siRNA沉默人类血红素敏感基因1对膀胱癌T24细胞的影响

目的 观察人类血红素敏感基因1(HRG-1)在膀胱正常组织和膀胱肿瘤组织中的表达情况,通过siRNA干扰技术抑制HRG-1在膀胱癌T24细胞的表达水平,观察其生长增殖的变化.方法 应用免疫组化SP法检测85例膀胱肿瘤(膀胱乳头状瘤25例,低级别膀胱癌30例,高级别膀胱癌30例)和20例膀胱正常组织石蜡标本中HRG-1表达情况,并进行相关临床病理分析.设计针对HRG-1基因的siRNA,转染膀胱癌T24细胞,应用免疫印迹法和RT-PCR分析HRG-1 siRNA的表达,MTT和流式细胞术(FCM)检测其增殖凋亡的变化.结果 正常膀胱组织和膀胱癌组织中HRG-1的表达水平比较差异有统计学意义(P＜0.05);HRG-1的阳性表达与膀胱肿瘤病例分级、临床分期相关(P＜0.05);转染siRNA后,HRG-1表达水平下降,MTT和FCM检测发现,细胞增殖曲线受到抑制,HRG-1-siRNA转染组与空载体转染组及未转染组比较,细胞凋亡比例增加,差异均具有统计学意义(P＜0.01).结论 siRNA-HRG-1处理T24细胞后,细胞的增殖受到抑制,同时凋亡比例增加,提示HRG-1基因可能参与了膀胱癌的发病.     

In vitro and in vivo anticancer activity evaluation of ursolic acid derivatives

Twenty-three ursolic acid (1) derivatives 2-24 (ten novel compounds 8-10, 14-17 and 22-24) modified at the C-3 and the C-28 positions were synthesized, and their structures were confirmed by IR, ¹H NMR, MS, and elemental analysis. The single crystals of compounds 15 and 17 were obtained. The cytotoxic activity of the derivatives was evaluated against HepG2, BGC-823, SH-SY5Y, HeLa and HELF cells by the MTT assay. The induction of apoptosis and affects on the cell cycle distribution with compound 14 were assessed by fluorescence microscopy, flow cytometry and the activity of caspase-3 in HepG2 cells. Compounds 14-17 had more significant antiproliferative ability against the four cancer cell lines and low cytotoxicity to human embryonic lung fibroblast cells (HELF). Compounds 11, 14-16, 21 and 23 were particularly active against HepG2 cell growth. Compound 14 was selected to investigate cell apoptosis and cell cycle distribution. Flow cytometric analysis and morphologic changes of the cell exhibited that treatment of HepG2 cells with compound 14 led to cell apoptosis accompanied by cell cycle arrest at the S phase in a dose-dependent manner. Furthermore, the activity of the caspase-3 enzyme was increased in the treated cells. In vivo studies using H22 xenografts in Kunming mice were conducted with compound 14 at doses of 50, 100 and 150 mg/kg body weight. The results revealed that the medium dosage group (100 mg/kg) showed significant anticancer activity (45.6 ± 4.3%) compared to the control group.     

Discovery of polyoxometalate-based HDAC inhibitors with profound anticancer activity in vitro and in vivo

We obtained 5 positive novel histone deacetylase inhibitors (HDACIs) from a polyoxometalate (POM) library by using a cell-based screening system targeting the p21 gene promoter. Among them, PAC-320, a new tri-organic-tin-substitute germanotungstate, displayed remarkable extracellular inhibitory activity. Meanwhile, the crystal structure of PAC-320 was characterized by X-ray crystallography. PAC-320 could stably exist under physiological conditions as revealed by UV spectrum, CV and TG. PAC-320 possessed a strong inhibitory effect to intracellular HDAC activity. More significantly, PAC-320 inhibited the growth of a variety of cancer cells, and exhibited remarkable anticancer effect in a hepatocarcinoma H22 cell mice model. This study revealed, for the first time, that the HDAC inhibitory activity is a mechanism by which POMs exert their anticancer effect.     

Anti-cancer activity of a novel palladium(II) complex on human breast cancer cells in vitro and in vivo

Anti-cancer effects of a newly-synthesized palladium(II) complex, [Pd(sac)(terpy)](sac)·4H₂O (sac = saccharinate, and terpy = 2,2′:6′,2″-terpyridine), were tested against human breast cancer cell lines, MCF-7 and MDA-MB-231. The Pd complex had a strong anti-growth effect in a dose- and time-dependent manner in vitro. This effect was also confirmed by the experiment performed on Balb/c mice in vivo. The IC₅₀ values were 0.09 μM for MDA-MB-231 and 3.05 μM for MCF-7. It was also very effective in disrupting the formation of MDA-MB-231 tubules on matrigel, indicative of a putative anti-invasive activity. It induced apoptosis via the cell death genes of DR4 and DR5. In conclusion, this newly-synthesized Pd (II) complex represents a potentially active novel drug for the breast cancer treatment.     

D. candidum has in vitro anticancer effects in HCT-116 cancer cells and exerts in vivo anti-metastatic effects in mice

BACKGROUND/OBJECTIVES

D. candidum is a traditional Chinese food or medicine widely used in Asia. There has been little research into the anticancer effects of D. candidum, particularly the effects in colon cancer cells. The aim of this study was to investigate the anticancer effects of D. candidum in vitro and in vivo.

MATERIALS/METHODS

The in vitro anti-cancer effects on HCT-116 colon cancer cells and in vivo anti-metastatic effects of DCME (Dendrobium canidum methanolic extract) were examined using the experimental methods of MTT assay, DAPI staining, flow cytometry analysis, RT-PCR, and Western blot analysis.

RESULTS

At a concentration of 1.0 mg/mL, DCME inhibited the growth of HCT-116 cells by 84%, which was higher than at concentrations of 0.5 and 0.25 mg/mL. Chromatin condensation and formation of apoptotic bodies were observed in cancer cells cultured with DCME as well. In addition, DCME induced significant apoptosis in cancer cells by upregulation of Bax, caspase 9, and caspase 3, and downregulation of Bcl-2. Expression of genes commonly associated with inflammation, NF-κB, iNOS, and COX-2, was significantly downregulated by DCME. DCME also exerted an anti-metastasis effect on cancer cells as demonstrated by decreased expression of MMP genes and increased expression of TIMPs, which was confirmed by the inhibition of induced tumor metastasis in colon 26-M3.1 cells in BALB/c mice.

CONCLUSIONS

Our results demonstrated that D. candidum had a potent in vitro anti-cancer effect, induced apoptosis, exhibited anti-inflammatory activities, and exerted in vivo anti-metastatic effects.     

ShRNA沉默基因DNA甲基转移酶3b的表达对膀胱癌T24细胞增殖的影响

目的体外研究重组质粒pshRNA-DNMT3b对膀胱癌T24细胞中DNMT3bmRNA和蛋白的沉默效应以及细胞增殖抑制的影响，初步探讨DNMT3b在膀胱肿瘤发生过程中的作用。方法实验分为空白对照组、HK组及pshRNA-DNMT3b组（24、48、72h）；常规培养T24细胞，用脂质体lipofectamine2000将重组质粒pshRNA-DNMT3b转染进入T24细胞，后进行RT．PCR、WesternBlot、MTY检测，观察pshRNA-DNMT3b对T24细胞中DNMT3bmRNA、蛋白质水平变化以及细胞增殖的影响。结果重组质粒pshRNA-DNMT3b成功的转染进入T24细胞；RT-PCR电泳结果用Gel-proanalyzer图像分析系统进行灰度分析，空白对照组、HK组、pshRNA-DNMT3b组（24、48、72h）条带的IOD相对比值（DNMT3／β-actin）分别是（99．56±1．24）％、（99．12±1．35）％、（75．77±1．42）％、（44．69±1．05）％和（20．52±0．89）％；Westernblot图像分析结果各组的相对比值分别是（99．43±1．28）％、（98．90±1．31）％、（67．83±1．02）％、（43．43±1．05）％和（21．92±0．89）％；在MTF检测中空白对照组和HK组之间差异无统计学意义（P〉0．05），pshRNA-DNMT3b组仅在72h后和前2组之间差异有统计学意义（P〈0．01），但抑制率仅增加0．45％左右。结论重组质粒pshRNA-DNMT3b能有效的降低膀胱癌1、24细胞中基因DNMT3bmRNA的转录及蛋白的表达，但在抑制细胞增殖上作用甚微。     

Effect of resveratrol on the metastasis of 4T1 mouse breast cancer cells in vitro and in vivo

We investigated the effects of resveratrol on metastasis in in vitro and in vivo systems. 4T1 cells were cultured in the presence of various concentrations (0-30 µmol/L) of resveratrol. For experimental metastasis, BALB/c mice were injected intravenously with 4T1 cells in the tail vein, and were orally administered various concentrations (0, 100, or 200 mg/kg Body weight) of resveratrol for 21 days. After resveratrol treatment, cell adhesion, wound migration, invasion, and MMP-9 activity were significantly decreased in a dose-dependent manner in 4T1 cells (P < 0.05). The numbers of pulmonary nodules were significantly decreased in mice fed the resveratrol (P < 0.05). The plasma MMP-9 activity was decreased in response to treatment with resveratrol in mice (P < 0.05). We conclude that resveratrol inhibits cancer metastasis both in vitro and in vivo, and this inhibition is likely due to the decrease in MMP-9 activity caused by resveratrol.     

转染人内皮抑素对卵巢癌细胞A2780体内外生长的影响

目的:探讨人内皮抑素(hum an endostatin,hES)对人卵巢癌细胞株A2780体内、外生长的影响及其可能机制。方法:高保真PCR从含有人内皮抑素基因的人肝脏组织中扩增出人内皮抑素编码区克隆入pcDNA3.1载体中,构建pcDNA3.1-ES重组质粒;将此重组质粒转染人卵巢癌细胞A2780中,观察A2780细胞生长。建立人卵巢癌裸鼠模型,随机将其分为两组:hES转染细胞组(A组,10只),对照组(B组,10只)。1月后,测量瘤体积、计算抑瘤率;标本行HE染色分析组织学形态;同时用W estern b lot检测hES、凋亡抑制蛋白bc l-2的表达;流式细胞仪检测转染对裸鼠移植瘤细胞凋亡率的影响。结果:hES转染A2780细胞后,细胞生长速率减慢。转染hES的裸鼠肿瘤生长较对照组明显减慢(P<0.001),抑瘤率为74%;HE染色及W estern b lot提示:转染后(A组)的组织学形态与转染前(B组)有显著差异;A组hES蛋白水平明显高于B组,而bc l-2蛋白水平明显低于B组;流式结果显示转染使肿瘤细胞的凋亡显著增加(P<0.05)。结论:转染hES体内、外均可抑制卵巢癌A2780细胞的生长,其机制与其促进了凋亡有关。     

抑癌候选基因NDRG2在膀胱肿瘤中的表达研究

目的 探讨N-myc下游调节基因(NDRG2)在膀胱正常组织和膀胱肿瘤组织中的表达情况,以及NDRG2的表达与膀胱肿瘤病理分级和临床分期的关系.方法 应用免疫组化SP法检测52例膀胱癌、10例膀胱乳头状肿瘤及10例膀胱正常组织石蜡标本中NDRG2表达情况,并进行相关临床病理分析.结果 NDRG2在正常膀胱组织阳性表达率为80.0％ (8/10),在膀胱肿瘤组织中阳性表达率为40.3％ (25/62),两者比较差异有统计学意义(x2=3.98,P＜0.05);NDRG2的阳性表达与膀胱肿瘤病理分级呈负相关(r=-0.288,P＜0.05),与C-myc表达呈负相关(r=-0.436,P＜0.01),与p53表达呈正相关(r=0.717,P＜0.01).结论 NDRG2基因在膀胱肿瘤组织中的表达水平随恶性程度的增高而降低,提示该基因可能对膀胱肿瘤病理分级和临床分期有一定的指导意义.     

莱菔硫烷对膀胱癌细胞增殖抑制作用

目的 研究丝裂原活化蛋白激酶(MAPKs)和磷脂酰肌醇激酶(PI3K)信号途径在莱菔硫烷(sulforaphane,SFN)抑制膀胱癌细胞增殖中的作用。方法噻唑蓝(MTT)法测定膀胱癌细胞的增殖作用,用实时定量PCR方法(re-al time-PCR)测定对环氧化酶(COX-2)mRNA的影响。结果 5～20μmol/L莱菔硫烷能明显抑制膀胱癌细胞的体外增殖、COX-2 mRNA表达;SB202190或LY294002预处理T24细胞,能明显降低莱菔硫烷对T24细胞增殖的抑制作用,提高细胞存活率;SB202190预处理T24细胞1h能完全阻断莱菔硫烷对COX-2 mRNA的抑制作用,而LY294002不影响莱菔硫烷对COX-2 mRNA的抑制作用。结论 莱菔硫烷可能是通过活化p38激酶途径和PI3K激酶途径抑制膀胱癌细胞生长,p38激酶信号途径在SFN抑制COX-2 mRNA中起关键作用。     

膀胱肿瘤细胞mRNA致敏的树突状细胞体外抗肿瘤免疫反应

目的:研究小鼠膀胱癌BTT739细胞mRNA致敏的树突状细胞(DC)诱导特异性的抗肿瘤免疫反应。方法:使用rmGM-CSF和rmIL-4培养诱导小鼠骨髓细胞分化为DC,从BTT739细胞中提取mRNA致敏DC,与小鼠淋巴细胞混合培养以诱导特异性的细胞毒性T淋巴细胞(CTL)。采用MTT比色法和软琼脂法检测CTL对BTT739细胞的杀伤作用。结果:MTT比色法和软琼脂法检测结果显示:在靶细胞∶效应细胞不同浓度组中,BTT739实验组和对照组的OD值和肿瘤集落形成个数都有显著性差异(P<0 01,P<0 05),在靶细胞∶效应细胞的比例为1∶5、1∶25、1∶50和1∶100时,其杀伤率分别为25. 0%、36 .1%、45 .9% 和58 .2%;当效应细胞作用于AGS细胞时,其OD值无显著差异。结论:以膀胱肿瘤细胞mRNA致敏DC诱导CTL可有效杀伤膀胱肿瘤细胞。     

Dietary risk factors in human bladder cancer

Retrospective data on dietary habits, employment history and tobacco use were obtained from 569 bladder cancer patients and 1025 age-matched controls admitted to Roswell Park Memorial Institute. Sex-adjusted relative risks revealed increases in risk for lower levels of an index of vitamin A intake. A similar pattern of risk elevation was associated with infrequent milk and carrot intake. Some elevation of risk was found for heavy coffee drinking but the apparent protective effect for milk consumption was not found to be a spurious result of lower coffee intake. Neither was the role of vitamin A explained by its relationship with smoking or employment in high risk occupations. Some association of bladder cancer with infrequent consumption of cruciferous vegetables was also observed. The findings of this investigation are consistent with tumor inhibition by retinoids in animal studies and the low risk associated with vitamin A in epidemiologic studies of lung cancer.     

Potential of resveratrol in anticancer and anti-inflammatory therapy

Phytochemicals present in food have shown significant prospects in the treatment and management of a vast array of human diseases. Resveratrol is a stilbene-type aromatic phytoalexin predominantly found in grapes, peanuts, berries, turmeric, and other food products. Resveratrol has been reported to exhibit several physiological activities including anticancer and anti-inflammatory activities in vitro and in experimental animal models, as well as in humans. Anticancer activity of this compound is mainly due to induction of apoptosis via several pathways, as well as alteration of gene expressions, all leading to a decrease in tumor initiation, promotion, and progression. Resveratrol exhibits anti-inflammatory activity through modulation of enzymes and pathways that produce mediators of inflammation and also induction of programmed cell death in activated immune cells. Resveratrol has been shown to produce no adverse effects, even when consumed at high concentrations. Hence, resveratrol possesses good potential to be used as an adjunctive or alternative therapy for cancer and inflammatory diseases.     

In vitro and in vivo antitrypanosomatid activity of 5-nitroindazoles

Previously, we have identified a series of 5-nitroindazoles with good antiprotozoal activities, against Trypanosoma cruzi epimastigotes and Trichomonas vaginalis. Most of them have shown very low unspecific toxicity on macrophage cell lines. In the present work, we assayed these compounds on T. cruzi bloodstream trypomastigotes and Leishmania promastigotes (Leishmania amazonensis, Leishmania braziliensis and Leishmania infantum). Derivatives 1, 2, 7 and 8 displayed remarkable trypanocidal activity (>80% lysis) equivalent to gentian violet. Derivatives 2 and 10, as Pentamidine, caused the complete lysis of promastigotes of Leishmania. An oxidative stress-mediated mechanism of action was confirmed for derivatives 1, 10 and 12 on T. cruzi epimastigotes. Supported by the in vitro activities, derivatives 1 and 2 were submitted to in vivo assays using an acute model of Chagas' disease and a short-term treatment. None of the animals treated with derivatives 1 and 2 died, unlike the untreated control and Benznidazole groups.     

Mechanism of selective anticancer activity of isothiocyanates relies on differences in DNA damage repair between cancer and healthy cells

Isothiocyanates (ITCs) are compounds derived from Brassica plants with documented anticancer activity. Molecular mechanisms of their selective activity aga     

结核杆菌抗原对γδT细胞体外增殖影响

目的 观察结核杆菌抗原(Mtb-Ag)对人γδT细胞体外增殖的影响,以解决医学研究中γδT细胞来源不足问题.方法 应用酸性改良罗氏培养基分离培养结核杆菌及丙酮酸钠液体培养基进行扩增,通过85℃水浴灭活,超声波细胞破碎制备Mtb-Ag.在体外以不同的剂量刺激人外周血单个核细胞(PBMC),采用四甲基偶氮噻唑蓝(MTT)法检测增殖程度,应用流式细胞仪检测γδT细胞增殖的百分率.结果 在一定范围内随着刺激剂量的增加PBMC增殖亦增加.流式细胞仪分析增殖结果显示,7 ug/ml刺激组γδT细胞的增殖率最高(约为49.08%).但当刺激剂量增加至9 ug/ml时,γδT细胞增殖比率开始降低(约为32.62%).而αβT细胞增殖比率增加明显(约为36.76%).结论 Mtb-Ag在合适刺激剂量时,可对人γδT细胞发挥优势扩增;当剂量过大时则对αβT细胞发挥优势扩增.     

芒柄花黄素诱导膀胱癌细胞凋亡作用

目的探讨芒柄花黄素诱导膀胱癌细胞凋亡的作用及机制。方法采用不同浓度(20、40、80 μmol/L)芒柄花黄素处理膀胱癌细胞, 采用噻唑蓝法观察芒柄花黄素对膀胱癌T-24和BIU-87细胞的增殖的抑制作用, 使用流式细胞术及Hoechst 33258染色检测T-24和BIU-87细胞凋亡率, 采用RT-PCR检测T-24细胞Bcl-2 mRNA表达;western blot检测T-24细胞p-p38蛋白激酶、Bcl-2蛋白含量变化。结果与对照组比较, 40、80 μmol/L芒柄花黄素组T-24和BIU-87细胞增殖率下降、细胞凋亡率明显升高, Hoechst33258染色显示T-24细胞呈凋亡状态;40、80 μmol/L 芒柄花黄素组T-24细胞Bcl-2 mRNA表达水平[(0.701±0.029)、(0.408±0.036)]均低于对照组, 差异均有统计学意义(P<0.01);与对照组比较, 20、40、80 μmol/L芒柄花黄素组T-24细胞p-p38蛋白激酶活性水平[(0.483±0.026)、(0.569±0.031)、(0.935±0.037)]均升高, Bcl-2蛋白含量[(0.998±0.034)、(0.591±0.030)、(0.426±0.027)]均降低,差异均有统计学意义(P<0.01)。结论芒柄花黄素可诱导膀胱癌T-24和BIU-87细胞凋亡、抑制细胞生长, 其机制可能与下调Bcl-2蛋白表达并活化p38蛋白激酶有关。     

Antioxidant effects of polyphenols in chocolate on low-density lipoprotein both in vitro and ex vivo

Cacao is rich in polyphenols such as (-)-epicatechin, and a colored component of cacao (cacao-red) is polyphenol, which is an antioxidant. These properties stimulated an investigation of the effects of cacao liquor polyphenols (CLP) on low-density lipoprotein (LDL) oxidation. The 2.2 '-azobis(4-methoxy-2,4-dimethylvaleronitrile) (AMVN-CH2O)-induced oxidizability of LDL was assessed by monitoring the absorbance at 234 nm. In vitro. 0.1-0.5 mg/dL CLP prolonged the oxidation lag time of LDL in a dose-dependent manner. Compared with the controls, it was prolonged 1.7-fold in the presence of 0.1 mg/dL CLP, 2.9-fold at 0.2 mg/dL, 3.8-fold at 0.3 mg/dL, 5.4-fold at 0.4 mg/dL, and 6.4-fold at 0.5 mg/dL. Furthermore, we enlisted 13 male volunteers to consume 35 g delipidated cocoa. Venous blood samples were taken before and at 2 h and 4 h after consuming the cocoa. The oxidation lag time of LDL before cocoa ingestion was 59.0 +/- 6.3 min, but it was prolonged at 2 h after cocoa (68.3 +/- 6.0 min); before returning to the initial lag time (61.7 +/- 5.7 min) before consumption. Thus we have shown that cocoa inhibited LDL oxidation both in vitro and ex vivo.