Identification of 3,6-disubstituted dihydropyrones as inhibitors of human lactate dehydrogenase

A series of 3,6-disubstituted dihydropyrones were identified as inhibitors of human lactate dehydrogenase (LDH)-A. Structure activity relationships were explored and a series of 6,6-spiro analogs led to improvements in LDHA potency (IC₅₀ <350 nM). An X-ray crystal structure of an improved compound bound to human LDHA was obtained and it illustrated additional opportunities to enhance the potency of these compounds, resulting in the identification of 51 (IC₅₀ = 30 nM).     

Binding of p110 Retinoblastoma Protein Inhibits Nuclear Import of Simian Virus SV40 Large Tumor Antigen

Nuclear import of the simian virus 40 large tumor antigen (T-ag) is dependent on its nuclear localization signal (NLS) within amino acids 126–132 that is recognized by the importin α/β1 heterodimer, as well as a protein kinase CK2 site at serine 112 upstream of the NLS, which enhances the interaction ∼50-fold. Here we show for the first time that T-ag nuclear import is negatively regulated by N-terminal sequences (amino acids 102–110), which represent the binding site (BS) for the retinoblastoma (Rb) tumor suppressor protein (p110^Rb). Quantitative confocal laser scanning microscopic analysis of the transport properties of T-ag constructs with or without Rb binding site mutations in living transfected cells or in a reconstituted nuclear transport system indicates that the presence of the RbBS significantly reduces nuclear accumulation of T-ag. A number of approaches, including the analysis of T-ag nuclear import in an isogenic cell pair with and without functional p110^Rb implicate p110^Rb binding as being responsible for the reduced nuclear accumulation, with the Ser¹⁰⁶ phosphorylation site within the RbBS appearing to enhance the inhibitory effect. Immunoprecipitation experiments confirmed association of T-ag and p110^Rb and dependence thereof on negative charge at Ser¹⁰⁶. The involvement of p110^Rb in modulating T-ag nuclear transport has implications for the regulation of nuclear import of other proteins from viruses of medical significance that interact with p110^Rb, and how this may relate to transformation.     

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.     

The role of exosomes in colorectal cancer disease progression and response to therapy

Colorectal cancer (CRC) is the second leading cause of cancer mortality in both men and women worldwide. Survival of patients is significantly associated with disease stage at diagnosis. Recent studies highlighted a role of exosomes in CRC development and progression, thus raising the interest on these nanosized vesicular structures as possible biomarkers. Exosomes contain a large variety of molecules, including proteins, lipids and nucleic acids, that are exchanged between cells either within tumor microenvironment or at distant sites from the primary tumor, where they prepare a suitable soil for tumor metastases. The present review summarizes the principal effects of exosomes on CRC development, progression, and provides an update of the most recent findings on the use of exosomal molecules as diagnostic, prognostic and predictive biomarkers in CRC.     

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5^cre:ZsGreen^l/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.     

The intracellular domain of cell adhesion molecule 1 is present in emphysematous lungs and induces lung epithelial cell apoptosis

Background

Pulmonary emphysema is characterized histologically by destruction of alveolar walls and enlargement of air spaces due to lung epithelial cell apoptosis. Cell adhesion molecule 1 (CADM1) is an immunoglobulin superfamily member expressed in lung epithelial cells. CADM1 generates a membrane-associated C-terminal fragment, αCTF, through A disintegrin- and metalloprotease-10-mediated ectodomain shedding, subsequently releasing the intracellular domain (ICD) through γ-secretase-mediated intramembrane shedding of αCTF. αCTF localizes to mitochondria and induces apoptosis in lung epithelial cells. αCTF contributes to the development and progression of emphysema as a consequence of increased CADM1 ectodomain shedding. The purpose of this study was to examine whether the ICD makes a similar contribution.

Results

The ICD was synthesized as a 51-amino acid peptide, and its mutant was synthesized by substituting seven amino acids and deleting two amino acids. These peptides were labeled with fluorescein isothiocyanate and were introduced into various cell lines. ICD peptide-derived fluorescence was well visualized in lung epithelial cells at the site of Mitotracker mitochondrial labeling, but was detected in locations other than mitochondria in other cell types. Mutant peptide-derived fluorescence was detected in locations other than mitochondria, even in lung epithelial cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays revealed that transduction of the ICD peptide increased the proportion of apoptotic cells 2- to 5-fold in the lung epithelial cell lines, whereas the mutant peptide did not. Abundance of the ICD was below the Western blot detection limit in emphysematous (n = 4) and control (n = 4) human lungs. However, the ICD was detected only in emphysematous lungs when it was immunoprecipitated with anti-CADM1 antibody (4/4 vs. 0/4, P = 0.029).

Conclusions

As the abundance of ICD molecules was sparse but present, increased CADM1 shedding appeared to contribute to the development of emphysema by generating αCTF and the ICD in lung epithelial cells.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0173-8) contains supplementary material, which is available to authorized users.     

中频交变电流抑制肿瘤细胞的体外增殖

目的:初步探究新型物理治疗技术中频交变电流对肿瘤细胞和正常细胞体外存活率的影响;设置不同实验参数(频率、电流、时间),观察影响效果;针对实验结果,分析中频交变电流抑制肿瘤细胞体外增殖的可能机制。方法:将对数生长期的细胞悬液接种于24孔培养板,贴壁后(24 h)施加不同参数的中频电流刺激,噻唑蓝(MTT)法检测细胞存活率;以人乳腺癌细胞MCF-7为例,观察中频交变电流对细胞生存环境温度和pH的影响,Annexin V-FITC/PI双染法流式细胞术定量检测细胞凋亡/死亡情况,扫描电子显微镜和透射电子显微镜观察细胞结构变化。结果:中频交变电流能降低肿瘤细胞的体外存活率,诱导其凋亡,但不影响正常细胞(L929)存活率;MCF-7细胞体外实验最适参数是100 kHz,50 mA,每天30 min;电刺激后MCF-7细胞表面微绒毛减少,细胞线粒体肿胀,有多泡体及溶酶体形成。结论:中频交变电流能抑制肿瘤细胞增殖,但不影响正常细胞(L929);抑制效果和电刺激的频率、电流和时间有关;抑制机制可能是中频交变电流能够诱导细胞凋亡,影响细胞膜表面蛋白功能,但不影响细胞生存环境。中频交变电流抑制肿瘤体外增殖为实体瘤等增生性疾病的治疗提供了新的思路。     

MMP-16与骨巨细胞瘤临床相关性的初步研究

目的:研究复发与未复发骨细胞瘤细胞中MMP-16的变化,初步探索MMP-16与骨巨细胞瘤复发之间的联系,进而讨论MMP-16对于骨巨细胞瘤的临床意义。方法:对临床中复发和未复发共50例骨巨细胞瘤患者切除的骨巨细胞瘤组织进行标本采集。首先应用免疫组化荧光染色直观观察复发组和未复发组骨巨细胞瘤组织中MMP-16的表达情况,再运用real-time PCR技术检测MMP-16 mRNA的表达情况,最后应用western blot技术测定MMP-16的含量。从形态、基因表达和蛋白三个方面全面比较复发组和未复发组骨巨细胞瘤组织中MMP-16的表达情况。结果:免疫组化结果显示复发组中MMP-16的表达要明显高于未复发组;real-timePCR结果显示复发组中的MMP-16 mRNA的表达量要明显高于未复发组;western blot结果显示复发组中MMP-16蛋白的表达量要高于未复发组(P0.05)。结论:MMP-16与骨巨细胞瘤存在密切联系,MMP-16的表达增强是骨巨细胞瘤的复发的重要标志,通过对MMP-16的测定对骨巨细胞瘤的预后判断和治疗方法的选择具有指导意义。     

细胞角蛋白20 在膀胱癌中的研究进展

膀胱肿瘤是最常见的泌尿系统肿瘤,其中上皮性肿瘤占95%以上,绝大多数为尿路移行上皮细胞癌。膀胱癌的早期症状不 明显,复发率较高,早期诊断和治疗对提高其疗效非常重要。近年来,诊断膀胱肿瘤的新方法不断出现,显著提高了膀胱肿瘤诊断 及预后预测水平。其中,膀胱肿瘤标记物检测已成为膀胱肿瘤的诊断新方法,具有十分重要的临床意义。研究发现,细胞角蛋白20 (cytokeratin 20,CK20)是中间纤维家族成员之一,在正常膀胱组织中特异性表达于伞细胞,在膀胱癌中特异性表达于膀胱移行细 胞癌,其诊断膀胱肿瘤的特异性和灵敏性均较高,且与膀胱肿瘤的临床分级、病理分期和转移均密切相关,因此可作为辅助诊断 膀胱肿瘤的检测标志物及治疗和预后评估指标。本文将就其在膀胱癌中的研究进展综述如下。     

纳米载药系统在肿瘤多药耐药中的应用

当今世界,肿瘤已经成为威胁人类健康的重大疾病。在肿瘤疾病中,化疗可控制肿瘤的生长和转移,增强放疗的疗效,是治疗肿瘤疾病的主要手段之一。而肿瘤多药耐药是影响化疗药物疗效、引起化疗失败的重要原因,影响肿瘤患者的治愈效果,降低生存率。如何提高化疗的疗效,延长肿瘤患者的寿命成为医学界的难题。纳米载药系统是生物医学领域研究的热点,相对于单一药物,纳米载药体现了许多优越性,具有良好的应用前景。纳米级颗粒更有利于药代动力学,这些纳米载药颗粒通过被动和主动的机制表现出在全身血液循环寿命延长,持续的药物释放动力,使其能更好的在肿瘤细胞中积累而发挥作用,提高化疗的疗效。本文综述了肿瘤多药耐药研究中主要的纳米载体以及它们在逆转多药耐药方面的应用,并展望载药系统的有更多更好的发展趋势。