二烯丙基三硫通过线粒体依赖性途径诱导人肝癌HepG2细胞凋亡

二烯丙基三硫(diallyl trisulfide,DATS)对多种肿瘤有抗癌作用,但机制尚不完全清楚．为探讨DATS对人肝癌细胞系HepG2细胞凋亡的影响,用丫啶橙/溴化乙锭(AO/EB)法观察细胞凋亡情况,在显微镜下计数凋亡细胞数．JC-1荧光染色观察线粒体膜电势变化．Western blot法检测细胞色素c蛋白分布,ELISA法检测caspase-3活性． 结果显示,DATS诱导HepG2细胞凋亡,用 50 μmol/L 与100 μmol/L DATS处理48 h,细胞凋亡率分别达到60.33%和93.67%,并引起HepG2细胞线粒体膜电势降低．Western blot显示,DATS能诱导胞浆细胞色素c增加,与此同时,线粒体细胞色素c减少,诱导HepG2细胞caspase-3活化．提示：DATS可通过降低线粒体膜电势,促进细胞色素c由线粒体膜释放到胞浆中,激活caspase-3途径诱导人肝癌细胞系HepG2细胞凋亡．     

预热诱导心肌细胞表达Hsp70对Fas通路活化后细胞凋亡变化的研究

目的:研究Hsp70对Fas通路活化后细胞凋亡的作用及其可能机制.方法:水浴预热诱导培养新生大鼠心肌细胞Hsp70表达,Fas抗体活化Fas通路,Western blot测定Hsp70的表达,流式细胞仪检测细胞凋亡率,特异性底物测定caspase-8及caspase-3的活力.结果:预热可以使心肌细胞Hsp70表达升高,Fas抗体降低细胞凋亡率,高表达Hsp70可以降低Fas通路活化后心肌细胞凋亡率,同时相应使caspase-8及caspase-3活力升高的幅度降低.结论:Hsp70可能通过抑制caspase-8及caspase-3的活力而降低Fas通路活化后培养新生大鼠心肌细胞凋亡的发生.     

调节TGF-β1/Smad信号通路对内质网应激状态下肝癌HepG2细胞凋亡的影响

目的: 探讨TGF-β1/Smad信号通路对内质网应激(ERS)状态下肝癌HepG2细胞凋亡的影响机制。方法: 首先建立内质网应激模型:以3 μmol/L的衣霉素(TM)处理人肝癌HepG2细胞株24 h,诱导细胞发生ERS。实验分为6组,每组3个复孔,实验重复3次,6组分别为:Untreated组(未处理组)、TM组(3 μmol/L TM处理组)、TM+NC组(3 μmol/L TM+si-TGF-β1阴性对照组)、TM+si-TGF-β1组(3 μmol/L TM+si-TGF-β1组)、TM+pEX-3组(3 μmol/L TM+质粒对照组)及TM+TGF-β1 pEX-3组(3 μmol/L TM+TGF-β1过表达质粒组),利用脂质体的方法将TGF-β1小干扰RNA(si-TGF-β1)及TGF-β1过表达质粒(TGF-β1 pEX-3)转染入HepG2细胞,转染24 h后,利用RT-qPCR和Western blot检测各组HepG2细胞TGF-β1/Smad信号通路相关因子TGF-β1、p-Smad2表达的情况;CCK-8和流式细胞术分别检测各组HepG2细胞增殖抑制率和凋亡率变化情况。结果: 与Untreated组相比,TM组细胞的TGF-β1及p-Smad2的表达明显降低(P＜0.05);与TM组相比,TM+si-TGF-β1组细胞的TGF-β1及p-Smad2的表达和细胞的增殖抑制率、凋亡率显著降低(P＜0.01),而TM+TGF-β1 pEX-3组细胞的TGF-β1及p-Smad2的表达和细胞增殖抑制率、凋亡率显著升高(P＜0.01)。结论: TGF-β1/Smad信号通路在肝癌HepG2细胞发生ERS后受到抑制,当该通路被激活后,ERS状态下肝癌HepG2细胞的凋亡率显著升高。     

半边旗二萜类成分PAG通过ROS诱导肝癌细胞凋亡的作用研究

探讨半边旗二萜类成分Pteisolic acid G(PAG)对人肝癌细胞HepG2增殖和凋亡的影响及作用机制。用不同浓度的PAG处理HepG2细胞后,采用MTT法检测细胞存活率;采用PI单染法检测细胞周期分布;采用Annexin V-FITC/PI双染法检测细胞凋亡率;采用RT-PCR和Western Blotting检测细胞内mRNA和蛋白表达情况;采用DCFH-DA法检测细胞内ROS水平,采用ROS抑制剂乙酰半胱氨酸(NAC)评价PAG细胞增殖抑制作用对ROS的依赖性。结果表明,在24 h、48 h和72 h时,PAG可剂量依赖性地抑制HepG2细胞的增殖(p0.05),IC_(50)分别为64.8μmol/L,38.5μmol/L和24.8μmol/L;用药24 h时PAG可剂量依赖性地使HepG2细胞阻滞在G_2/M期,同时增加HepG2细胞凋亡率(p0.05);PAG可剂量依赖性地降低HepG2细胞内Bcl-2 mRNA和caspase 3、PARP、Bcl-2蛋白的表达(p0.05),增加Bax mRNA和actived-caspase 3、cleaved-PARP、Bax蛋白的表达(p0.05)。当使用1 mmol/L的ROS抑制剂NAC预处理HepG2细胞时,PAG对HepG2细胞增殖抑制作用被显著阻断。上述结果表明,半边旗二萜类成分PAG可提高Bax/Bcl-2的基因和蛋白表达比值,从而诱导肝癌细胞HepG2凋亡,该作用可能是通过升高细胞内ROS水平来实现的。     

JKA97诱导HepG2凋亡及其分子机制研究

目的:研究药物JKA97对人肝癌细胞HepG2增殖的抑制作用及其分子机制.方法:采用MTT法观察药物JKA97对细胞增殖的影响;倒置显微镜观察细胞形态变化;流式细胞术检测细胞凋亡;Western blot方法检测线粒体融合蛋白mfn2表达水平变化.结果:药物JKA97抑制人肝癌细胞HepG2细胞增殖,5、10、20 μmol/L作用组24 h抑制率分别为34.26%、43.08%、54.02%;药物JKA97诱导人肝癌细胞HepG2凋亡,10、20 μmol/L JKA97作用HepG2细胞24h,细胞凋亡率分别为22.9%、72.9%;线粒体融合蛋白mfn2表达水平显著降低.结论:药物JKA97对人肝癌细胞HepG2生长具有明显的增殖抑制作用,诱导细胞凋亡;线粒体融合蛋白mfn2表达水平降低可能是其重要的分子机制之一.     

NF-kB拮抗剂PDTC对HepG2细胞的抑制及对caspase-3表达的影响

目的：研究NF-kB拮抗剂PDTC诱导人肝癌HepG2细胞凋亡及其对凋亡相关基因caspase-3表达的影响,初步探讨其诱导凋亡的可能机制。方法：以不同浓度的PDTC处理人肝癌HepG2细胞,利用MTT法检测对人肝癌HepG2细胞凋亡的影响;利用RT-PCR和Western-blot检测caspase-3mRNA和蛋白的表达。结果：不同浓度PDTC作用人肝癌HepG2细胞不同时间后,能够显著抑制HepG2细胞的生长增殖,存在剂量和时间依赖性（P〈0.05）;PDTC能够上调HepG2细胞中caspase-3mRNA和蛋白的表达。结论：NF-kB拮抗剂PDTC对人肝癌HepG2细胞产生显著抑制,并能上调HepG2细胞中caspase-3mRNA和蛋白的表达。     

NF-kB 拮抗剂PDTC 对HepG2 细胞的抑制及对caspase-3 表达的影响

目的:研究NF-kB拮抗剂PDTC诱导人肝癌HepG2细胞凋亡及其对凋亡相关基因caspase-3表达的影响,初步探讨其诱导凋亡的可能机制。方法:以不同浓度的PDTC处理人肝癌HepG2细胞,利用MTT法检测对人肝癌HepG2细胞凋亡的影响;利用RT-PCR和Western-blot检测caspase-3mRNA和蛋白的表达。结果:不同浓度PDTC作用人肝癌HepG2细胞不同时间后,能够显著抑制HepG2细胞的生长增殖,存在剂量和时间依赖性(P<0.05);PDTC能够上调HepG2细胞中caspase-3mRNA和蛋白的表达。结论:NF-kB拮抗剂PDTC对人肝癌HepG2细胞产生显著抑制,并能上调HepG2细胞中caspase-3mRNA和蛋白的表达。     

姜黄素通过MAPK信号通路诱导人肝癌SMMC-7721细胞凋亡

本文阐述了姜黄素(Curcumin)对体外培养的人肝癌SMMC-7721细胞增殖和凋亡的影响,并探讨了其诱导凋亡的信号转导机制。采用MTT法和细胞计数法检测不同浓度姜黄素对人肝癌细胞株SMMC-7721增殖的影响,利用流式细胞术检测姜黄素对人肝癌SMMC-7721细胞凋亡的影响,通过RT-PCR及Western blot检测姜黄素对人肝癌SMMC-7721细胞中凋亡相关蛋白Caspase-3、Survivin、Bcl-2和Bax表达的影响,最后通过检测MAPK的磷酸化水平分析姜黄素诱导SMMC-7721细胞凋亡的信号转导机制,通过MAPK抑制剂实验进一步证实诱导凋亡的分子机制。研究结果显示,姜黄素呈时间和剂量依赖性抑制人肝癌SMMC-7721细胞的增殖,其中40μmol/L姜黄素可明显诱导SMMC-7721细胞的凋亡,并呈时间依赖性上调促凋亡蛋白Caspase-3和Bax的表达、下调抗凋亡蛋白Survivin和Bcl-2的表达,姜黄素对凋亡相关蛋白表达的调节及诱导凋亡可以通过激活JNK、抑制ERK和p38 MAPK信号通路实现。表明姜黄素可诱导人肝癌SMMC-7721细胞凋亡,其机制与姜黄素激活JNK、抑制ERK和p38 MAPK信号通路从而上调Caspase-3和Bax的表达,下调Survivin和Bcl-2的表达有关。     

灵芝肽诱导人肝癌HepG2细胞凋亡的研究

研究了灵芝肽(GLP)在体外对人肝癌HepG2细胞凋亡的影响,并初步探讨了其作用机制。结果显示,透射电镜下可见细胞染色质浓缩、聚集于核边缘成块状,形成典型的凋亡小体;GLP使HepG2细胞阻滞于G0/G1期,随着GLP浓度升高,其G0/G1期的细胞比例随之增加;同时细胞的早期、晚期和总的凋亡率亦均随之增加,存在剂量-效应关系;Western blotting检测结果显示,抑制凋亡基因bcl-2和survivin表达下调,而促凋亡基因p53表达上调,并且都存在剂量依赖性;细胞凋亡的关键蛋白酶caspase-3被激活,并且caspase-3酶活性与GLP浓度亦有剂量依赖性。提示GLP体外可诱导人肝癌HepG2细胞凋亡,其作用机制可能与bcl-2和survivin表达下调、p53表达上调及Caspase-3被激活有关。     

PIG11与热休克蛋白60的相互作用介导肝癌HepG2细胞凋亡

为了探讨候选肝癌抑癌蛋白PIG11(p53-induced gene 11,PIG11)诱导细胞凋亡的机制,首次在HepG2细胞株中鉴定了11个PIG11结合蛋白,热休克蛋白60(heat shock protein 60,Hsp60)为其中之一．采用免疫共沉淀联合Western blot 技术对Hsp60进行了验证．用Western blot检测其蛋白质表达,结果显示：pLXSN-PIG11-HepG2细胞中Hsp60蛋白表达较pLXSN-HepG2、HepG2细胞组下调(n=3,P < 0.01)．选取与Hsp60关系密切的Bax蛋白进行研究,Western blot结果显示PIG11高表达可引起胞浆Bax向线粒体转位．以上结果表明,PIG11蛋白能与HepG2细胞中的Hsp60结合,促进Hsp60-Bax的分离,引起Bax从胞液到线粒体转位,激活线粒体凋亡途径,这可能是其诱导HepG2细胞凋亡的主要机制之一．     

Curcumin Mitigates the Intracellular Lipid Deposit Induced by Antipsychotics In Vitro

Scope

First- and second-generation antipsychotics (FGAs and SGAs, respectively), both inhibit cholesterol biosynthesis and impair the intracellular cholesterol trafficking, leading to lipid accumulation in the late endosome/lysosome compartment. In this study we examined if curcumin, a plant polyphenol that stimulates exosome release, can alleviate antipsychotic-induced intracellular lipid accumulation.

Methods

HepG2 hepatocarcinoma cells were treated with antipsychotics or placebo and DiI-labelled LDL for 18 h and then exposed to curcumin for the last 2 h. Cells and media were collected separately and used for biochemical analyses, electron microscopy and immunocytochemistry. Exosomes were isolated from the incubation medium by ultracentrifugation.

Results

Curcumin treatment reduced the number of heterolysosomes and shifted their subcellular localization to the periphery, as revealed by electron microscopy, and stimulated the release of lysosomal β-hexosaminidase and exosome markers flotillin-2 and CD63 into the media. The presence of DiI in exosomes released by cells preloaded with DiI-LDL demonstrated the endolysosomal origin of the microvesicles. Furthermore, curcumin increased the secretion of cholesterol as well as LDL-derived DiI and [³H]-cholesterol, in association with a decrease of intracellular lipids. Thus, the disruption of lipid trafficking induced by FGAs or SGAs can be relieved by curcumin treatment. This polyphenol, however, did not mitigate the reduction of cholesterol esterification induced by antipsychotics.

Conclusion

Curcumin stimulates exosome release to remove cholesterol (and presumably other lipids) accumulated within the endolysosomal compartment, thereby normalizing intracellular lipid homeostasis. This action may help minimize the adverse metabolic effects of antipsychotic treatment, which should now be evaluated in clinical trials.     

The COX-2 inhibitor Celecoxib enhances the sensitivity of KB/VCR oral cancer cell lines to Vincristine by down-regulating P-glycoprotein expression and function

Previous studies have indicated that long-term chemotherapy decreases the sensitivity of oral cancer cells to chemotherapeutics while simultaneously increasing resistance to these drugs. COX-2 inhibitors are known to enhance the toxic action of anti-tumor drugs against cancer cells. Using the MTT method, we investigated the influence of the COX-2 selective inhibitor Celecoxib on the proliferation of KB/VCR oral cancer cell lines and analyzed the effect of Celecoxib on the regulation of P-glycoprotein (P-gp) expression and function. Western blot analysis was employed to detect the expression of P-gp, and flow cytometry was used to evaluate P-gp function by detecting the accumulation of the active P-gp functional fluorescence substrate within KB/VCR cells. The results revealed that a low dose of Celecoxib (10 μmol/L) showed no growth inhibitory effects on KB/VCR cell lines. When the concentration of Celecoxib was greater than or equal to 20 μmol/L, the inhibitory effect on KB/VCR cells was significantly enhanced in a time- and dose-dependent manner. The lower dose of Celecoxib (10 μmol/L) significantly enhanced the toxicity of Vincristine (VCR) against KB/VCR cell lines. After the application of Celecoxib plus VCR (10 μmol/L+1.5μmol/L, respectively) treatment for 24, 48 or 72 h, the growth inhibition rates of KB/KBV cells were 37.82 ± 1.60%, 47.84 ± 1.29% and 54.43 ± 2.35%, respectively, which were significantly higher than the rates in the cells treated only with Celecoxib (10 μmol/L) or VCR (1.5 μmol/L) (all P<0.01). P-gp expression levels in KB/KBV cells treated with Celecoxib plus VCR (10 μmol/L+1.5 μmol/L, respectively) were markedly lower than the levels in control cells and those treated with VCR (1.5 μmol/L) (all P<0.01). In addition, the intensity of Rho123 fluorescence of KB/KBV cells in cells treated with Celecoxib plus VCR (10 μmol/L+1.5 μmol/L, respectively) or Celecoxib alone (10 μmol/L) was significantly higher than the intensity observed in control cells and those treated with VCR alone (1.5 μmol/L) (all P<0.01). The underlying mechanism of these phenomena is likely correlated with the down-regulation of the expression and function of P-gp due to Celecoxib, thereby increasing the amount of VCR accumulated in KB/VCR cells.     

Downstream caspases are novel targets for the antiapoptotic activity of the molecular chaperone hsp70

The response of cancer cells to apoptosis-inducing agents can be characterized by 2 opposing factors, the proapoptotic caspase cascade and the antiapoptotic stress protein Hsp70. We show here that these factors interact in U-937 leukemia cells induced to apoptosis with anticancer drugs, etoposide and adriamycin (ADR). The protective effect of Hsp70 was verified using 2 approaches: mild heat stress and transfection-mediated overexpression of the Hsp70 gene. The increase in Hsp70 levels attained by these 2 methods was found to postpone caspase activation for 12-18 hours. An in vitro assay was developed using mouse myeloma NS0/1 cells, which lack the expression of Hsp70. Measurement of DEVD-ase activity in extracts of apoptotic NS0/1 cells incubated with purified Hsp70 showed that Hsp70 reduced caspase activity by up to 50% of its control value in a dose-dependent manner. The hypothesis that the inhibitory effect of Hsp70 on caspase-3/7 activity related to a direct interaction between Hsp70 and the caspases was tested by reciprocal immunoprecipitations and Far-western analyses. These tests were performed with extracts of Hsp70-overexpressing, control, and ADR-treated U-937 cells and using anti-caspase-3, caspase-7, and anti-Hsp70 antibodies, and the data clearly showed that Hsp70 was able to interact with the proforms of these caspases in cell lysates and with reconstituted purified proteins but did not bind the activated forms of either caspase-3 or -7. This association was also corroborated by a novel, enzyme-linked immunosorbent assay-like assay, protein interaction assay, that combined the advantages of immunoprecipitation and immunoblotting in a 96-well microplate-based assay. Thus, Hsp70 may act to suppress caspase-dependent apoptotic signaling through binding the precursor forms of both caspase-3 and caspase-7 and preventing their maturation.     

Curcumin-induced degradation of ErbB2: A role for the E3 ubiquitin ligase CHIP and the Michael reaction acceptor activity of curcumin

We investigated the molecular mechanism underlying curcumin depletion of ErbB2 protein. Curcumin induced ErbB2 ubiquitination but pretreatment with proteasome inhibitors neither prevented curcumin depletion of ErbB2 protein nor further accumulated ubiquitinated ErbB2. Curcumin increased association of endogenous and ectopically expressed CHIP, a chaperone-dependent ubiquitin ligase, with ErbB2. In COS7 cells cotransfected with ErbB2 and various CHIP plasmids followed by curcumin treatment, CHIP-H260Q (a mutant lacking ubiquitin ligase activity) promoted less curcumin-induced ErbB2 ubiquitination than did wild type CHIP, and CHIP-K30A (a mutant incapable of binding Hsp90 and Hsp70) neither associated with ErbB2 nor promoted its ubiquitination. ErbB2 mutants lacking the kinase domain failed to associate with CHIP and were completely resistant to ubiquitination and depletion induced by curcumin. Finally, curcumin's Michael reaction acceptor functionality was required for both covalent association of curcumin with ErbB2 and curcumin-mediated ErbB2 depletion. These data suggest (1) that CHIP-dependent ErbB2 ubiquitination is implicated in curcumin-stimulated ErbB2 depletion, and (2) that covalent modification of ErbB2 by curcumin is the proximal signal which initiates this process.     

Homocysteine-induced caspase-3 activation by endoplasmic reticulum stress in endothelial progenitor cells from patients with coronary heart disease and healthy donors

Previous studies have suggested an association of hyperhomocysteinemia-induced vascular pathology with enhanced apoptotic potential of endothelial progenitor cells in patients with coronary heart disease. Our results indicate that 500 μmol/L homocysteine induced endothelial progenitor cell apoptosis and activation of caspase-3, both of which were abolished by 100 μmol/L and 200 μmol/L salubrinal, an agent that prevents endoplasmic reticulum stress-induced apoptosis. The addition of 500 μmol/L homocysteine caused a release of Ca(2+) from intracellular stores, and enhanced phosphor-eukaryotic initiation factor 2α phosphorylation at Ser51 and the expression of a glucose-regulated protein of 78 kDa and a C/EBP homologous protein independently of extracellular Ca(2+). These effects of homocysteine on endothelial progenitor cells were significantly greater in patients with coronary heart disease than in healthy donors. These findings suggest that homocysteine induces endoplasmic reticulum stress-mediated activation of caspase-3 in endothelial progenitor cells, an event that is enhanced in patients with coronary heart disease. Furthermore, enhanced endoplasmic reticulum stress-mediated activation of caspase-3 in endothelial progenitor cells might be involved in hyperhomocysteinemia-associated vascular pathology.     

Stereoselective Regulation of P-gp Activity by Clausenamide Enantiomers in Caco-2, KB/KBv and Brain Microvessel Endothelial Cells

The (−)- and (+)-clausenamide (CLA) enantiomers have different pharmacokinetic effects in animals, but their association with putative stereoselective regulation of P-glycoprotein (P-gp) remains unclear. Using three cells expressing P-gp—Caco-2, KBv and rat brain microvessel endothelial cells(RBMEC), this study investigated the association of CLA enantiomers with P-gp. The results showed that the rhodamine 123 (Rh123) accumulation, an indicator of P-gp activity, in Caco-2, KBv and RBMECs was increased by (−)CLA (1 or 5 μmol/L) at 8.2%–28.5%, but reduced by (+)CLA at 11.7%–25.9%, showing stereoselectivity in their regulation of P-gp activity. Following co-treatment of these cells with each CLA enantiomer and verapamil as a P-gp inhibitor, the (+)-isomer clearly antagonized the inhibitory effects of verapamil on P-gp efflux, whereas the (−)-isomer had slightly synergistic or additive effects. When higher concentrations (5 or 10 μmol/L) of CLA enantiomers were added, the stimulatory effects of the (+)-isomer were converted into inhibitory ones, leading to an enhanced intracellular uptake of Rh123 by 24.5%–58.2%; but (−)-isomer kept its inhibition to P-gp activity, causing 30.0%–63.0% increase in the Rh123 uptake. The biphasic effects of (+)CLA were confirmed by CLA uptake in the Caco-2 cells. (+)CLA at 1 μmol/L had significantly lower intracellular uptake than (−)CLA with a ratio[(−)/(+)] of 2.593, which was decreased to 2.167 and 1.893 after CLA concentrations increased to 2.5 and 5 μmol/L. Besides, in the non-induced KB cells, (+)CLA(5 μmol/L) upregulated P-gp expression at 54.5% relative to vehicle control, and decreased Rh123 accumulation by 28.2%, while (−)CLA(5 μmol/L) downregulated P-gp expression at 15.9% and increased Rh123 accumulation by 18.0%. These results suggested that (−)CLA could be a P-gp inhibitor and (+)CLA could be a modulator with concentration-dependent biphasic effects on P-gp activity, which may result in drug—drug interactions when combined with other P-gp substrate drugs.     

Binding partners for curcumin in human schwannoma cells: Biologic Implications

Curcumin (diferuloylmethane) is a potent anti-inflammatory and anti-tumorigenic agent that has shown preclinical activity in diverse cancers. Curcumin up-regulates heat shock protein 70 (hsp70) mRNA in several different cancer cell lines. Hsp70 contributes to an escape from the apoptotic effects of curcumin by several different mechanisms including prevention of the release of apoptosis inducing factor from the mitochondria and inhibition of caspases 3 and 9. Previously we showed that the combination of curcumin plus a heat shock protein inhibitor was synergistic in its down-regulation of the proliferation of a human schwannoma cell line (HEI-193) harboring an NF2 mutation, possibly because curcumin up-regulated hsp70, which also binds merlin, the NF2 gene product. In order to determine if curcumin also interacts directly with hsp70 and to discover other binding partners of curcumin, we synthesized biotinylated curcumin (bio-curcumin) and treated HEI-193 schwannoma cells. Cell lysates were prepared and incubated with avidin-coated beads. Peptides pulled down from this reaction were sequenced and it was determined that biotinylated curcumin bound hsp70, hsp90, 3-phosphoglycerate dehydrogenase, and a β-actin variant. These binding partners may serve to further elucidate the underlying mechanisms of curcumin’s actions.     

The polysaccharides from Ganoderma lucidum: Are they always inhibitors on human hepatocarcinoma cells?

The antitumor activity of intracellular polysaccharides from submerged fermentation of Ganoderma lucidum was investigated focusing on the inhibition on human liver cancer cells. The polysaccharides inhibited human hepatocarcinoma cell HepG2 during earlier phase with lower dosage but obviously became less functional in later phase regardless of the dosage applied. However, apoptosis of the drugged HepG2 cells appeared in later incubation phase with high dosage, and the apoptosis could be enhanced by supplemental dose of the intracellular polysaccharides. Nevertheless, the intracellular polysaccharides inhibited other human hepatocarcinoma cells such as BEL-7402 and Huh-7 but luckily stimulated human normal liver cell L02 only in a positive dose- and time-dependent manner; so did the sulfated extracellular polysaccharides when it inhibited HepG2 and L02 cells. However, the toxicity of sulfated extracellular polysaccharides to L02 cells can be eliminated by the intracellular polysaccharides.     

Homocysteine-Induced Caspase-3 Activation by Endoplasmic Reticulum Stress in Endothelial Progenitor Cells from Patients with Coronary Heart Disease and Healthy Donors

Previous studies have suggested an association of hyperhomocysteinemia-induced vascular pathology with enhanced apoptotic potential of endothelial progenitor cells in patients with coronary heart disease. Our results indicate that 500 μmol/L homocysteine induced endothelial progenitor cell apoptosis and activation of caspase-3, both of which were abolished by 100 μmol/L and 200 μmol/L salubrinal, an agent that prevents endoplasmic reticulum stress-induced apoptosis. The addition of 500 μmol/L homocysteine caused a release of Ca²⁺ from intracellular stores, and enhanced phosphor-eukaryotic initiation factor 2α phosphorylation at Ser51 and the expression of a glucose-regulated protein of 78 kDa and a C/EBP homologous protein independently of extracellular Ca²⁺. These effects of homocysteine on endothelial progenitor cells were significantly greater in patients with coronary heart disease than in healthy donors. These findings suggest that homocysteine induces endoplasmic reticulum stress-mediated activation of caspase-3 in endothelial progenitor cells, an event that is enhanced in patients with coronary heart disease. Furthermore, enhanced endoplasmic reticulum stress-mediated activation of caspase-3 in endothelial progenitor cells might be involved in hyperhomocysteinemia-associated vascular pathology.