Inhibitory effects of oleoylethanolamide (OEA) on H2O2-induced human umbilical vein endothelial cell (HUVEC) injury and apolipoprotein E knockout (ApoE-/-) atherosclerotic mice

Atherosclerosis (AS) is initiated by vascular endothelial cell injury, which is induced by lipid and protein oxidation. Oleoylethanolamide (OEA), a dietary fat-derived lipid, has shown atheroprotective effect. In vitro studies demonstrated that OEA showed cytoprotective effects on H₂O₂-induced primary cultured human umbilical vein endothelial cell (HUVEC) injury model. Further investigation of the cytoprotective effects of OEA demonstrated that OEA exerted its function by scavenging for reactive oxygen species, as well as increasing anti-oxidative enzymes, reducing lipid peroxidation, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells and apoptosis-related proteins expression. The in vivo study using an ApoE-/- mouse model fed with high-fat diet for 8 weeks showed that OEA (10 mg/kg/day, i.g.) administration reduced blood lipid levels, prevented endothelial cell damage and inhibited early AS plaque formation. In conclusion, our results suggested that OEA exerted a pharmacological effect on ameliorating atherosclerotic plaque formation through the inhibition of oxidative stress-induced endothelial cell injury and therefore OEA can be a potential candidate drug for anti-atherosclerosis.     

6-磷酸果糖激酶-2/果糖-2，6-二磷酸酶3对人脐静脉内皮细胞管腔形成的影响

文题释义： 6-磷酸果糖激酶-2/果糖-2,6-二磷酸酶3(PFKFB3)：糖酵解作为血管内皮细胞中提供能量的主要方式,而6-磷酸果糖激酶-2/果糖-2,6-二磷酸酶作为糖酵解中重要的刺激剂,在肿瘤新生血管方面具有重要作用,在此次实验中主要用于说明在高糖环境下对血管内皮的作用。 糖尿病性视网膜病变：是糖尿病中微血管严重并发症之一,一旦产生视网膜新生血管,将严重影响工作人群的视力,此次实验主要研究脐静脉内皮细胞在高糖环境中PFKFB3的作用,以期找到缓解视网膜新生血管的新靶点。 背景：糖尿病性视网膜病变中关于新生血管的研究多局限于血管内皮生长因子,而6-磷酸果糖激酶-2/果糖-2,6-二磷酸酶(phosphofructokinase-2/fructose-2,6-bisphosphatase,PFKFB3)也有一定的作用。 目的：探讨PFKFB3小干扰RNA(siRNA)对高糖环境下人脐静脉内皮细胞的作用及机制。 方法：将人脐静脉内皮细胞分为4组：正常糖组(5.5 mmol/L 葡萄糖)、正常糖+PFKFB3-siRNA组、高糖组(30 mmol/L葡萄糖)、高糖+PFKFB3-siRNA组。正常糖+PFKFB3-siRNA组和高糖+PFKFB3-siRNA组以PFKFB3-siRNA转染人脐静脉内皮细胞。采用Western blot检测PFKFB3的沉默表达效果,并选取沉默效果最佳者进行后续实验。体外管腔形成实验检测细胞成管能力,实时荧光定量PCR检测PFKFB3的mRNA表达,Western blot检测PFKFB3及磷酸化AKT/AKT的蛋白表达。 结果与结论：①PFKFB3-siRNA能明显抑制PFKFB3的蛋白表达(P < 0.01);②与正常糖组相比,正常糖+ PFKFB3-siRNA组管腔形成总长度增加(P < 0.05),高糖组管腔形成总长度明显减少(P < 0.01),高糖+ PFKFB3-siRNA组管腔形成总长度无明显变化(P > 0.05);与高糖组相比,高糖+PFKFB3-siRNA组管腔形成能力明显增加(P < 0.05);与正常糖组相比,高糖组中PFKFB3的mRNA及蛋白表达均明显升高(均P < 0.01);与高糖组相比,高糖+PFKFB3-siRNA组的PFKFB3蛋白表达降低(P < 0.05);③与正常糖组相比,正常糖+PFKFB3-siRNA组与高糖组中磷酸化AKT/AKT比值均降低(P < 0.01),而高糖组+PFKFB3-siRNA组磷酸化AKT/AKT蛋白比值无明显变化(P > 0.05);与高糖组相比,高糖+PFKFB3-siRNA组磷酸化AKT/AKT蛋白比值升高(P < 0.01)。提示采用siRNA沉默PFKFB3基因表达可抑制人脐静脉内皮细胞中PFKFB3的表达,促进管腔形成能力,其机制可能与下调AKT表达有关。 ORCID: 0000-0001-9070-3292(曹阳) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Semaphorin 3A过表达对H_2O_2诱导的人脐静脉内皮细胞损伤的影响

目的:探讨semaphorin 3A(Sema 3A)对过氧化氢(H_2O_2)诱导的人脐静脉内皮细胞(HUVECs)损伤的影响。方法:构建Sema 3A过表达载体,以脂质体转染法转染HUVECs,过表达效果以qPCR和Western blot法验证;待测细胞以200μmol/L H_2O_2处理4 h;qPCR法检测炎性细胞因子水平;乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)和丙二醛(MDA)水平以相应比色法检测;细胞活力以MTT法检测;流式细胞术检测细胞凋亡,凋亡相关蛋白cleaved caspase-3及Bcl-2水平以Western blot法检测。结果:Sema 3A过表达能显著增加H_2O_2诱导的HUVECs凋亡、炎性细胞因子分泌以及LDH和MDA含量,同时显著抑制SOD活性和细胞活力;Sema 3A对未经H_2O_2处理的HUVECs没有损伤效应,即其对HUVECs的损伤具有H_2O_2依赖性。结论:Sema 3A能显著加重H_2O_2诱导的HUVECs损伤,在氧化应激所致的内皮细胞损伤过程中发挥促进作用。     

15-deoxy-delta(12,14)-prostaglandin J2 inhibits IFN-gamma-induced galectin-9 expression in cultured human umbilical vein endothelial cells

BACKGROUND: Galectin-9 is involved in chemotaxis and adhesion of eosinophils, and is induced in vascular endothelial cells by interferon-gamma (IFN-gamma). 15-deoxy-delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is a ligand for peroxisome proliferator-activated receptor-gamma (PPAR-gamma), and known to modulate the expression of various genes. METHODS: We have studied the effect of 15d-PGJ(2) on the IFN-gamma-induced galectin-9 expression in human umbilical vein endothelial cells (HUVEC) in culture. RESULTS: 15d-PGJ(2) inhibited the IFN-gamma-induced galectin-9 expression in a PPAR-gamma-independent manner, and also inhibited the adhesion of EoL-1 cells to an HUVEC monolayer treated with IFN-gamma. 15d-PGJ(2) partially inhibited IFN-gamma-induced phosphorylation of STAT-1 in HUVEC. CONCLUSIONS: 15d-PGJ(2) may regulate inflammatory reactions through the inhibition of galectin-9 expression.     

DNA修复蛋白MGMT基因的克隆及转导脐血CD23^＋细胞后耐药特征的研究

目的：增强脐血ＣＤ３４＾＋造血细胞对化疗药物的耐药表型,探讨逆转录病毒介导的基因转移效率和耐药基因特性,以及在脐血造血干细胞保护性基因治疗中的作用和意义。方法：应用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）从人肝细胞中获得编码六氧甲基鸟嘌呤－ＤＮＡ－甲基转移酶（Ｏ＾６－ｍｅｔｈｙｌｇｕａｎｉｎｅ－ＤＮＡ－ｍｅｔｈｙｌｔｒａｎｓｆｅｒａｓｅ,ＭＧＭＴ）ｃＤＮＡ;利用基因重组技术,将其克隆于ｐＧＥＭ－Ｔ质粒载体并构建了逆转录病毒载体Ｇ１Ｎａ－ＭＧＭＴ;应用脂质体ＬｉｐｏｆｅｃｔＡＭＩＮＥ基因转移法将后者导入ＧＰ＋Ｅ８６和ＰＡ３１７病毒包装细胞,以卡氮芥１,３－Ｂｉｓ（２－Ｃｈｌｏｒｏｅｔｈｙｌ）－１－Ｎｉｔｒｏｓｏｕｒｅａ（ＢＣＮＵ）加压筛选后的阳性克隆上清经乒乓效应后继而感染脐血ＣＤ３４＾＋细胞。应用ＰＣＲ,Ｓｏｕｔｈ     

血管紧张素(1-7)通过抑制JAK/STAT信号通路对抗高糖诱导的人脐静脉内皮细胞损伤

目的:研究血管紧张素(1-7)[Ang-(1-7)]能否通过抑制JAK/STAT信号通路对抗高糖诱导的人脐静脉内皮细胞(HUVECs)损伤。方法:CCK-8检测细胞存活率;Western blot法检测内皮细胞JAK2、STAT3、pJAK2、p-STAT3、cleaved caspase-3和内皮型一氧化氮合酶(e NOS)的蛋白水平;Hoechst 33258染色荧光显微镜照相法检测内皮细胞凋亡数量;DCFH-DA染色荧光显微镜照相法检测内皮细胞内活性氧簇(ROS)的水平。结果:应用高糖(40 mmol/L)处理HUVECs 12~48 h能明显上调JAK2的磷酸化水平,于24 h达最高峰;在24~48 h能明显上调STAT3的磷酸化水平,于36 h最高;在12~24 h能明显上调cleaved caspase-3的表达,在3~48 h随着时间延长,e NOS的表达逐渐降低。2μmol/L的Ang-(1-7)或20μmol/L的JAK/STAT通路抑制剂AG490预处理0.5 h可显著抑制由高糖引起的内皮细胞损伤,表现为p-STAT3、p-JAK2及cleaved caspase-3蛋白水平降低、细胞存活率及e NOS表达升高,细胞凋亡数量和胞内ROS生成减少。结论:Ang-(1-7)能通过抑制JAK/STAT通路对抗高糖诱导的人脐静脉内皮细胞损伤。     

氧化砷诱导食管癌细胞凋亡线粒体形态改变

通过研究食管癌细胞株ＳＨＥＥＣＩ细胞凋亡早期线粒体的形态改变和ｂｃｌ－２ｂａｘ的表达,以了解三氧化二砷（Ａｓ２Ｏ３）作用于食管癌细胞的机制。方法食管癌细胞株ＳＨＥＥＣ１用１、２、３μｍｏｌ／Ｌ Ａｓ２Ｏ３作用２、４、６、１２、２４ｈ。用Ａｎｎｅｘｉｎ－Ｖ荧光探针,流式细胞仪和ＤＮＡ组方图检早期凋亡细胞;光镜和电镜检测凋亡细胞的形态学改变;用罗达明（Ｒｈｏｄａｍｉｎ１２３）荧光探针检查活细胞线粒体的     

硫代硫酸钠标准溶液(0.1 mol/L)标定的不确定度评定方法研究

建立硫代硫酸钠标准溶液(0.1mol/L)标定的不确定度评定方法.以国家标准为依据,建立数学模型,分析不确定度来源,探讨不确定度的评定方法.硫代硫酸钠标准溶液(0.1mol/L)标定的不确定度评定,对产品质量检测结果的可信度表达有重要意义.     

Salidroside protects human fibroblast cells from premature senescence induced by H(2)O(2) partly through modulating oxidative status

Although salidroside and salidroside-like compounds are considered as most critical constitutes needed and responsible for multiple therapeutic benefits of Rhodiola rosea L., including anti-aging, direct demonstration regarding the role of salidroside in anti-aging process is still deficient. In this study, we selected the H(2)O(2)-induced premature senescence model in human fetal lung diploid fibroblasts to investigate the protection of salidroside against aging in vitro and associated molecular mechanisms. We found that salidroside considerably reversed senescence-like phenotypes in the oxidant challenged model, including alterations of morphology, cell cycle, SA-β-gal staining, DNA damage, as well as related molecules expression such as p53, p21 and p16. The protection occurred in a dose-dependent manner, with 5μM offering best efficacy. The proposed antioxidant property of the compound was confirmed in this cellular system, and thus at least partially accounted for the protection of the compound against premature senescence. Similar protection of salidroside against replicative senescence was observed as well. Interestingly, the regulation of senescence-related molecules by salidroside involved ROS-irrelevant mechanisms in both models. This finding presents salidroside as an attractive agent with potential to retard aging and attenuate age-related diseases in humans.     

A pro-drug of the green tea polyphenol (−)-epigallocatechin-3-gallate (EGCG) prevents differentiated SH-SY5Y cells from toxicity induced by 6-hydroxydopamine

Regular consumption of green tea benefits people in prevention from cardiovascular disorders, obesity as well as neurodegenerative diseases. (−)-Epigallocatechin-3-gallate (EGCG) is regarded as the most biologically active catechin in green tea. However, the stability and bioavailability of EGCG are restricted. The purpose of the present study was to investigate whether a pro-drug, a fully acetylated EGCG (pEGCG), could be more effective in neuroprotection in Parkinsonism mimic cellular model. Retinoic acid (RA)-differentiated neuroblastoma SH-SY5Y cells were pre-treated with different concentrations of EGCG and pEGCG for 30 min and followed by incubation of 25 μM 6-hydroxydopamine (6-OHDA) for 24 h. We found that a broad dosage range of pEGCG (from 0.1 to 10 μM) could significantly reduce lactate dehydrogenase release. Likewise, 10 μM of pEGCG was effective in reducing caspase-3 activity, while EGCG at all concentrations tested in the model failed to attenuate caspase-3 activity induced by 6-OHDA. Furthermore, Western-blot analysis showed that Akt could be one of the specific signaling pathways stimulated by pEGCG in neuroprotection. It was demonstrated that 25 μM of 6-OHDA significantly suppressed the phosphorylation level of Akt. Only pEGCG at 10 μM markedly increased its phosphorylation level compared to 6-OHDA alone. Taken together, as pEGCG has higher stability and bioavailbility for further investigation, it could be a potential neuroprotective agent and our current findings may offer certain clues for optimizing its application in future.     

红细胞生成素3'-增强子片段对内皮细胞缺氧时环加氧酶-2基因表达的影响

目的和方法：体外合成红细胞生成素（Ｅｐｏ）３′－增强子野生型及突变型片段，借助脂质体，转入内皮细胞，用半定量ＲＴ－ＰＣＲ方法测定分离细胞在常氧或缺氧条件下培养４ｈ的环加氧酶２（ＣＯＸ－２）ｍＲＮＡ。结果：（１）大鼠主动脉内皮细胞、肺微血管内皮细胞在常氧下培养，有ＣＯＸ－２基因表达；（２）缺氧能诱导这两种细胞ＣＯＸ－２基因表达增加；（３）野生型Ｅｐｏ３′－增强子片段能阻断缺氧对内皮细胞ＣＯＸ－２基因表达的诱导作用，而突变片段则无此作用。结论：在ＣＯＸ－２基因序列中，可能存在Ｅｐｏ３′－端增强子片段，它参与了内皮细胞的缺氧反应     

Clustering the adhesion molecules VLA-4 (CD49d/CD29) in Jurkat T cells or VCAM-1 (CD106) in endothelial (ECV 304) cells activates the phosphoinositide pathway and triggers Ca2+ mobilization

Ligation of very late antigen (VLA)-4 (α₄β₁ integrin) with a cross-linked anti-α₄ subunit monoclonal antibody (mAb) triggered a biphasic Ca²⁺ response in Jurkat cell populations and in peripheral human lymphocytes. Cross-linking vascular cell adhesion molecule (VCAM)-1 (the counter-receptor of VLA-4) in ECV 304 endothelial cells generated a biphasic Ca²⁺ response. Tumor necrosis factor-α-primed human umbilical cord vascular endothelial cells also responded to the cross-linked mAb with a biphasic Ca²⁺ profile. Ligated VLA-4 (Jurkat cells) or VCAM-1 (ECV 304) stimulated the production of myo-inositol 1,4,5-trisphosphate. ECV 304 cells induced a biphasic Ca²⁺ response in Fura2-loaded Jurkat cells, whereas a transient response was observed when Jurkat cells were added to Fura2-loaded ECV 304 cells. The Ca²⁺ responses in these experiments involved VLA-4/VCAM-1 interactions since they were significantly reduced (～ 80%) by prior treatment of the target cells with the relevant noncross-linked mAb. Close contact between the cells triggered mutual Ca²⁺ signaling as shown by spectrofluorimetric and confocal microscopy time-dependent recordings. Fibronectin and its CS-1 fragment (V25) triggered a sustained Ca²⁺ response in Jurkat cells (confocal microscopy). Our results suggest that the VLA-4 and VCAM-1 adhesion molecules can transduce a signal that involves activation of the phosphoinositide pathway and the mobilization of Ca²⁺.     

CD4~+CD25~+调节T细胞对内皮细胞炎性因子分泌的影响

目的:探讨CD4+CD25+调节性T细胞(Tregs)对氧化型低密度脂蛋白(ox-LDL)诱导人脐静脉内皮细胞(HU-VECs)VCAM-1、MCP-1、IL-6表达的影响。方法:磁性细胞分离器(MACS)分离CD4+CD25+T细胞及CD4+CD25-T细胞。在ox-LDL作用下,将内皮细胞分别与anti-CD3mAb激活的CD4+CD25+T细胞,CD4+CD25-T细胞共培养24小时。分别应用流式细胞术、ELISA、real-timePCR测定Tregs对ox-LDL诱导损伤HUVECs炎性因子VCAM-1、MCP-1、IL-6表达的影响。应用Transwell小室及中和抗体实验观察Tregs作用于HUVECs的具体机制。结果:与对照组比较,Tregs细胞可显著抑制ox-LDL诱导损伤HU-VECs炎性因子VCAM-1、MCP-1、IL-6的表达;被Transwell隔离或加入中和性抗体anti-IL-10/anti-TGF-β后,Tregs对HUVECs的抑制作用可被部分逆转。结论:Tregs细胞可显著抑制ox-LDL诱导损伤HUVECs炎性因子的表达,其作用机制既依赖于细胞直接接触,又依赖于细胞因子。     

Cytokine-stimulated human lung alveolar epithelial cells release eotaxin-2 (CCL24) and eotaxin-3 (CCL26).

Asthma is a complex inflammatory disease characterized by a prolonged underlying airway inflammation resulting from cytokine-orchestrated signaling between many types of cells, including airway epithelial cells. Trafficking, recruitment, and activation of cells in airway disease are, in part, modulated by the newly discovered CC subfamily of chemokines, eotaxin (CCL11), eotaxin-2 (CCL24) and eotaxin-3 (CCL26), which transduce signals by acting as agonists for the CCR3 receptor. The specific cytokine stimuli that modulate CCL24 and CCL26 release in airway epithelial cells remain poorly defined. Thus, human 549 alveolar type II epithelium-like cells were stimulated singly and with combinations of 1-100 ng/ml tumor necrosis-factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-4, cytokines known to be elevated in the airways of asthmatics. Release of CCL11, CCL24, and CCL26 was quantified by ELISA, and CCR3 receptors monitored by immunocytochemistry and FACS analysis. Results suggest that epithelial cells release CCL11 during the first 24 h of stimulation, in contrast to a significant increase in CCL24 and CCL26 release after 24-48 h of stimulation. Differential release of the eotaxins in response to cytokine combinations was noted. The alveolar type II epithelial cells were found to possess constitutive CCR3 receptors, which increased after proinflammatory cytokine stimulation. The airway epithelium CCR3 receptor/eotaxin ligand signal transduction system may be an important target for development of novel mechanism-based adjunctive therapies designed to interrupt the underlying chronic inflammation in allergic and inflammatory disorders.     

Signaling through CD50 (ICAM-3) stimulates T lymphocyte binding to human umbilical vein endothelial cells and extracellular matrix proteins via an increase in β1 and β2 integrin function

Regulated adhesion of T lymphocytes to antigen-presenting cells, endothelial cells and extracellular matrix proteins is crucial in T lymphocyte activation and migration to the sites of injury. In this study, we show that three monoclonal antibodies (mAb) recognizing different epitopes on the CD50 (ICAM-3) molecule increase T lymphocyte adhesion to tumor necrosis factor (TNF)-stimulated human umbilical vein endothelial cells and extracellular matrix proteins. These phenomena are mediated by an increase in β1 and β2 integrin avidity since (a) CD50-induced adhesion to endothelial cells was abrogated by simultaneous blocking of β1- and β2-mediated adhesion pathways but not by interfering with either one individually, (b) CD50 mAb increased β1 integrin-mediated adhesion to extracellular matrix proteins and to fibronectin-derived synthetic peptides, (c) CD50 mAb enhanced T lymphocyte binding to ICAM-1 transfectants, and (d) CD50 mAb did not modify surface expression patterns of β1 or β2 integrins on T lymphocytes. Our data suggest that constitutively expressed CD50 (ICAM-3) can play a pivotal role in initiating a cascade of adhesion events which may be crucial in immune activation and in the development of inflammatory lesions.     

Synthesis of Poly(lauryl acrylate) by Single-Electron Transfer/Degenerative Chain Transfer Living Radical Polymerization Catalyzed by Na2S2O4 in Water

Living radical polymerization of lauryl acrylate was achieved by SET/DTLRP in water catalyzed by sodium dithionite. The work describes the synthesis of a highly hydrophobic and polar monomer in aqueous medium. The plots of versus conversion and ln[M]₀/[M] versus time are linear, indicating a controlled polymerization. This method leads to α,ω-diiodopoly(lauryl acrylate)s that can be further functionalized. The MWDs were determined using a combination of three detectors: RALLS, DV, and RI. The method studied in this work represents a possible route to prepare well-tailored macromolecules made of LA in environment friendly reaction medium. The syndiotactic content is 75%.

     

S100A9-induced release of interleukin (IL)-6 and IL-8 through toll-like receptor 4 (TLR4) in human periodontal ligament cells

S100A8, S100A9, and calprotectin (the S100A8/S100A9 complex) are calcium-binding proteins that promote extracellular pro-inflammatory functions and may play an important role in periodontal disease. Both toll-like receptor 4 (TLR4) and the receptor for advanced glycation end-products (RAGE) are thought to be important receptors for S100A8, S100A9, and calprotectin, but the specific pathways in periodontal ligament (PDL) cells are not yet clear. Our study was designed to identify the specific receptors for S100A9 in human PDL cells. Additionally, we investigated the specific pathways that activate the secretion of pro-inflammatory cytokines interleukins (IL)-6 and IL-8 in PDL cells. The role of nuclear factor (NF)-κB, mitogen-activated protein kinase (MAPK) and reactive oxygen species (ROS) in S100A9-induced pro-inflammatory cytokines were investigated through western blot analysis, dichlorodihydrofluorescein diacetate (H₂DCFDA) probe and the application of specific pathway inhibitors. Our results suggest that the S100A9-induced release of IL-6 and IL-8 from human PDL cells is dependent on TLR4, but not RAGE. We provide evidence that S100A9 promotes the secretion of IL-6 and IL-8 through different pathways. Specifically, S100A9 up-regulates the secretion of IL-6 from human PDL cells through NF-κB and p38 pathways and up-regulates the release of IL-8 from human PDL cells through the NF-κB, extracellular-regulated kinase (ERK) 1/2, c-Jun amino-terminal kinase (JNK) 1/2, and p38 signaling pathways. In addition, the release of both cytokines depends on ROS production. The release of both cytokines depends on ROS production. These results suggest that S100A9 promotes pro-inflammatory responses in PDL cells through the TLR4-mediated NF-κB and MAPK signaling pathways.     

3-cinnamoyl-4-hydroxy-6-methyl-2H-pyran-2-one (CHP) inhibits human ovarian cell proliferation by inducing apoptosis

Coumarins induce apoptosis by activating mitochondrial pathway and caspase-3-dependent apoptotic pathway. In the present study, we first time investigated the effect of 3-cinnamoyl-4-hydroxy-6-methyl-2H-pyran-2-one (CHP) on induction of apoptosis in human ovarian carcinoma cells. The data from MTT assay revealed a significant inhibitory effect on cell viability at 30 (87%) and 50 μM (74%) concentration of CHP in OVCAR-3 and OVCAR-420 cells, respectively after 72 h. Apoptosis analysis using annexin V/PI double staining followed by flow cytometry showed 59 and 52% binding to annexin V-FITC in OVCAR-3 and OVCAR-420 cells respectively. propidium iodide (PI) staining and flow cytometry examination indicated a significant increase in percentage of cells in G2/M phase after treatment with CHP compared to DMSO control group. Reactive oxygen species (ROS) assay kit showed increase in levels of ROS. We used rhodamine-123 (Rh-123) staining and flow cytometry assay to determine changes in mitochondrial membrane potential (ΛΨm). The results revealed that CHP significantly decreased MMP to 85.65 ± 1.2443% & 49.78 ± 1.6554% at 10 and 30 μM respectively in OVCAR-3 compared to 95.97 ± 2.1243% in control group. Western blot analysis clearly indicated a significant increase in the expression of Caspase-3, Bax, and release of Cytochrome c and decrease in Bcl-2, CDK1 and Cyclin B1 expression on treatment with CHP. Therefore, CHP may become a potential candidate for the treatment of human ovarian cancers.     

Detection of ABCC2, CYP2B6 and CYP3A4 in human peripheral blood mononuclear cells using flow cytometry

ABCC2 has a wide tissue distribution and can mediate the efflux of a number of therapeutic compounds from cells and contribute to potential treatment failure. Its diverse expression and ability to efflux a number of substrates imply a number of physiological and pharmacological roles. CYP2B6 and CYP3A4 are responsible for the metabolism of a number of therapeutic compounds. Reports on the expression of these proteins in various cells and tissues have been contradictory mainly due to differences in experimental approach and cell type studied. With the advances in commercially available antibodies we describe here a simplified technique for the detection of ABCC2, CYP2B6 and CYP3A4 in human peripheral blood mononuclear cells (PBMC) by flow cytometry. Results are expressed as mean increase in fluorescence compared to isotypically matched controls. Using these assays we confirmed the expression of these proteins in human PBMC. These methods are rapid and reproducible and have potential use for both in vitro and clinical applications.