括子代HBV－DNA、HBV－RNA和抗原（HBsAg或HBcAg/HBeAg)表达的减少。结果表明,所有设计的核酶对HBV有＞70％的抑制活性,而tRNA包埋核酶的活性略高于裸露核酶。特别值得提出的是,由8个或12个相同核酶连接的鸟枪型质粒（8RS3、12RS3和12RsS3）具有很高的抑制HBV活性,达到＞9－％的抑制。     

共轭亚油酸对HepG2细胞脂质沉积的影响

目的:研究共轭亚油酸(conjugated linoleic acid,CLA)对HepG2细胞脂肪沉积的影响,以探讨其对肝脏脂肪沉积的可能机制.方法:体外培养HepG2细胞,不同浓度CLA(0、10、50、100 μmol.1-1)作用24小时后,100 nmol.1-1胰岛素作用1小时.以比色法测定培养液中游离脂肪酸的含量,油红O染色观察细胞脂肪沉积,Western-blot检测Akt和P-Akt蛋白表达.结果:随着CLA浓度增加,培养液中的游离脂肪酸含量显著降低,细胞中红染脂滴增多,Western-blot结果发现,Akt蛋白的磷酸化水平增高.结论:CLA可以增加细胞内脂肪合成和细胞中Akt蛋白的磷酸化水平.     

Mesoionic Nucleosides 3: Studies on Mesoionic Imidazo [2,1-b]-1/3-thiazine Derivatives

Abstract

Anhydro 1- (2', 3', 5’ -tri-Oacetyl-β-D-ribofuranosyl) -5-hydroxy-7-oxoimidazo[2, 1-b]-1, 3-thiazinium hydroxide, a mesoionic imidazothiazine nucleoside, was prepared by the condensation of the appropriately acetylated ribofuranosyl imidazoline-2-thione with carbon suboxide. Although stable at or below O° C, the product rapidly underwent ring-opening at room temperature to afford the precursor nucleoside. A possible scheme for this ring-opening reaction is proposed based on the results of a model study employing anhydro 1-methyl-5-hydrcxy-7-oxoimidazo[2, 1-b]-1, 3-thiazinium hydroxide.     

The Synthesis of (1,3,4-Oxadiazol-2-yl)Acrylic Acid Derivatives with Antibacterial and Protistocidal Activities

A series of new 1,3,4-oxadiazol-2-yl-acrylic acids was synthesized by cyclization of 4-(2-R-hydrazino)- 4-oxo-2-butenic acids, and their antibacterial and protistocidal activities were studied. The p-substituted benzyl derivatives in the Z-form were shown to exhibit a high protistocidal activity, which exceeded that of the reference drug Baycox (toltrazuril) by several times, whereas the 3-hydroxy-2-naphthyl derivative, in addition to a very high protistocidal activity, also exhibited a moderate antibacterial activity.     

Amino Derivatives of Natural Epoxyalantolactone: Synthesis and Cytotoxicity toward Tumor Cells

Reactions of natural epoxyalantolactone with primary and secondary amines, which lead to hydrated benzo[g]furo[4,3,2-cd]indolones and eudesmane-type amino derivatives, respectively, have been studied. Epoxyalantolactone conjugates have been synthesized with the involvement of pharmacophoric amines, and their cytotoxic activity toward some tumor cell lines has been tested. The involvement of the signaling pathway of protein p53 in the death of tumor cells, the contribution of oxidative stress, and the induction of apoptosis have been investigated in experiments in vitro.     

槲皮素下调hTERT表达对肝癌HepG2细胞生长影响研究

目的观察槲皮素对肝癌HepG2细胞生长及对hTERT基因表达的影响。方法以台盼蓝拒染法计数肝癌细胞的生长抑制率,透射电镜从形态变化上了解凋亡的发生,流式细胞术检测细胞周期变化,western-blot、RT-PCR检测hTERT基因表达改变,PCR-TRAP法检测端粒酶活性。结果台盼蓝拒染法计数显示槲皮素抑制肝癌HepG2细胞增殖的作用明显,且呈浓度和时间依赖性,槲皮素处理48h后的Ic50为25.5μm。形态学检测显示出细胞凋亡的特征变化,流式细胞仪检测表明经10～20μm/L的槲皮素处理,肝癌HepG2细胞周期阻滞于G0/G1期,且HepG2细胞hTERT蛋白和mRNA表达降低,端粒酶活性受抑制。结论槲皮素能抑制肝癌细胞的生长,呈时间、剂量依赖性,能诱导HepG2细胞发生凋亡,其抑制增生与诱导凋亡的机制可能与下调hTERT基因表达,抑制端粒酶活性,破坏端粒稳定性有关。     

Cytotoxicity and transcriptional activation of stress genes in human liver carcinoma cells (HepG2) exposed to cadmium chloride

To evaluate the role of lipid oxidation in atherogenesis the levels of lipid- and protein-bound products of peroxidation in normal and atherosclerotic areas of human aorta were investigated. The level of fluorescent (360/430 nm) lipid products was measured in chloroform-methanol extracts of aortic tissue. Normal intima, initial lesions and fatty streaks had a similar content of fluorescent substances. On the other hand, high level of fluorescent products was found in atherosclerotic plaques. Cholesterol covalently bound to proteins, which serve as a marker of lipoperoxidation, was measured by high performance liquid chromatography after mild alkaline hydrolysis of delipidated tissue protein samples. The levels of protein-bound cholesterol in initial lesions and fatty streaks were close to its content in uninvolved intima (59 ± 18 and 92 ± 18 vs 70 ± 13 nmol/g protein). The content of covalently bound cholesterol in atherosclerotic plaques was dramatically higher (90-fold) than in the normal tissue. In addition to protein-bound cholesterol, considerable amount of lipofuscin was revealed in the cells of atherosclerotic plaques, but not in the cells of normal intima, initial lesions or fatty streaks. Thus, the contents of all investigated lipid- and protein-bound products of lipoperoxidation in earlier atherosclerotic lesions were similar to their levels in normal tissue. It can be due to a low rate of oxidized product formation and/or high rate of its degradation in or elimination from the vessel wall.     

Differential Cytotoxicity of Acetaminophen in Mouse Macrophage J774.2 and Human Hepatoma HepG2 Cells: Protection by Diallyl Sulfide

Non-steroidal anti-inflammatory drugs (NSAIDs), including acetaminophen (APAP), have been reported to induce cytotoxicity in cancer and non-cancerous cells. Overdose of acetaminophen (APAP) causes liver injury in humans and animals. Hepatic glutathione (GSH) depletion followed by oxidative stress and mitochondrial dysfunction are believed to be the main causes of APAP toxicity. The precise molecular mechanism of APAP toxicity in different cellular systems is, however, not clearly understood. Our previous studies on mouse macrophage J774.2 cells treated with APAP strongly suggest induction of apoptosis associated with mitochondrial dysfunction and oxidative stress. In the present study, using human hepatoma HepG2 cells, we have further demonstrated that macrophages are a more sensitive target for APAP—induced toxicity than HepG2 cells. Using similar dose- and time-point studies, a marked increase in apoptosis and DNA fragmentation were seen in macrophages compared to HepG2 cells. Differential effects of APAP on mitochondrial respiratory functions and oxidative stress were observed in the two cell lines which are presumably dependent on the varying degree of drug metabolism by the different cytochrome P450s and detoxification by glutathione S-transferase enzyme systems. Our results demonstrate a marked increase in the activity and expression of glutathione transferase (GST) and multidrug resistance (MDR1) proteins in APAP-treated HepG2 cells compared to macrophages. This may explain the apparent resistance of HepG2 cells to APAP toxicity. However, treatment of these cells with diallyl sulfide (DAS, 200 μM), a known chemopreventive agent from garlic extract, 24 h prior to APAP (10 μmol/ml for 18h) exhibited comparable cytoprotective effects in the two cell lines. These results may help in better understanding the mechanism of cytotoxicity caused by APAP and cytoprotection by chemopreventive agents in cancer and non-cancerous cellular systems.     

Modulation of Lipid Metabolism by Deep-Sea Water in Cultured Human Liver (HepG2) Cells

It has been found that deep-sea water was associated with lower serum lipid in animal model studies. Herein, we investigated whether DSW exerted a hypolipidemic activity and further elucidated how DSW modulated lipid metabolism in HepG2 cells. Preliminary animal studies showed that DSW exhibited potency to decrease serum total cholesterol, triglycerides, and LDL cholesterol, and increase HDL cholesterol, and the hepatic lipid contents were also significantly lower in the DSW group. When DSW was added to HepG2 cells, it decreased the lipid contents of hepatocyte through the activation of AMP-activated protein kinase, thus inhibiting the synthesis of cholesterol and fatty acid. Besides, LDL receptor was upregulated by activation of sterol regulatory element-binding protein-2. In addition, the levels of apolipoprotein AI and cholesterol 7-alpha-hydroxylase were also raised. Our investigation provided mechanisms by which DSW modulated lipid metabolism and indicated that DSW was worthy of further investigation and could be developed as functional drinking water in the prevention and treatment of hypolipidemic and other lifestyle-related diseases.     

Selective Cytotoxicity of Amidinopiperidine Based Compounds Towards Burkitt's Lymphoma Cells Involves Proteasome Inhibition

Serine proteases have proven to be promising pharmacological targets in contemporary drug discovery for cancer treatment. Since azaphenylalanine-based compounds manifest cytotoxic activity, we have selected serine protease inhibitors designed and synthesized in-house with large hydrophobic naphthalene moiety for screening. The cytotoxic potential of screened molecules was correlated to modifications of R(1) residues. The most cytotoxic were compounds with greater basicity; amidinopiperidines, piperidines and benzamidines. Amidinopiperidine-based compounds exert cytotoxicity in low μM range, with IC(50) 18 μM and 22 μM for inhibitors 15 and 16 respectively. These compounds exhibited selective cytotoxicity towards the Burkitt's lymphoma cells Ramos and Daudi, and proved nontoxic to PMBC, Jurkat and U937. They induce caspase-dependent apoptotic cell death, as demonstrated by the use of a pan-caspase inihibitor, zVADfmk, which was able to rescue Ramos cells from compound(s)-induced apoptosis. We confirm a disruption of the pro-survival pathway in Burkitt's lymphoma through NFκB inhibition. The accumulation of phosphorylated precursor (p105) and inhibitory (IκB) molecules with no subsequent release of active NFκB implicated the involvement of proteasome. Indeed, we show that the amidinopiperidine-based compounds inhibit all three proteolytical activities of the human 20S proteasome, with the most prominent effect being on the trypsin-like activity. Consistently, treatment of Ramos cells with these compounds led to an increase in ubiquitinated proteins. The amidinopiperidine-based serine protease inhibitors presented are, as selective inducers of apoptosis in Burkitt's lymphoma cells, promising leads for the development of novel chemotherapeutics.     

PCNA 泛素化对Hela 细胞苯并芘损伤敏感性的影响

目的:观察PCNA泛素化修饰对Hela细胞损伤敏感性的影响。方法:Western blot法检测His-PCNA及His-mutant PCNA(mPCNA,K164R)在Hela细胞中的表达。DNA损伤剂苯并芘(BaP)和依托泊苷(VP-16)分别处理Hela细胞后,MTT法检测不同细胞系对DNA损伤药物的敏感性;Western blot法检测细胞PCNA的泛素化修饰。结果:Western blot结果显示His-PCNA和His-mPCNA在Hela细胞中稳定高表达。MTT结果显示,苯并芘损伤后,稳定高表达mPCNA的细胞系与野生型及高表达PCNA细胞系相比,其细胞存活率呈明显下降趋势,而VP-16作用后,三种细胞存活率无明显差异。Western blot结果显示苯并芘损伤可特异性诱导PCNA发生泛素化修饰。结论:苯并芘损伤能够诱导PCNA发生泛素化修饰,从而降低Hela细胞对苯并芘损伤的敏感性。     

Cell Specific Cytotoxicity and Structure-Activity Relationship of Lipophilic 1-B-D-Arabinofuranosylcytosine (Ara-C) Derivatives

Abstract

Lipophilic derivatives of ara-C were developed with the aim to improve drug penetration and retention in solid tumors. Ara-C was esterified at the 5′-position with fatty acids (16–22 C-atoms, 0–3 double bonds). The derivatives were inactive in cell lines with various forms of ara-C and 2′,2′-difluorodeoxycytidine (dFdC, gemcitabine) resistance, including deoxycytidine kinase (dCK) deficiency. The activity in the parent cell lines correlated negatively with chain length and positively with double bonds.     

重组荞麦胰蛋白酶抑制剂对人肝癌细胞的凋亡及半胱氨酸天冬酶活性的影响

先前的研究表明,基因重组荞麦胰蛋白酶抑制剂 (rBTI) 具有诱导不同肿瘤细胞凋亡的作用.为了揭示其诱导肿瘤细胞凋亡的可能机理,从基因水平上探讨与凋亡有关的分子事件,本研究用不同浓度的 rBTI 体外作用于人肝癌细胞 HepG2 后,采用 MTT 比色法检测抑制剂对epG2 细胞的抑制率,用 DNA 凝胶电泳和细胞核的形态学观察检测 HepG2 细胞的凋亡.结果表明,rBTI 在体外能够明显抑制 HepG2 细胞的增长,并诱导细胞凋亡.另外,细胞凋亡与Bcl-2/Bax mRNA 水平有关.通过 RT-PCR 检测发现,细胞经过rBTI处理后,抗凋亡基因Bcl-2 mRNA 水平下调,促凋亡基因 Bax mRNA 有所上调,而对照 GAPDH 无变化.对 HepG2细胞中 Fas/Fas 配体及半胱氨酸天冬酶（caspase）的研究证明,细胞经过 rBTI 处理后,对死亡受体 Fas mRNA没有影响; rBTI 可明显激活caspase-3 和 caspase-9 酶活性, 对caspase-8 活性几乎无影响.上述结果表明,rBTI 对HepG2 细胞具有明显的诱导凋亡作用,其诱导细胞凋亡的机制与 caspase-3 依赖性凋亡调节信号通路有关,未涉及 Fas/Fas 配体途径.     

Alkaline Ceramidase 2 (ACER2) and Its Product Dihydrosphingosine Mediate the Cytotoxicity of N-(4-Hydroxyphenyl)retinamide in Tumor Cells

Increased generation of dihydrosphingosine (DHS), a bioactive sphingolipid, has been implicated in the cytotoxicity of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) in tumor cells. However, how 4-HPR increases DHS remains unclear. Here we demonstrate that 4-HPR increases the expression of ACER2, which catalyzes the hydrolysis of dihydroceramides to generate DHS, and that ACER2 up-regulation plays a key role in mediating the 4-HPR-induced generation of DHS as well as the cytotoxicity of 4-HPR in tumor cells. Treatment with 4-HPR induced the accumulation of dihydroceramides (DHCs) in tumor cells by inhibiting dihydroceramide desaturase (DES) activity, which catalyzes the conversion of DHCs to ceramides. Treatment with 4-HPR also increased ACER2 expression through a retinoic acid receptor-independent and caspase-dependent manner. Overexpression of ACER2 augmented the 4-HPR-induced generation of DHS as well as 4-HPR cytotoxicity, and 4-HPR-induced death in tumor cells, whereas knocking down ACER2 had the opposite effects. ACER2 overexpression, along with treatment with GT11, another DES inhibitor, markedly increased cellular DHS, leading to tumor cell death, whereas ACER2 overexpression or GT11 treatment alone failed to do so, suggesting that both ACER2 up-regulation and DES inhibition are necessary and sufficient to mediate 4-HPR-induced DHS accumulation, cytotoxicity, and death in tumor cells. Taken together, these results suggest that up-regulation of the ACER2/DHS pathway mediates the cytotoxicity of 4-HPR in tumor cells and that up-regulating or activating ACER2 may improve the anti-cancer activity of 4-HRR and other DHC-inducing agents.     

TFPI-2对人肝癌细胞生长增殖、凋亡及AFP合成的影响

目的: 探讨TFPI-2基因对人肝癌细胞Hep3B生长增殖、凋亡及甲胎蛋白AFP表达的影响。 方法: 将重组质粒PCDNA3.1-TFPI-2转染Hep3B细胞并经G418稳定筛选后,RT-PCR和Western blot检测转染前后TFPI-2 mRNA和蛋白表达水平,采用CCK-8法、生长曲线观察TFPI-2对人肝癌细胞Hep3B生长增殖的影响,通过平板克隆形成实验观察单个细胞的增殖能力,RT-PCR检测AFP mRNA的表达,并用电化学发光法测定培养上清液中甲胎蛋白AFP含量,流式细胞仪检测细胞早晚期凋亡情况。 结果: 转染成功的Hep3B细胞检测到TFPI-2 mRNA和蛋白的表达;与转染空载体及未转染的细胞相比,转染TFPI-2的细胞生长增殖能力明显减弱;AFP mRNA表达抑制率为16.51%,AFP蛋白分泌明显低于对照组(P<0.01);流式细胞术检测转染TFPI-2的Hep3B细胞早期凋亡率明显增加(24.03%±7.28% vs 8.77%±3.66%)。 结论: TFPI-2表达可显著抑制肝癌细胞生长和AFP的表达,同时还能诱导细胞早期凋亡。为进一步探讨靶向TFPI-2的肝癌基因治疗提供了实验依据。     

NPAS2基因敲除的HepG2细胞系构建及其对肝癌细胞凋亡的影响

目的:利用CRISPR/Cas9基因编辑技术构建生物节律基因NPAS2敲除的HepG2肝癌细胞系,并初步探讨NPAS2基因敲除对肝癌细胞凋亡的影响。方法:利用sgRNA在线设计工具,针对NPAS2设计两条sgRNA;利用PX459质粒构建分别含有两条sgRNA的敲除载体PX459-sgRNA1;PX459-sgRNA2;利用T7核酸内切酶I检测两条sgRNA活性;将活性较高的打靶载体瞬时转染HepG2细胞,经过药物筛选,克隆化培养及基因测序后得到NPAS2敲除的HepG2肝癌细胞系;利用Western blot检测NPAS2蛋白的表达和凋亡相关蛋白Caspase3的活化;利用流式细胞仪检测敲除细胞系的凋亡水平。结果:成功构建了针对NPAS2的打靶载体;并筛选得到了活性较高的打靶载体;经过药物筛选和克隆化培养得到的NPAS2敲除肝癌细胞系未检测到NPAS2蛋白的表达;进一步发现NPAS2敲除的肝癌细胞Caspase3明显活化,凋亡水平显著升高。结论:利用CRISPR/Cas9基因编辑技术成功构建了NPAS2基因敲除的HepG2肝癌细胞系,并发现NPAS2敲除可以促进肝癌细胞凋亡,为进一步研究生物节律基因NPAS2及其它相关基因在肝癌发生发展中的作用机制提供了有力的工具。