Gliotoxin Inhibits Proliferation and Induces Apoptosis in Colorectal Cancer Cells

The discovery of new bioactive compounds from marine natural sources is very important in pharmacological research. Here we developed a Wnt responsive luciferase reporter assay to screen small molecule inhibitors of cancer associated constitutive Wnt signaling pathway. We identified that gliotoxin (GTX) and some of its analogues, the secondary metabolites from marine fungus Neosartorya pseufofischeri, acted as inhibitors of the Wnt signaling pathway. In addition, we found that GTX downregulated the β-catenin levels in colorectal cancer cells with inactivating mutations of adenomatous polyposis coli (APC) or activating mutations of β-catenin. Furthermore, we demonstrated that GTX induced growth inhibition and apoptosis in multiple colorectal cancer cell lines with mutations of the Wnt signaling pathway. Together, we illustrated a practical approach to identify small-molecule inhibitors of the Wnt signaling pathway and our study indicated that GTX has therapeutic potential for the prevention or treatment of Wnt dependent cancers and other Wnt related diseases.     

Lipid-Soluble Ginseng Extract Induces Apoptosis and G0/G1 Cell Cycle Arrest in NCI-H460 Human Lung Cancer Cells

This study was performed to elucidate the anticancer mechanism of a lipid-soluble ginseng extract (LSGE) by analyzing induction of apoptosis and arrest of cell cycle progression using the NCI-H460 human lung cancer cell line. Proliferation of NCI-H460 cells was potently inhibited by LSGE in a dose-dependent manner. The cell cycle arrest at the G0/G1 phase in NCI-H460 cells was induced by LSGE. The percentage of G0/G1 phase cells significantly increased, while that of S phase cells decreased after treatment with LSGE. The expression levels of cyclin-dependent kinase2 (CDK2), CDK4, CDK6, cyclin D3 and cyclin E related to G0/G1 cells progression were also altered by LSGE. In addition, LSGE-induced cell death occurred through apoptosis, which was accompanied by increasing the activity of caspases including caspase-8, caspase-9 and caspase-3. Consistent with enhancement of caspase activity, LSGE increased protein levels of cleaved caspase-3, caspase-8, caspase-9, and poly-ADP-ribose polymerase (PARP). These apoptotic effects of LSGE were inhibited by the pan-caspase inhibitor Z-VAD-fmk. These findings indicate that LSGE inhibits NCI-H460 human lung cancer cell growth by cell cycle arrest at the G0/G1 phase and induction of caspase-mediated apoptosis.     

6-Bromoisatin Found in Muricid Mollusc Extracts Inhibits Colon Cancer Cell Proliferation and Induces Apoptosis,Preventing Early Stage Tumor Formation in a Colorectal Cancer Rodent Model

Muricid molluscs are a natural source of brominated isatin with anticancer activity. The aim of this study was to examine the safety and efficacy of synthetic 6-bromoisatin for reducing the risk of early stage colorectal tumor formation. The purity of 6-bromoisatin was confirmed by ¹H NMR spectroscopy, then tested for in vitro and in vivo anticancer activity. A mouse model for colorectal cancer was utilized whereby colonic apoptosis and cell proliferation was measured 6 h after azoxymethane treatment by hematoxylin and immunohistochemical staining. Liver enzymes and other biochemistry parameters were measured in plasma and haematological assessment of the blood was conducted to assess potential toxic side-effects. 6-Bromoisatin inhibited proliferation of HT29 cells at IC₅₀ 223 μM (0.05 mg/mL) and induced apoptosis without increasing caspase 3/7 activity. In vivo 6-bromoisatin (0.05 mg/g) was found to significantly enhance the apoptotic index (p ≤ 0.001) and reduced cell proliferation (p ≤ 0.01) in the distal colon. There were no significant effects on mouse body weight, liver enzymes, biochemical factors or blood cells. However, 6-bromoisatin caused a decrease in the plasma level of potassium, suggesting a diuretic effect. In conclusion this study supports 6-bromoisatin in Muricidae extracts as a promising lead for prevention of colorectal cancer.     

火炬树叶浸提液对小麦根尖细胞的遗传毒性

为了解入侵植物火炬树对小麦根尖细胞的遗传毒性,用不同浓度的火炬树叶浸提液(0.5‰、1‰、2‰、5‰、1%、3%、5%、7%、10%)处理萌发期的小麦根尖6、12、24和36h,观察其有丝分裂的变化和染色体行为的异常。结果表明,除低浓度(<2‰)浸提液外,火炬树叶浸提液对小麦根尖细胞产生了明显的毒害作用。随着处理浓度的升高和处理时间的延长,有丝分裂指数逐渐降低,同时诱发了微核、多核和染色体畸变的出现,畸变率随处理浓度的提高和处理时间的延长而逐渐升高。     

Saquayamycin B1 Suppresses Proliferation,Invasion, and Migration by Inhibiting PI3K/AKT Signaling Pathway in Human Colorectal Cancer Cells

Moromycin B (Mor B), saquayamycin B₁ (Saq B₁), saquayamycin B (Saq B), and landomycin N (Lan N), four angucyclines produced by the marine-derived actinomycete Streptomyces sp., are a class of polyketone compounds containing benzanthracene. Here, the structure–activity relationship of these four compounds was analyzed in human colorectal cancer (CRC) cells. Saq B₁, which showed the strongest cytotoxicity with an IC₅₀ of 0.18–0.84 µM for CRC cells in MTT assays, was employed to test underlying mechanisms of action in SW480 and SW620 cells (two invasive CRC cell lines). Our results showed that Saq B₁ inhibited CRC cell proliferation in a dose- and time-dependent manner. Notably, lower cytotoxicity was measured in normal human hepatocyte cells (QSG-7701). Furthermore, we observed proapoptosis, antimigration, and anti-invasion activities of Saq B₁ in CRC cells. At the same time, the protein and mRNA expression of important markers related to the epithelial–mesenchymal transition (EMT) and apoptosis changed, including N-cadherin, E-cadherin, and Bcl-2, in Saq B₁-treated CRC cells. Surprisingly, the PI3K/AKT signaling pathway was shown to be involved in Saq B₁-induced apoptosis, and in inhibiting invasion and migration. Computer docking models also suggested that Saq B₁ might bind to PI3Kα. Collectively, these results indicate that Saq B₁ effectively inhibited growth and decreased the motor ability of CRC cells by regulating the PI3K/AKT signaling pathway, which provides more possibilities for the development of drugs in the treatment of CRC.     

Palmitic Acid and Ergosta-7,22-dien-3-ol Contribute to the Apoptotic Effect and Cell Cycle Arrest of an Extract from Marthasterias glacialis L. in Neuroblastoma Cells

We describe the effect of a chemically characterized lipophilic extract obtained from Marthasterias glacialis L. against human breast cancer (MCF-7) and human neuroblastoma (SH-SY5Y) cell lines. Evaluation of DNA synthesis revealed that both cell lines were markedly affected in a concentration-dependent way, the SH-SY5Y cell line being more susceptible. Cell cycle arrest was observed, an effect induced by the sterol, ergosta-7,22-dien-3-ol, present in the extract. Morphological evaluation of treated cells showed the advent of lipid droplets and chromatin condensation compatible with apoptosis, which was confirmed by the evaluation of caspase-3 and -9 activities. Palmitic acid was the main compound responsible for this apoptotic effect by a ceramide-independent mechanism that involved endoplasmic reticulum (ER)-stress with upregulation of CCAAT/-enhancer-binding protein homologous protein (CHOP).     

生长温度对烤烟生物碱含量及烟碱代谢相关酶基因表达的影响

以云烟87为材料,采用人工气候室模拟云南江川、贵州遵义和河南襄县的气温条件,分别在烟草移栽-团棵、团棵-现蕾及成熟期进行不同温度处理,研究不同生长温度对烟碱、新烟碱、降烟碱和假木贼碱含量以及烟碱代谢关键酶基因表达的影响。结果表明:在3个不同生育期中,较低的生长温度均促进了烟碱的生物合成及其在根系中的积累。在移栽-团棵期,低温处理下根系中的烟碱含量升高至0 d时的1.54倍,而同时期叶片中的烟碱含量显著低于根系;进入团棵-现蕾期以及成熟期后,较高的生长温度则更有利于叶片中烟碱的积累,其烟碱含量较0 d时高约1.80~4.38倍;新烟碱、降烟碱和假木贼碱的含量变化趋势与烟碱基本相似。总体而言,在不同生育期,烟碱的合成和器官水平上的分布不同程度地受到生长温度的影响,成熟期温度对烟碱量的影响最大,具有相对较高生长温度条件的河南襄县更有利于叶片中烟碱的积累。本研究结果揭示了温度调节烟草生物碱含量及烟碱代谢的作用基础,在一定程度上解释了3个地区烟叶香型风格存在差异的原因。     

qRT-PCR分析莱茵衣藻氮、硫同化相关基因的表达

实验发现,缺氮、缺硫的环境有利于莱茵衣藻油脂的积累,说明与氮、硫同化相关的基因也与油脂合成有着密切的关系。分析了氮、硫同化通路相关基因,选择氮同化相关的6个基因(NAR3、NAR1.1、NII1、NIA1、NIT2、AOX1)以及硫同化相关的6个基因(SULP1、SIR1、SLT1、ARS1、SULTR1、ATS1)进行q RT-PCR分析。对莱茵衣藻CC124以及CC425藻株分别进行缺氮、缺硫处理,Trizol法连续提取4 d的RNA作为模板,进行实时荧光定量PCR检测基因的表达水平。结果发现,这12种基因均较对照上调表达,其中,莱茵衣藻氮、硫运载体基因上调表达非常显著。缺氮条件下NAR3、NAR1.1较正常最高上调表达200倍;缺硫条件下SLT1较对照有1 000~2 000倍的m RNA表达量,SULTR2也最高能增高450倍的表达量。     

响应面法优化超声波辅助乙醇提取余甘多酚的工艺研究

以提高余甘果肉中的多酚提取率为目的,采用超声波辅助乙醇提取。以乙醇浓度80%为固定条件,通过4组单因素试验探讨了超声波提取温度、功率、提取时间及料液比对余甘多酚提取效果的影响;运用Box-Behnken设计,通过3因素3水平的响应面分析法优化了超声波辅助乙醇提取余甘多酚的工艺。结果表明：在乙醇浓度80％,料液比1 g ∶ 25 mL条件下,余甘多酚的最佳超声波辅助乙醇提取的工艺条件为超声波提取温度43.80 ℃,功率250.96 W,提取时间23.90 min。按此工艺条件提取的余甘多酚提取率为17.21％。     

西藏青稞品种喜玛拉雅10号干旱胁迫的SSH文库构建及干旱诱导表达基因分析

为了研究西藏青稞抗旱的分子机制,以西藏耐旱青稞(Hordeum vulgare L.var.nudum HK.f.)品种喜玛拉雅10号为材料,利用抑制性差减杂交技术(SSH)构建青稞苗期叶片干旱胁迫诱导基因表达的正向差减文库。挑取600个阳性克隆进行PCR验证,并对验证后的单克隆进行测序分析,共获得420个有效序列。去除冗余序列和嵌合序列后,获得220条高质量EST序列,其中183条singlets,37条contigs。BlastN分析表明,其中189个EST序列可以在GenBank的unigene中找到同源序列,31个未能找到unigene同源序列;BlastX分析表明,62个EST序列unigene与未知功能蛋白或假定蛋白有较高的相似性;158条EST序列与已知功能蛋白有较高的同源性。获得的EST序列多数不仅与植物的非生物胁迫相关,也与植物的生物胁迫反应相关,说明植物在胁迫反应中有明显的共同机制。其中与大麦表达序列高度同源的EST占58%;这些基因涉及参与信号转导和转录调节的基因(11.96%),编码保护、防御和胁迫耐受蛋白的基因(9.78%),跨膜转运相关基因(5.43%),光合作用基因(6.52%),参与结构和功能代谢合成途径中的一些酶(30.43%)等代谢过程。实验结果表明,青稞在干旱复水条件下可诱导一系列特异基因的表达,如参与抗旱反应相关酶和蛋白基因锌指蛋白、衰老相关蛋白、CAS、细胞色素P450、热激蛋白、胁迫诱导蛋白,以及LEA蛋白、脱水蛋白、P5CS等保护蛋白基因。用KOBAS系统将56个EST定位到32个Pathways中,初步分析发现,谷氨酸盐代谢途径(Glutamate metabolism)、精氨酸和脯氨酸代谢途径(Arginine and proline metabolism)、泛素蛋白介导的蛋白质水解代谢(Ubiquitin mediated proteolysis)途径可能与青稞抗旱相关性较大。