苦参素抑制人膀胱癌T24细胞生长作用研究

目的探讨苦参素对人膀胱癌T24细胞的抑制作用及其可能机制。方法MTT法检测T24细胞的生长抑制率;流式细胞仪检测细胞周期改变和免疫组化,SP法检测Bcl 2和Bax蛋白表达的变化。结果2 mg•mL 1苦参素作用24和72 h后细胞生长抑制率为14.10%和49.67%; 8 mg•mL 1苦参素作用24和72 h后细胞生长抑制率为35.40%和80.50%。随着苦参素浓度的增加,G0/G1 期T24细胞所占比例逐渐上升,G2/M期细胞所占比例逐渐下降;且细胞内Bcl 2表达下调,Bax表达上调。结论苦参素通过诱导癌细胞周期阻滞于G0/G1期来抑制人膀胱癌T24细胞的生长增殖。其机制可能与下调Bcl 2和上调Bax蛋白表达,诱导癌细胞凋亡有关。     

Paclitaxel (Taxol) Inhibits the Arylamine N‐Acetyltransferase Activity and Gene Expression (mRNA NAT1) and 2‐Aminofluorene‐DNA Adduct Formation in Human Bladder Carcinoma Cells (T24 and TSGH 8301)

Abstract: Acetylator polymorphism in man results from differential expression of human liver N‐acetyltransferase. N‐Acetyltransferase enzyme activity has been demonstrated to be involved in some types of chemical carcinogenesis. Paclitaxel (taxol) had been shown to affect N‐acetyltransferase activity of human lung cancer cells. In this study, paclitaxel was chosen to investigate the effects of arylamine N‐acetyltransferase activity (N‐acetylation of substrate), gene expresssion and 2‐aminofluorene‐DNA adduct formation in human bladder carcinoma cell lines (T24 and TSGH 8301). The N‐acetyltrasnsferase activity (N‐acetylation of substrates) was determined by high performance liquid chromatography assaying for the amounts of acetylated 2‐aminofluorene and p‐aminobenzoic acid and nonacetylated 2‐aminofluorene and p‐aminobenzoic acid. Intact human bladder carcinoma T24 and TSGH 8301 cells were used for examining N‐acetyltransferase activity, gene expression and 2‐aminofluorene‐DNA adduct formation. The results demonstrated that the N‐acetyltransferase activity, gene expression (NAT1 mRNA) and 2‐aminofluorene‐DNA adduct formation in intact human bladder carcinoma cells were inhibited and decreased by paclitaxel in a dose‐dependent manner. The effects of paclitaxel on the apparent values of Km and Vmax of N‐acetyltransferase enzyme from intact human bladder carcinoma cells were also determined in these cell lines. A marked influence of paclitaxel was observed on the decreasing apparent values of K_m and V_max from intact human bladder carcinoma cells (T24 and TSGH 8301). Thus, paclitaxel is an uncompetitive inhibitor to the NAT enzyme.     

聚维酮对人膀胱癌T24细胞的抑制作用研究

目的 探讨聚维酮(PVP)对人膀胱癌T24细胞的抑制作用。 方法 采用MTT法检测PVP对T24细胞生长的抑制作用，流式细胞仪检测PVP对T24细胞周期的影响和黏附作用。结果 2.5%PVP作用24和72 h，对人膀胱癌T24细胞生长抑制率分别为（15.11±2.36）%和（49.57±7.07）%；浓度为7.5%时24和72 h抑制率分别（35.42±5.55）%和（79.66±19.92）%。5.0%PVP作用24和72 h时，细胞G0/G1期所占比例分别为（74.17±0.91）%和（46.69±3.76）%；G2/M期细胞所占比例分别为（14.63±0.47）%和（41.88±1.50）%。PVP能提高抗鼠IgG1对T24细胞的黏附作用。结论 PVP能通过诱导人膀胱癌T24细胞周期阻滞于G2/M期和作为化疗药的黏附性载体而抑制癌细胞的生长增殖。     

Sensitization of Hepatocellular Carcinoma Cells to Apo2L/TRAIL by a Novel Akt/NF‐κB Signalling Inhibitor

Hepatocellular carcinoma (HCC) cells are intrinsically resistant to tumour necrosis factor‐related apoptosis ligand (Apo2L/TRAIL), in part, due to the compensatory activation of nuclear factor‐kappaB (NF‐κB). To broaden the clinical utilization of Apo2L/TRAIL in HCC, OSU‐A9, a potent indole‐3‐carbinol‐derived Akt/NF‐κB signalling inhibitor was used to overcome the intrinsic resistance. The antitumour effects of OSU‐A9, Apo2L/TRAIL and the therapeutic combination were assessed by MTT assay, caspase activation and PARP cleavage, and the synergistic interactions were determined by Calcusyn analysis. NF‐κB reporter gene and RT‐PCR were tested for the activation of NF‐κB and the expression of death receptors (DR)4 and 5. OSU‐A9 could sensitize HCC cells to Apo2L/TRAIL with high potency through down‐regulation of Akt/NF‐κB signalling. OSU‐A9 dose‐dependently reduced Akt phosphorylation and the expression and nuclear localization of RelA/p65, accompanied by parallel decreases in the expression of NF‐κB target products, including Bcl‐xL, Mcl‐1, cIAP1, cIAP2 and survivin. Moreover, OSU‐A9 increased DR5 expression through a reactive oxygen species (ROS)‐dependent mechanism. Concertedly, these mechanisms underlie the synergistic interaction between OSU‐A9 and Apo2L/TRAIL in mediating apoptotic death in HCC cells. The ability of OSU‐A9 to accentuate Apo2L/TRAIL‐induced apoptosis by inactivating Akt/NF‐κB signalling might foster a promising therapeutic strategy for HCC.     

Inhibition of NF‐κB and metastasis in irinotecan (CPT‐11)‐resistant LoVo colon cancer cells by thymoquinone via JNK and p38

Clinically used chemotherapeutics can effectively eliminate most tumor cells. However, they cause unwanted side effects and result in chemoresistance. To overcome such problems, phytochemicals are now used to treat cancers by means of targeted therapy. Thymoquinone (TQ) is used to treat different cancers (including colon cancer) and is an NF‐κB inhibitor. Irinotecan resistant (CPT‐11‐R) LoVo colon cancer cell line was previous constructed by step‐wise CPT‐11 challenges to un‐treated parental LoVo cells and expresses EGFR/IKKα/β/NF‐κB pathway. TQ resulted in reduced total and phosphorylation of IKKα/β and NF‐κB and decreased metastasis in CPT‐11‐R cells. TQ not only reduced activity of ERK1/2 and PI3K but also activated JNK and p38. Furthermore, TQ was also found to suppress metastasis through activation of JNK and p38. Therefore, TQ suppressed metastasis through NF‐κB inhibition and activation of JNK and p38 in CPT‐11‐R LoVo colon cancer cells. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 669–678, 2017.     

The Inhibitory Effect of Intravesical Fisetin against Bladder Cancer by Induction of p53 and Down‐Regulation of NF‐kappa B Pathways in a Rat Bladder Carcinogenesis Model

Intravesical chemotherapy after transurethral resection has been widely used as an adjuvant therapy to prevent recurrence and progression of superficial bladder cancer. Due to the insufficiency of the current chemotherapeutics, there is an urgent need to search for more novel, effective and safe intravesical agents. Previously, we have proved the in vitro apoptotic effects of fisetin, a dietary flavonoid, on bladder carcinoma cells. In the present study, we have further explored its intravesical efficacy and investigated the underlying mechanisms of its inhibitory effect of bladder cancer through an autochthonous rat model of bladder cancer induced by intravesical N‐methyl‐N‐nitrosourea (MNU). We found that fisetin‐induced apoptosis in bladder cancer is mediated via modulation of two related pathways: up‐regulation of p53 and down‐regulation of NF‐κB pathway activity, causing changes in the ratio of pro‐ and antiapoptotic proteins. Meanwhile, administration of fisetin significantly reduced the incidence of MNU‐induced bladder tumours by suppressing NF‐κB activation and modulating the expression of NF‐κB target genes that regulate cell proliferation and cell apoptosis. Our study suggests that the activation of p53 and inhibition of the NF‐κB pathway may play important roles in the fisetin‐induced apoptosis in bladder cancer. Furthermore, intravesical fisetin effectively inhibited, without any toxicity, the carcinogenesis of bladder cancer in MNU‐initiated rats. These findings identify the in vivo chemopreventive efficacy of fisetin and suggest that fisetin could be used as a novel, effective and safe intravesical agent for bladder cancer.     

Synthesis of Novel Heterocyclic Ring‐Fused 18β‐Glycyrrhetinic Acid Derivatives with Antitumor and Antimetastatic Activity

Glycyrrhetinic acid (GA) is one of the most important triterpenoic acids shows many pharmacological effects, especially antitumor activity. GA triggers apoptosis in various tumor cell lines. However, the antitumor activity of GA is weak, thus the synthesis of new synthetic analogs with enhanced potency is needed. By introducing various five‐member fused heterocyclic rings at C‐2 and C‐3 positions, 18 novel GA derivatives were obtained. These compounds were evaluated for their inhibitory activity against the growth of eight different tumor cell lines using a SRB assay. The most active compound 37 showed IC₅₀ between 5.19 and 11.72 μm , which was about 11‐fold more potent than the lead compound GA. An apoptotic effect of GA and 37 was determined using flow cytometry and trypan blue exclusion assays. We also demonstrated here for the first time that GA and the synthetic derivatives exhibited inhibitory effect on migration of the tested tumor cells, especially 37 which was about 20‐fold more potent than GA on antimetastatic activity.     

NF‐κB as the mediator of metformin's effect on ageing and ageing‐related diseases

Ageing can be defined as the progressive failure of repair and maintenance systems with a consequent accumulation of cellular damage in nucleic acids, proteins, and lipids. These various types of damage promote ageing by driving cellular senescence and apoptosis. The nuclear factor‐kappa B (NF‐kB) pathway is one of the key mediators of ageing and this pathway is activated by genotoxic, oxidative and inflammatory stress, and regulates expression of cytokines, growth factors, and genes that regulate apoptosis, cell‐cycle progression, and inflammation. Therefore, NF‐kB is increased in a variety of tissues with ageing, thus the inhibition of NF‐kB leads to delayed onset of ageing‐related symptoms and pathologies such as diabetes, atherosclerosis, and cancer. Metformin is often used as an anti‐diabetic medication in type 2 diabetes throughout the world and appears to be a potential anti‐ageing agent. Owing to its antioxidant, anticancer, cardio‐protective and anti‐inflammatory properties, metformin has become a potential candidate drug, improving in the context of ageing and ageing‐related diseases. An inappropriate NF‐kB activation is associated with diseases and pathologic conditions which can impair the activity of genes involved in cell senescence, apoptosis, immunity, and inflammation. Metformin, inhibiting the expression of NF‐kB gene, eliminates the susceptibility to common diseases. This review underlines the pleiotropic effects of metformin in ageing and different ageing‐related diseases and attributes its effects to the modulation of NF‐kB.     

Doses and Time‐Dependent Effects of 3′‐Azido‐3′‐deoxythymidine on T47D Human Breast Cancer Cells in vitro

The objective of the present work was to study the effects of 3′‐azido‐3′‐deoxythymidine (azidothymidine, Zidovudine^®) on human breast cancer cells by using, as a model, the T47D cell line (typified as oestrogen‐dependent and p53‐mutated). Low azidothymidine doses (3.125 μM) increase the percentage of cells in S‐phase, with the effect reversing after 24 hr of incubation; as azidothymidine doses increase, the magnitude and duration of its effect increase proportionally, although, even with the highest concentrations (50–100 μM) the effects decline after 48 hr of incubation. If media (containing azidothymidine or vehicle) are daily renewed, the azidothymidine effects (accumulation of cells in S‐phase) are higher and decline later than when media and drug are not changed during the whole culture period, thereby suggesting that the reversion of azidothymidine effects could be related with a degradation of the drug or accumulation in media of substances which counteract its effects. Azidothymidine inhibits T47D cell proliferation at concentrations higher than 50 μM. The exposure to 50 or 100 μM azidothymidine induced cell apoptosis after 48 hr or more of incubation. We conclude that: a) azidothymidine, with appropriate doses and duration of treatment, synchronizes cells in S‐phase, inhibits proliferation, and induces apoptosis, b) the discontinuous application of the drug rather than continuous exposure to it increases its efficiency to synchronize the T47D cell cycle. This in vitro anti‐breast cancer activity suggests that a possible clinical usefulness of azidothymidine, either alone or associated with other drugs with cycle‐specific antitumoural activity circumscribed to the S‐phase of cell cycle, is worthy of investigation.     

Design and optimize N‐substituted EF24 as effective and low toxicity NF‐κB inhibitor for lung cancer therapy via apoptosis‐to‐pyroptosis switch

As NF‐κB signaling pathway is constitutively activated in lung cancer, targeting NF‐κB has a potential for the treatment. EF24 has been proved to be a NF‐κB inhibitor with good antitumor activity, while whose toxicity possibly became one of the obstacles to enter into clinical application. In order to find high efficiency and low toxicity NF‐κB inhibitors, EF24 was modified and 13d was screened out. It was proved that 13d possessed an effective combination of inhibiting NF‐κB pathway and showing lower cytotoxicity on normal cells as well as less toxicity in acute toxicity experiment compared with the lead compound of EF24. In addition, 13d was found to inhibit cell vitality, arrest cell cycle in G2/M phase, promote cell apoptosis, and suppress the xenograft tumor growth. Furthermore, 13d was elucidated to induce pyroptosis developing from apoptosis, which was associated with the inhibition of NF‐κB. Taken together, it was suggested that 13d was a potent antitumor agent.     

Investigation of Ornithine Decarboxylase Activity and Two-Dimensional Electrophoretic Protein Profile Following Exposure of T24 Bladder Carcinoma Cells to Tumor Promoter and Carcinogen

Summary: To develop appropriate screening tools for biomarkers of effects of exposure to occupational chemical insult. Changes were investigated in T24 human bladder carcinoma cell ornithine decarboxylase activity and protein profiles by quantitative two-dimensional polyacrylamide gel electrophoresis (2D PAGE), biochemical events potentially altered by an established human bladder carcino gen and tumor promoter. A unique chromatographic approach was used to demonstrate that in vitro exposure of T24 cells for 6 h to varying concentrations of the carcinogen 4-aminobiphenyl elevates enzyme activity 5.3- to 5.9-fold. As a second method to identify potential biomarkers of exposure, two-dimensional gel electrophoresis was used to compare the protein pattern of vehicle control-treated T24 to 4-aminobiphenyl or tumor promoter (12-o-tetradecanoylphorbol-13-acetate)treated cells. Changes in abundance and modification of proteins are determined using the Kepler software package to analyze and compare gels across treatment groups. With this technology, protein markers are identified by significant alterations in spot density (mean ratio of Coomassie Blue intensity; p. 001, Student's t test) following T24 treatment with the carcinogen or the tumor promoter. Fifteen protein spots from a detectable pool of 542 demonstrate two-fold or greater changes in intensity. The results illustrate the potential of automated two-dimensional gel analysis for classifying different gel patterns, an approach that can be applied to patterns whose differences are obscured by the minor changes in spot intensity that arise between separate cell cultures. In addition to the ornithine decarboxylase assay, 2D PAGE offers much promise to evaluate potential biomarkers for occupational and environmental carcinogens. These results will be used to further develop NIOSH efforts in the molecular epidemiology of occupational bladder carcinogenesis. [K]Key [K]Words: biomarker, bladder carcinogenesis, occupational exposure, ODC, 2D PAGE.     

Synthesis and SAR Studies of Dual AKT/NF‐κB Inhibitors Against Melanoma

The protein Kinase B alpha (AKT) and nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) pathways are central regulators of cellular signaling events at the basis of tumor development and progression. Both pathways are often up‐regulated in different tumor types including melanoma. We recently reported the identification of compound 1 (BI‐69A11) as inhibitor of the AKT and the NF‐κB pathways. Here, we describe SAR studies that led to novel fluorinated derivatives with increased cellular potency, reflected in efficient inhibition of AKT and IKKs. Selected compounds demonstrated effective toxicity on melanoma, breast, and prostate cell lines. Finally, a representative derivative showed promising efficacy in an in vivo melanoma xenograft model.     

飞燕草素对人膀胱癌T24细胞增殖、凋亡的影响及机制研究

目的 探讨飞燕草素对人膀胱癌细胞株T24的增殖抑制作用、诱导凋亡作用及其相关的分子机制。方法 体外培养T24细胞,利用CCK-8试验检测飞燕草素对人膀胱癌T24细胞的杀伤作用;利用流式细胞术试验检测细胞凋亡情况、活性氧水平变化;利用Western blotting检测细胞凋亡相关蛋白和信号通路相关蛋白的表达。结果 飞燕草素能够有效抑制人膀胱癌T24细胞增殖,IC₅₀为（42.37±2.82）μmol·L^-1;飞燕草素能够通过线粒体依赖性途径诱导T24细胞发生凋亡,使Cyt-c、cleaved-Caspase-3、PARP和Bax蛋白表达量升高,Bcl-2表达量降低;飞燕草素能够降低T24细胞中的活性氧水平,并激活JNK/STAT3信号通路。结论 飞燕草素对人膀胱癌T24细胞具有良好的杀伤作用,并能够通过降低T24细胞内活性氧水平,进而调控JNK/STAT3信号通路来诱导T24细胞发生线粒体依赖性凋亡。     

Different Effects of Scopolamine and Inhibition of Prolyl Oligopeptidase on Mnemonic and Motility Functions of Young and 8‐ to 9‐Month‐Old Rats in the Radial‐Arm Maze

Abstract: Prolyl oligopeptidase (POP) has been connected to memory and mood through regulation of the brain levels of its biologically active peptide substrates and phosphatidylinositol system. This is the first study in a radial‐arm maze of the effects of a single dose of a novel potent prolyl oligopeptidase inhibitor, KYP‐2047 (5 mg/kg, dissolved in 5% Tween 80), on memory and learning of scopolamine‐treated (0.4 mg/kg, dissolved in saline) rats. Habituated (days 1 and 2) and trained (days 3–11) young (3 months) and old (8–9 months) male Wistar rats were given (i) saline + Tween, (ii) saline + KYP‐2047, (iii) scopolamine + Tween or (iv) scopolamine + KYP‐2047 30 min. prior to testing their memory. Food rewards located in four randomly chosen arms of the maze. The rat had 10 min. to find and eat the rewards. Time spent in the maze, visits to each arm and number of eaten rewards were measured. Old rats made generally more errors, spent more time and visited fewer arms per minute in the maze than young rats. The memory‐ and function‐impairing effects of scopolamine were also seen more clearly in old than young rats. KYP‐2047 had no or only a marginal effect on memory of either age group, but when given without scopolamine, it slightly increased the maze motility of young rats and decreased the motility of old rats. In a separate locomotor activity test, KYP‐2047 enhanced the motility of young rats supporting a suggested role of POP in motor functions.