Cannabis-Derived Compounds Cannabichromene and Δ9-Tetrahydrocannabinol Interact and Exhibit Cytotoxic Activity against Urothelial Cell Carcinoma Correlated with Inhibition of Cell Migration and Cytoskeleton Organization

Cannabis sativa contains more than 500 constituents, yet the anticancer properties of the vast majority of cannabis compounds remains unknown. We aimed to identify cannabis compounds and their combinations presenting cytotoxicity against bladder urothelial carcinoma (UC), the most common urinary system cancer. An XTT assay was used to determine cytotoxic activity of C. sativa extracts on T24 and HBT-9 cell lines. Extract chemical content was identified by high-performance liquid chromatography (HPLC). Fluorescence-activated cell sorting (FACS) was used to determine apoptosis and cell cycle, using stained F-actin and nuclei. Scratch and transwell assays were used to determine cell migration and invasion, respectively. Gene expression was determined by quantitative Polymerase chain reaction (PCR). The most active decarboxylated extract fraction (F7) of high-cannabidiol (CBD) C. sativa was found to contain cannabichromene (CBC) and Δ9-tetrahydrocannabinol (THC). Synergistic interaction was demonstrated between CBC + THC whereas cannabinoid receptor (CB) type 1 and type 2 inverse agonists reduced cytotoxic activity. Treatments with CBC + THC or CBD led to cell cycle arrest and cell apoptosis. CBC + THC or CBD treatments inhibited cell migration and affected F-actin integrity. Identification of active plant ingredients (API) from cannabis that induce apoptosis and affect cell migration in UC cell lines forms a basis for pre-clinical trials for UC treatment.     

高效钙钛矿太阳能电池吸光层的设计与优化

本文设计了多元混合钙钛矿吸光层涂布液配方,并采用两步法组装了钙钛矿电池器件。由于两步法制备钙钛矿吸光层存在配方和工艺相对复杂的问题,导致电池器件性能难以提升,因此,本文采用正交实验的方法,筛选出影响电池器件性能的关键因素。针对关键因素设计了系列实验,通过观察钙钛矿层薄膜形貌,测试钙钛矿太阳能电池器件的光电性能,并采用多种表征手段研究了钙钛矿层薄膜性质,确定了关键因素的添加量,最后优选出钙钛矿电池吸光层的最佳配方,得到高质量的钙钛矿薄膜,电池的光电转换效率达到21.1%。     

原子力显微镜对中性粒细胞与K562细胞超微结构及机械性能的探测

采用原子力显微镜在纳米尺度下对正常中性粒细胞与白血病细胞株K562细胞的表面形貌及细胞的硬度、粘附力进行定性定量分析.结果表明,相比正常中性粒细胞的平均粗糙度(Ra=5.31±1.52 nm),K562细胞的超微结构更为复杂,细胞表面平均粗糙度显著升高(Ra=26.54±8.01 nm).此外,细胞的生物机械特性也有显著差别:中性粒细胞的硬度为9.5±1.3 kPa,AFM针尖与中性粒细胞的非特异性粘附力为135±23.4 pN;K562细胞的硬度为3.0±0.8 kPa,AFM针尖与K562细胞的非特异性粘附力为95±15.6 pN.AFM在单细胞水平上的探测表明,中性粒细胞和K562细胞的超微结构和机械特性均有明显差异.通过对细胞表面超微结构和力学特性的探测可以诊断慢性粒细胞白血病,原子力显微镜有望成为临床肿瘤诊断的工具.     

Determination and distribution of N-acetyl- and N-glycolylneuraminic acids in culture media and cell-associated glycoconjugates from human malignant mesothelioma and adenocarcinoma cells

Sialic acids containing glycoconjugates are very common in human neoplasias and their expression frequently correlates with malignant phenotype and the tumor grade. The majority of tumor markers containing sialic acids in man involve changes in the amount of total sialic acids and in the presence of the two main sialic acid types, Neu5Ac and Neu5Gc, and their derivatives. The aim of the present study was to examine whether malignant mesothelioma cell lines synthesize sialic acid containing glycoconjugates at both the extracellular and cell membrane levels and particularly whether the type and the content of Neu5Ac and Neu5Gc are of biological importance for mesothelioma cell differentiation and evaluation of its prognosis. The study was performed in three human malignant mesothelioma cell lines, two with a fibroblast like phenotype (STAV-FCS and Vester) and one of epithelial differentiation (STAV-AB), which developed from the pleural effusions of patients with malignant mesothelioma and in one human adenocarcinoma cell line (Wart). Neu5Ac and Neu5Gc were determined following a mild hydrolysis step and a sample clean-up procedure. The determination was performed by reversed-phase HPLC after the NeuAc and NeuGc had been converted to per-O-benzoylated derivatives. It was found that Neu5Gc is the major sialic acid in the culture media of all cell lines examined. Molar ratios of Neu5Ac to Neu5Gc showed that Neu5Gc is the predominant sialic acid in the culture medium of the fibroblast-like mesothelioma cells. Neu5Ac is almost undetectable in the cell membrane, whereas Neu5Gc is present in considerable amounts. The obtained results suggest that the type and the content of Neu5Ac and Neu5Gc in culture media are of biological importance for mesothelioma cell differentiation and may be of value in the evaluation of prognosis.     

药物与细胞膜相互作用的毛细管电泳方法研究

以细胞膜在毛细管内构成假固定相,建立一种基于毛细管电泳测定药物与细胞膜相互作用参数的方法．以西酞普兰和兔红细胞膜为相互作用模型,以不同浓度的细胞膜混悬液为电泳缓冲液,采用峰漂移法,并结合Scatchard分析,测得西酞普兰与兔红细胞膜的结合常数为0．977g^-1·L．该方法简单、快速,为研究药物与细胞膜的相互作用提供了新的技术手段,为高通量筛选药物膜通透性和活性,以及评价药物在体内吸收提供一种新的方法．     

Cell attachment and detachment on micropattern-immobilized poly(N-isopropylacrylamide) with gelatin

To investigate by microscopic observation the effects on cell behaviour of immobilized polymers, a micropattern-immobilization technique using a photo-mask was employed. Poly(acrylic acid) or poly(N-isopropylacrylamide-co-acrylic acid) was coupled with azidophenyl groups to form a photo-reactive polymer. The photo-reactive polymer was coated, with or without gelatin, on a cell-culture polystyrene plate and photo-irradiated through a micropatterned photo-mask. Mouse fibroblast STO cells were cultured on the micropattern-immobilized plate. The surface wettability of the immobilized plate was examined by measurement of the contact angle in the cell culture medium. The attachment of cells on the plate was significantly affected by the surface properties. Although the poly(acrylic acid) has the same effect on cell adhesion as a bare polystyrene surface, co-immobilization with gelatin significantly enhanced cell adhesion, while poly(N-isopropylacrylamide) reduced it. However, co-immobilization with gelatin enhanced cell adhesion, and, on the co-immobilized surface, cell detachment was observed by lowering the temperature. Micropattern immobilization was useful for comparing the effects of materials on cell behavior and for constructing biochips.     

The temperature effect during pulse application on cell membrane fluidity and permeabilization

Cell membrane permeabilization is caused by the application of high intensity electric pulses of short duration. The extent of cell membrane permeabilization depends on electric pulse parameters, characteristics of the electropermeabilization media and properties of cells exposed to electric pulses. In the present study, the temperature effect during pulse application on cell membrane fluidity and permeabilization was determined in two different cell lines: V-79 and B16F-1. While cell membrane fluidity was determined by electron paramagnetic resonance (EPR) method, the cell membrane electropermeabilization was determined by uptake of bleomycin and clonogenic assay. A train of eight rectangular pulses with the amplitude of 500 V/cm, 700 V/cm and 900 V/cm in the duration of 100 micros and with repetition frequency 1 Hz was applied. Immediately after the pulse application, 50 microl droplet of cell suspension was maintained at room temperature in order to allow cell membrane resealing. The cells were then plated for clonogenic assay. The main finding of this study is that the chilling of cell suspension from physiological temperature (of 37 degrees C) to 4 degrees C has significant effect on cell membrane electropermeabilization, leading to lower percent of cell membrane permeabilization. The differences are most pronounced when cells are exposed to electric pulse amplitude of 900 V/cm. At the same time with the decreasing of temperature, the cell membranes become less fluid, with higher order parameters in all three types of domains and higher proportion of domain with highest order parameter. Our results indicate that cell membrane fluidity and domain structure influence the electropermeabilization of cells, however it seems that some other factors may have contributing role.     

The Effect of Beta Adrenoreceptor Blockers on Viability and Cell Colony Formation of Non-Small Cell Lung Cancer Cell Lines A549 and H1299

Beta adrenoblockers are a large class of drugs used to treat cardiovascular diseases, migraines, glaucoma and hyperthyroidism. Over the last couple of decades, the anticancer effects of these compounds have been extensively studied. However, the exact mechanism is still not known, and more detailed studies are required. The aim of our study was to evaluate the anticancer activity of beta adrenoblockers in non-small cell lung cancer cell lines A549 and H1299. In order to find the relationship with their selectivity to beta adrenoreceptors, selective (atenolol, betaxolol, esmolol, metoprolol) and non-selective (pindolol, propranolol and timolol) beta blockers were tested. The effect on cell viability was evaluated by MTT assay, and the activity on cell ability to form colonies was tested by clonogenic assay. The type of cell death was evaluated by cell double staining with Hoechst 33342 and Propidium iodide. The most active adrenoblockers against both tested cancer cell lines were propranolol and betaxolol. They completely inhibited lung cancer cell colony formation at 90% of the EC₅₀ (half-maximal effective concentration) value. Most tested compounds induced cell death through apoptosis and necrosis. There was no correlation established between beta adrenoblocker anticancer activity and their selectivity to beta adrenoreceptors.     

Dose-dependent effect of resveratrol on proliferation and apoptosis in endothelial and tumor cell cultures

Experimental data suggest that Resveratrol, a compound found in grapes and other fruits may influence cell proliferation and apoptosis. The aim of our experiments was to study the effect of Resveratrol on tumor cell cultures and an endothelial cell culture in order to examine the effect of various doses of this compound on active cell death and cell proliferation. Human tumor (HT-29, SW-620, HT-1080) and endothelial (HUV-EC-C) cells were treated with various doses of (0.1 to 100.0 microg/ml) Resveratrol in vitro. Cell number, apoptotic and mitotic index was measured 24, 48 and 72 h after treatment. Low doses (0.1-1.0 microg/ml) of Resveratrol enhance cell proliferation, higher doses (10.0-100.0 microg/ml) induce apoptosis and decrease mitotic activity, which is reflected in changes of cell number. Resveratrol influences dose dependently the proliferative and apoptotic activity of human tumor and endothelial cells. The possible role of formaldehyde in the mechanism of action of Resveratrol is discussed.     

Design,synthesis, and anticancer evaluation of acetamide and hydrazine analogues of pyrimidine

A library of acetamide and hydrazine analogues were generated on the pyrimidine ring through a multistep reaction starting from 5-nitro-pyrimidine-4,6-diol and pyrimidine-4,6-diol, respectively. The synthesized analogues were screened for in vitro cytotoxic activity against various human cancer cell lines like HCT-1 and HT-15 (colon), MCF-7(breast), PC-3 (prostrate), SF268 (CNS) using MTT method. From the bioassay results, it was observed that even though many of the synthesized derivatives exhibited a good potency against various screened cancer cell lines, compound 14a from the acetamide series was found to show potent anticancer activity on all the tested cancer cell lines with IC₅₀ value of 0.36μM on CNS cell line and 1.6μM on HT-21 cell line, and compound 19xxi from hydrazine series of pyrimidine showed potent activity against three tested cancer cell lines with IC₅₀ value of 0.76μM on HT-29 cell line, 2.6μM on HCT-15, and 3.2μM on MCF-7 cell line.     

Monitoring cell survival after extraction of a single subcellular organelle using optical trapping and pulsed-nitrogen laser ablation

This paper characterizes cell viability in three different cell lines--Chinese hamster ovary cells (CHO), neuroblastoma cells fused with glialoma cells (NG108-15) and murine embryonic stem cells (ES-D3)--after N2 laser disruption of the cell membrane and removal, via optical trapping, of a single subcellular organelle. Morphological changes and viability (as determined by live/dead fluorescent stains) of the cell were monitored every half hour over a 4-h period postsurgery. The ability of the cell to survive organelle extraction was found to depend both on the conditions under which surgery was performed and on the cell type. The average viability after surgery for CHO cells was approximately 80%, for NG 108 cells it was approximately 30% and for ES-D3 cells postsurgery viability was approximately 10%. From over 600 surgeries we found the survival of the cell is determined almost exclusively within the first hour postsurgery regardless of cell line. The optimal pulse energy for N2 laser ablation was approximately 0.7 microJ. The N2 pulse produced an approximately 1-3 microm hole in the cell membrane and proved to be the primary source of cell death in those cells that did not survive the procedure.     

Investigating the morphology and mechanical properties of blastomeres with atomic force microscopy

Atomic force microscopy (AFM) was used to directly investigate the morphology and mechanical properties of blastomeres during the embryo development. With AFM imaging, the surface topography of blastomeres from two‐cell, four‐cell, and eight‐cell stages was visualized, and the AFM images clearly revealed the blastomere's morphological changes during the different embryo developmental stages. The section measurements of the AFM topography images of the blastomeres showed that the axis of the embryos nearly kept constant during the two‐cell, four‐cell, and eight‐cell stages. With AFM indenting, the mechanical properties of living blastomeres from several embryos were measured quantitatively under physiological conditions. The results of mechanical properties measurements indicated that the Young's modulus of the two blastomeres from two‐cell embryo was different from each other, and the four blastomeres from the four‐cell embryo also had variable Young's modulus. Besides, the blastomeres from two‐cell embryos were significantly harder than blastomeres from four‐cell embryos. These results can improve our understanding of the embryo development from the view of cell mechanics. Copyright © 2013 John Wiley & Sons, Ltd.     

Optimization and characterization of immobilized E. coli for engineered thermostable xylanase excretion and cell viability

There are many parameters that may have influenced the properties of cell during immobilization process. Particularly, the immobilization methods, carrier materials, and enzyme loading amount that have been proved to be important for immobilization process. The physiological responses of microorganisms are depending on the immobilization technique used. Typical alterations to the micro-environment of the immobilized cell involved the altered water activity, presence of ionic charges, cell confinement and modified surface tension. In this study, the graphene oxide was selected as a suitable carrier for immobilization process of recombinant E.coli and adsorption was chosen as an appropriate method to improve the production of engineered thermostable xylanase. High level production of thermostable xylanase by immobilized recombinant cell in the 5 L bioreactor was studied by using optimum research surface methodology (RSM) conditions was studied. The immobilization of E. coli onto nanoparticle matrix manages to improve the cell performance by improving the protein expression, reduced the occurrences of cell lysis as well as improved the plasmid stability of the host cell. Thus, immobilization contributes a physical support for both whole cells as well as enzymes to develop a better operative achievement system for industrialized fields and give rise to the biological advancement existing enzyme for instance xylanase.     

Cu(II)、Ni(II)配合物的合成、抗肿瘤作用及其对细胞周期分布的影响

以2-氨基-5-氯苯酚和2-喹啉甲醛合成的席夫碱作为配体,分别与氯化镍、氯化铜反应合成了2个金属配合物C1和C2,其结构通过单晶X-射线衍射进行了解析。采用MTT法测试了配合物C1和C2对不同人肝癌细胞系和正常肝细胞系HL-7702增殖抑制活性,结果表明C1、C2对人肝癌细胞系的抑制活性均优于顺铂,且对正常肝细胞系HL-7702的毒性要弱于顺铂。通过活性氧实验、细胞周期等实验,可以推断出配合物C1、C2抗肿瘤机制是通过产生活性氧造成肿瘤细胞的氧化损伤,以及使细胞周期停滞在G0/G1期阻滞细胞复制。     

Synthesis and distribution of glycosaminoglycans in human leukemic B- and T-cells and monocytes studied using specific enzymic treatments and high-performance liquid chromatography

Identification of glycosaminoglycans (GAGs) synthesized by three human leukaemic cell lines-Jurkat (T-cell leukaemia), Daudi (Burkitt's lymphoma, B-cell leukaemia) and THP-1 (acute monocytic leukemia)-and normal peripheral blood mononuclear cells (PBMC) and their distribution among cell membrane and culture medium were studied. GAGs were isolated using ion-exchange chromatography on DEAE-Sephacel and their composition and fine chemical structure were studied using high-performance liquid chromatography with radiochemical detection. All cell lines synthesize chondroitin sulphate (CS) and heparan sulphate (HS) in both cell membrane and culture medium. No hyaluronan was detected using treatment with specific lyases and highly sensitive HPLC methodology. CS is the major secreted GAG in all cell lines tested and the major cell retained GAG in Jurkat and Daudi. HS is the major GAG in the cell membrane of THP-1. The amounts of distinct GAGs synthesized by all cancer cell lines differ from those produced by normal PBML indicating a major role of GAGs in malignant transformation of human lymphocytes and monocytes.     

Engineering Photocleavable Protein-decorated Hydrogels to Regulate Cell Adhesion and Migration

Cell adhesion and migration play essential roles in tissue development and maintenance, and abnormal cell migration is involved in life-threatening diseases, including vascular disease, tumor formation, and metastasis. The advances in hydrogel-based 3D cell culture development facilitated the investigation of cell motility behavior, including cell-cell and cell-matrix adhesion and cell migration in a microenvironment more related to in vivo situations. Establishing advanced methods for these in vitro studies is thus necessary. Photo-sensitive proteins show advantages in remote and non-invasive regulation of hydrogels' properties, and thus are of great potential in regulating 3D cultured cells' behavior. In the presented study, we engineered photocleavable protein(PhoCl)-decorated hydrogels to regulate cell adhesion and migration of MDA-MB-231. The integrin-binding motif RGD was fused to the PhoCl and was decorated on the hydrogel. After being exposed to light at 405 nm, the PhoCl was cleaved and the RGD motif was released, resulting in detachment of the binding cells. The regulatory effect of the light illumination showed a time-dependent and cell density-dependent manner. Furthermore, the elimination of RGD by patterned light exposure completely suspended the cell migration to the corresponding region, suggesting a controllable regulation of the cell migration direction.     

微流控芯片单细胞进样和溶膜

单细胞分析对重大疾病的早期诊断、治疗和药物筛选以及细胞生理、病理过程的研究有重要意义.将毛细管电泳用于单细胞多组分的测定已取得一些成果,但受毛细管的一维结构限制,单细胞进样和溶膜操作较复杂.微流控分析芯片的网络结构和微米级的通道尺寸使简化单细胞分析成为可能.     

Differential interference contrast microscopy for real-time dynamics and manipulation of single cells in microchannels

Nomarski differential interference contrast (DIC) microscopy was used for real-time dynamics of intact single cells in various microchannels for adaptation to microfluidic chip application. The cheek cell was chosen as a model, single cell and the dynamics was measured at the microchannels. The image resolution of single cell was shaper and more distinct in DIC than in conventional microscopy. The individual single living cells were also manipulated by both hydrodynamic and electrokinetic flow-driving forces at the microchannels. The DIC contrast was enhanced according to the order of round-, square-, and rectangle-type microchannels. The velocity of the single living cell was consistently increased with increasing electric field strength and pH. However, the velocity of cell was decreased with increasing run buffer concentration. The driving direction of the individual single cell was simply controlled by changing the polarity of the applied voltage and the electric field strength. The cells were consistently manipulated in the microchannel under the co-application of the low electric field of 2.44 V/cm, instead of the solo application of the hydrodynamic force.     

Synthesis of chitosan-stabilized copper nanoparticles (CS-Cu NPs): Its catalytic activity for C-N and C-O cross-coupling reactions and treatment of bladder cancer

Herein, we report the synthesis of copper nanoparticles at ambient conditions using biopolymer, chitosan, as a protecting and stabilizing agent and hydrazine as a reducing agent. The obtained nanoparticles (CS-Cu NPs) were characterized using XRD, FT-IR, FE-SEM, EDS, TEM and UV–Vis spectroscopy. This nanocomposite was utilized as an efficient heterogeneous nanocatalyst for the aryl and heteroaryl C–N and C–O cross coupling reactions with excellent yields at mild conditions. The nanocatalyst were isolated and reused for 10 times with reproducible catalytic activity. Cell viability of nanocomposite was very low against bladder cancer (UM-UC-3 (Transitional cell carcinoma), SCaBER (Squamous cell carcinoma), and TCCSUP (Grade IV, transitional cell carcinoma)) cell lines without any cytotoxicity on the normal cell line. The best anti-human bladder cancer properties of nanocomposite against the above cell lines was in the case of TCCSUP cell line. According to the above findings, the nanocomposite may be administrated for the treatment of several types of human bladder cancer in humans.     

Study of the peak broadening due to detection in the electrophoretic separation of DNA by CE and microchip CE and the application of image sensor for ultra‐small detection cell length

Narrow peaks are important to high‐resolution and high‐speed separation of DNA fragments by capillary electrophoresis and microchip capillary electrophoresis. Detection cell length is one of the broadening factors, which is often ignored in experiments. However, is it always safe to neglect detection cell length under any condition? To answer this question, we investigated the influence of detection cell length by simulation and experiments. A parameter named as detection cell length ratio was proposed to directly compare the detection cell length and the spatial length of sample band. Electrophoretic peaks generated by various detection cell length ratios were analyzed. A simple rule to evaluate the peak broadening due to detection cell length was obtained. The current states of the detection cell length of detection system and their reliabilities in capillary electrophoresis and microchip capillary electrophoresis were analyzed. Microchip capillary electrophoresis detection with an ultra‐small detection cell length of 0.36 μm was easily achieved by using an image sensor.