In vitro antitumor activity of broccolini seeds extracts

Broccolini (Brassica oleracea Italica × Alboglabra) is a hybrid of broccoli and kai‐lan, Chinese broccoli. To date, no report on antitumor activity of Broccolini (NOT Broccoli) is available. In this study, we evaluated the antiproliferative effects of broccolini seeds extract (BSE) on human lung and ovarian cancer cells. It was found that BSE induces A549 and OVCAR‐3 cells apoptosis in a dose‐dependent manner by using MTT assay. The IC₅₀ values of BSE in A549 and OVCAR‐3 cells were estimated to be 81.94 and 78.6 µg/ml, respectively. Furthermore, the phase contrast microscope showed that in high‐dose group (90～120 µg/ml), the morphology structure of OVCAR‐3 cells become irregular and exhibited characteristics of apoptosis such as cell membrane shrinkage, condensation and fragmentation of nuclear chromatin as well as formation of apoptotic bodies. SCANNING 33:402–404, 2011. © 2011 Wiley Periodicals, Inc.     

Study of morphological and mechanical features of multinuclear and mononuclear SW480 cells by atomic force microscopy

This article studies the morphological and mechanical features of multinuclear and mononuclear SW480 colon cancer cells by atomic force microscopy to understand their drug‐resistance. The SW480 cells were incubated with the fullerenol concentrations of 1 mg/ml and 2 mg/ml. Morphological and mechanical features including the height, length, width, roughness, adhesion force and Young's modulus of three multinuclear cell groups and three mononuclear cell groups were imaged and analyzed. It was observed that the features of multinuclear cancer cells and mononuclear cancer cells were significantly different after the treatment with fullerenol. The experiment results indicated that the mononuclear SW480 cells were more sensitive to fullerenol than the multinuclear SW480 cells, and the multinuclear SW480 cells exhibited a stronger drug‐resistance than the mononuclear SW480 cells. This work provides a guideline for the treatments of multinuclear and mononuclear cancer cells with drugs.     

TNFalpha increases in vitro migration of human HPV18-positive SW756 cervical carcinoma cells.

TNFalpha has been associated with both, tumor survival and apoptosis. This cytokine is also involved in promoting cell migration during wound healing and tumorigenesis. SW756 is a HPV18-positive cervical carcinoma cell line, which has been used to study different mechanisms of cervical cancer progression. An in vitro assay of scratch wound healing onto monolayers of SW756 cells was used to assess the effect of TNFalpha on cell migration into a wound space. It was found that SW756 cells have the ability to migrate, but not proliferate in response to scratch wounding in a serum-free medium supplemented with TNFalpha. RT-PCR analysis showed that SW756 cells express TNFalpha mRNA when incubated in medium with and without serum. Wound closure and migration rate of SW756 cells were significantly increased in the presence of serum-free media supplemented with TNFalpha (10 ng/mL) as compared to serum-free media, and media supplemented with either anti-TNFalpha antibody or both TNFalpha and anti-TNFalpha antibody (p < 0.05). The results showed a stimulatory effect of TNFalpha on the migration of SW756 cervical carcinoma cells, suggesting a novel and important role for TNFalpha in cervical cancer progression.     

Lysophosphatidylcholine acyltransferase 1 is involved in the regulation of exosome secretion and uptake in colorectal cancer cells

Lysophosphatidylcholine acyltransferase 1 (LPCAT1) is a phospholipid acyltransferase that promotes phospholipid synthesis and plasma membrane reconstruction. Exosomes play an important role in tumor metastasis. The release and uptake of exosomes are key steps of their functions and depend on plasma membrane fusion and plasma membrane receptors, respectively. The purpose of this study was to explore whether LPCAT1-induced plasma membrane remodeling would change the secretion and uptake behavior of exosomes in tumor cells. We first confirmed the abnormally high expression of LPCAT1 in colorectal cancer cells by quantitative real-time PCR (qPCR) and Western blot analysis. Then, SW620 cells were used as exosome source cells, and SW480 cells were used as exosome receiver cells. Exosomes from SW620 cells could effectively promote the migration of SW480 cells. When LPCAT1 expression was reduced via siRNA knockdown in the source cells, the secretion of exosomes was downregulated, thus weakening the pro-migratory effects of exosomes on target cells. Conversely, when LPCAT1 was knocked down in target cells, the uptake of exosomes in target cells also decreased sharply. These results undoubtedly revealed that LPCAT1 is functionally associated with the release and internalization of exosomes in colorectal cancer cells and could affect the paracrine effects of exosomes, preliminarily extending the classical metabolic function of LPCAT1 to exosome-related pathways.     

Adaptive control of a vehicle-seat-human coupled model using quasi-zero-stiffness vibration isolator as seat suspension

We propose the quasi-zero-stiffness (QZS) vibration isolator as seat suspension to improve vehicle vibration isolation performance. The QZS vibration isolator is composed of vertical spring and two symmetric negative stiffness structures used as stiffness correctors. A vehicle-seat-human coupled model considering the QZS vibration isolator is established as a three degree-of-freedom (DOF) model; it is composed of a quarter car model and a simplified 1 DOF model combined vehicle seat and human body. This model considers the changing mass of the passengers and sets the total mass of the vehicle seat and human body as an uncertain parameter, which investigates the overload and unload conditions in practical engineering. To further improve the vehicle ride comfort, a constrained adaptive backstepping controller law based on the barrier Lyapunov function (BLF) is presented. The dynamic characteristic of the active vehicle-seathuman coupled model under shock excitation was analyzed using numerical method. The results show that the designed controller law can isolate the shock excitation transmitted from the road to the passengers effectively, and both the vehicle and seat suspension strokes remain in the allowed stroke range.     

Interaction of IL-22/IL-22R1 promotes cell proliferation and suppresses apoptosis of colorectal cancer via phosphorylation of STAT3

Interleukin-22 (IL-22) is a member of IL-10 cytokine family which is expressed in activated T cells predominantly and in activated natural killer cells at lower levels. Previous studies have demonstrated the link between elevated levels of IL-22 and disease severity of psoriasis, Crohn’s disease, rheumatoid arthritis and interstitial lung diseases. However, the function of IL-22 in the development and progression of colorectal cancer (CRC) remains elusive. In this study, we first evaluated the IL-22/IL-22R1 level in CRC patients, and found that tumor tissues have more active expression of IL-22 and IL-22R1 than normal tissues, presenting correlation with the degree of differentiation of tumor tissues. Subsequently, Caspase and cell viability assays were performed on SW-480 cell line which expresses high level of IL-22R1 to examine if the supplementation of IL-22 has an impact on apoptosis and proliferation. In comparison with treatment of 5-FU, supplementation of IL-22 promoted cell proliferation and ameliorated apoptosis. To unveil signal transduction upon activation of IL-22R, we examined the phosphorylation of STAT3 in SW-480 cell line following supplementation of IL-22. The treatment of IL-22 also increased the level of p-Akt, an essential component in PI3K/Akt pathway. Although the link between STAT3 phosphorylation and PI3K/ Akt activation remains to be explored, our study revealed the mechanism underlying the effects of IL-22R activation on apoptosis as well as tumor differentiation, indicating the prognostic value of IL-22/IL-22R.     

一种新型的串联中医推拿机械臂的运动学分析

针对中医的按、揉、滚、振4种推拿手法,进行了运动学和动力学特征的分析,基于分析结果进行了机械臂的选型,提出一种新型的混合型推拿机器人,并对该机械臂的运动学正反解进行了计算和分析。利用Pro/E对机械臂进行建模,通过Mechanism/Pro模块导入ADAMS/view模块中进行运动学仿真与分析。结果表明该机械臂能较好地模拟中医推拿师的推拿动作,且具有良好的运动特性。     

Detection of ROS and translocation of ERP-57 in apoptotic induced human neuroblastoma (SH-SY5Y) cells

Several toxic compounds are known to induce apoptosis in mammalian cell lines. The human neuroblastoma cells (SH-SY5Y) were exposed to the phosphatase inhibiting toxin okadaic acid (OA) or hydrogen peroxide (H2O2) to induce apoptosis as well as generate reactive oxygen species (ROS). Mitoxantrone (MXT) was used as a positive control for apoptosis. The SH-SY5Y cells were transfected with eukaryotic expression plasmid pHyPer-dMito encoding mitochondrial-targeted fluorescent or pHyPer-dCito encoding cytoplasmic-targeted fluorescent sensor for hydrogen peroxide (HyPer). The ERp57, also called GRP58 (Glucose-regulated protein 58), is a stress protein induced in conditions like glucose starvation and viral infection. Recently ERp57 was shown to translocate from the endoplasmatic reticulum to the cell surface in anthracycline-induced apoptotic cells. ERp57 co-translocation together with calreticulin has been suggested to be crucial for recognizing tumor cells to induce immunogenic cell death. ERp57 translocation after exposure to okadaic acid was studied using immunofluorescence and confocal microscopy. These studies indicated that okadaic acid has induced the translocation of ERp57 to the cellular membrane.     

Changes in macrophage morphology and prolonged cell viability following exposure to polyethylene particulate in vitro

The interaction of macrophages and ultra-high molecular weight polyethylene (PE) wear plays an important role in perpetuating chronic inflammation at the bone implant interface, leading to peri-implant osteolysis and mechanical failure of the implant. A model to study the interaction of human mature macrophages with orthopaedic biomaterial wear has been previously developed. With the use of the model, in this study, the mature human monocyte-derived macrophages (MDMs) were observed with light, fluorescent, and scanning electron microscopy (SEM), as well as transmission electron microscopy (TEM). The cell viability was investigated using calcein and ethidium staining. Following exposure to PE particulate, the morphology of the human MDMs was heterogeneous: rounded, flattened, and elongated. There was no morphological evidence of cytotoxicity or apoptosis. The MDM viability was not influenced by phagocytosis of PE particulate in a negative fashion. In fact, more prolonged cell viability was observed in the human MDMs exposed to PE particulate when compared to controls.     

Overexpression of inhibin α (1-32) fusion protein promotes apoptosis and cell cycle arrest in a cervical cancer cell model (Hela cells)

Inhibins play important roles in the reproductive system. To evaluate whether inhibin α (1-32) fusion protein plays a role in cervical cancer growth, the plasmid pVAX-inhα was constructed and its effect on proliferation and apoptosis of the human cervical cancer cell line (Hela) was checked by flow cytometry and real-time PCR. The expression and localization of inhibin α protein were detected by RT-PCR and confocal microscopy which showed that inhibin α protein was expressed and localized in the nucleus of Hela cells. Over expression of inhibin α gene significantly induced cell apoptosis and ceased S phase of cell cycle. Furthermore, cell proliferation was significantly suppressed 96 h post-transfection and mRNA level of anti-apoptosis genes (Bcl-2, NFκB) were decreased but pro-apoptosis genes (Bax, wild type p53) and inhibin co receptor (TGFβR3) were increased, indicating that inhibin, through its co-receptor, might activate apoptotic and cell growth cascades which regulate proliferation and apoptosis in Hela cells. These results suggest that inhibin α (1-32) fusion protein, located in the cell nucleus, can regulate Hela cells growth and apoptosis by induction of apoptotic pathways such as NFκB, Bcl-2 and p53 families. These findings may have a significant impact on future research regarding cervical cancer cell lines     

Programmed neuronal cell death induced by HIV-1 tat and methamphetamine

Apoptosis and autophagy are the two major types of programmed cell death (PCD) in neurons. Homeostatic autophagy often precedes apoptosis, and when apoptosis is blocked, the failure to keep homeostasis will lead to necrosis instead. It has been reported that human immunodeficiency virus (HIV) infected methamphetamine (Meth) abusers represent greater neuropathological abnormalities than Meth abusers or HIV-positive non-Meth users. Recent publications suggest that Tat and Meth when administered together result in greater neuronal damage than when administered separately. However, the cellular events of the combined Tat-Meth effect have not yet been fully characterized. Therefore, we investigated the effects of Tat and/or Meth on apoptosis and autophagy to elucidate whether PCD was involved in Tat and/or Meth-induced neuronal damage. Annexin-V-FITC/PI staining assay was used to detect cellular apoptosis using a neuroblastoma cell line SH-SY5Y. Cellular ultrastructural changes were observed under transmission electron microscopy (TEM). Flow-cytometric data showed apoptosis following Meth treatment, and more extensive apoptosis with Tat + Meth treatment. The most important finding was that the autophagosome and/or multilamellar bodies (MLBs) were most pronounced with Tat + Meth treatment, were less so with Meth treatment, and infrequent with Tat treatment. This suggests the involvement of autophagy and apoptosis in Tat with Meth-elicited cell damage. However, the relation between apoptosis and autophagy remains unknown in this experiment. Further research is needed to analyze the relation among related molecules. A thorough understanding of this multifaceted relationship will be critical for the assessment of therapeutic modalities for patients with HIV with drug abuse.     

Tumor necrosis factor-alpha induced DNA cleavage in human articular chondrocytes may involve multiple endonucleolytic activities during apoptosis

Apoptosis has been documented in chondrocytes both in the growth plates of young, healthy cartilages and in osteoarthritic cartilages; little, however, is known about apoptosis in chondrocytes of normal adult articular cartilage. For the current study, apoptosis in adult chondrocytes was evaluated by labeling DNA fragments using the ISEL in situ end labeling of 3'-recessed strand breaks) or TUNEL (5'-recessed or blunt-ended strand breaks with terminal deoxynucleotidyl transferase-mediated nick end labeling) techniques in primary cultures of chondrocytes in monolayer. Apoptosis was induced in the chondrocytes by either Tumor Necrosis Factor alpha (TNF alpha), Interleukin 1-beta (IL-1 beta), or anti-Fas antibody but only after 48 hours in culture. At 4 and 24 hours, there was no detectable DNA fragmentation. With TNF alpha, IL1 beta, and anti-Fas antibody, chondrocytes show evidence of at least two types of DNA strand breaks within the same cell (as assessed by simultaneous labeling with ISEL and TUNEL). Therefore, some pathways leading to apoptosis in chondrocytes appear to involve more than one type of endonuclease activity. When the chondrocytes were cultured as explants with the articular matrix intact (ex vivo), neither IL-1 beta, TNF alpha, the anti-Fas antibody, nor fibronectin fragments were able to induce apoptosis in the chondrocytes. In normal human adult cartilage that was untreated and uncultured (in situ), DNA fragmentation was undetectable; however, a significant number of chondrocytes in osteoarthritic cartilage did contain strand breaks. These data suggest that apoptosis occurs in chondrocytes in which the matrix has been disrupted experimentally or destroyed by the osteoarthritic disease process. The results of these studies suggest that the ECM may be an essential survival factor for chondrocytes.     

Synergistic anticancer activity of curcumin and catechin: an in vitro study using human cancer cell lines

The most practical approach to reduce morbidity and mortality of cancer is to delay the process of carcinogenesis by usage of anticancer agents. This necessitates that safer compounds are to be critically examined for anticancer activity especially, those derived from natural sources. A spice commonly found in India and the surrounding regions, is turmeric, derived from the rhizome of Curcuma longa and the major active component is a phytochemical termed curcumin. Green tea is one of the most popular beverages used worldwide, produced from the leaves of evergreen plant Camellia sinensis and the major active ingredients are polyphenolic compounds known as catechins. In this study, synergistic anticancer activity of curcumin and catechin was evaluated in human colon adenocarcinoma HCT 15, HCT 116, and human larynx carcinoma Hep G-2 cell lines. Although, both curcumin or catechin inhibited the growth of above cell lines, interestingly, in combination of both these compounds highest level of growth control was observed. The anticancer activity shown is due to cytotoxicity, nuclear fragmentation as well as condensation, and DNA fragmentation associated with the appearance of apoptosis. These results suggest that curcumin and catechin in combination can inhibit the proliferation of HCT 15, HCT 116, as well as Hep G-2 cells efficiently through induction of apoptosis.     

An automatic algorithm for the segmentation and morphological analysis of microvessels in immunostained histological tumour sections

A fully automatic segmentation and morphological analysis algorithm for the analysis of microvessels from CD31 immunostained histological tumour sections is presented. Development of the algorithm exploited the distinctive hues of stained vascular endothelial cells, cell nuclei and background, to provide the seeds for a 'region-growing' method for object segmentation in the 3D hue, saturation, value (HSV) colour model. The segmented objects, identified as microvessels by CD31 immunostaining, were post-processed with three morphological tasks: joining separate objects that were likely to belong to a single vessel, closing objects that had a narrow gap around their periphery, and splitting objects with multiple lumina into individual vessels. The automatic segmentation was validated against a hand-segmented set of 44 images from three different SW1222 human colorectal carcinomas xenografted into mice. 96.3 ± 0.9% of pixels were found to be correctly classified. Automated segmentation was carried out on a further 53 images from three histologically distinct mouse fibrosarcomas (MFs) for morphological comparison with the SW1222 tumours. Four morphometric measurements were calculated for each segmented vessel: vascular area (VA), ratio of lumen area to vascular area (lu/VA), eccentricity (e), and roundness (ro). In addition, the total vascular area relative to tumour tissue area (rVA) was calculated. lu/VA, e and ro were found to be significantly smaller in MF tumours than in SW1222 tumours (p < 0.05; unpaired t-test). The algorithm is available through the website http://www.caiman.org.uk where images can be uploaded, processed and sent back to users. The output from CAIMAN consists of the original image with boundaries of segmented vessels overlaid, the calculated parameters and a Matlab file, which contains the segmentation that the user can use to derive further results.     

Murine double minute gene 2 (MDM2) promoted hepatocellular carcinoma (HCC) cell growth by targeting fructose-1,6-bisphosphatase (FBP1) for degradation

To study the roles and association of murine double minute gene 2 (MDM2) and fructose-1,6-biphosphatase (FBP1) in human hepatocellular carcinoma (HCC), growth response of human HCC cells was assessed using proliferation and apoptosis assay. Pro-survival AKT signaling associated proteins (p-AKT, survivin and cleaved caspase 3) were assessed using western blotting. The correlation between MDM2 and FBP1 was assessed using co-immunoprecipitation combined with ubiquitination assay. Our data suggested that low expression of FBP1 was correlated with high levels of MDM2 in HCC cell lines (Huh7 and Hep3B). Overexpression of FBP1 resulted in anti-proliferation, pro-apoptosis, the up-regulation of cleaved caspase 3 while the downregulation of survivin and phosphor (p)-AKT, however, knockdown of FBP1 led to the opposite. Furthermore, overexpression of MDM2 potently reversed FBP1-induced proliferation inhibition and apoptosis, while Nutlin-3 (an MDM2 inhibitor) reversed the change trends induced by FBP1 knockdown in the aforementioned events. Lastly, but not least importantly, our data elucidated that MDM2 binds directly to FBP1 and promotes FBP1 ubiquitination. In conclusion, our data firstly suggested the involvement of FBP1 and its association with MDM2 in HCC cell growth. MDM2-FBP1-regulated HCC cell growth and the activation of AKT were mediated, at least in part, through FBP1 degradation.     

Apoptosis Induction of K562 Cells by Lymphocytes: An AFM Study

Antitumor immunotherapies, as a prospective approach for local cancer treatment, are attracting increasing interests. To detect the reacting course of immune and tumor cells, we have observed the process of K562 cells (a human erythroleukemic cell line) coculturing with peripheral lymphocytes, and the morphological and ultrastructural alterations of K562 cells and lymphocytes were investigated as well using atomic force microscopy (AFM). AFM morphological imaging revealed that after coculture the apoptosis‐like structures such as blebbing, pores, and apoptotic bodies were observed on the K562 cells. Also, the cell‐surface roughness decreased significantly, which implied the changes in chemical composition of cell membranes. Moreover, the lymphocytes were damaged to some extent induced by the coculture. The data demonstrated that K562 cells could be attacked and induced apoptosis by lymphocytes, and they would make damages to lymphocytes to escape the surveillance of immune system. SCANNING 35:7‐11, 2013. © 2012 Wiley Periodicals, Inc.     

L-Selenocystine induce HepG2 cells apoptosis through ROS-mediated signaling pathways

At present, Hepatocarcinoma is one of the main causes of tumor related death all over the world. However, there are still many clinical restrictions on the treatment of liver cancer. Recently, L-Selenocystine has been shown to be a novel treatment for tumors, especially human glioma cells. But, the mechanism of L-Selenocystine against hepatocellular carcinoma remains unclear. Therefore, the main objective of this study was to investigate the effects of L-Selenocystine on HepG2 cell proliferation and activation of reactive oxygen species (ROS) mediated signaling pathway. L-Selenocystine can significantly inhibit HepG2 cell proliferation by activating caspase-3 and cleaving PARP to induce apoptosis. Moreover, the excessive production of ROS and the influence of Bax signaling pathway which can promote cell apoptosis are key factors for L-Selenocystine to induce HepG2 cell apoptosis. Therefore, the date of this study suggest that ROS mediated signal transduction mechanism may provide certain reference significance for L-Selenocystine induced HepG2 cell apoptosis.     

Long-term monitoring of live cell proliferation in presence of PVP-Hypericin: a new strategy using ms pulses of LED and the fluorescent dye CFSE

During fluorescent live cell imaging it is critical to keep excitation light dose as low as possible, especially in the presence of photosensitizer drugs, which generate free radicals upon photobleaching. During fluorescent imaging, stress by excitation and free radicals induces serious cell damages that may arrest the cell cycle. This limits the usefulness of the technique for drug discovery, when prolonged live cell imaging is necessary. This paper presents a strategy to provide gentle experimental conditions for dynamic monitoring of the proliferation of human lung epithelial carcinoma cells (A549) in the presence of the photosensitizer Polyvinylpyrrolidone-Hypericin. The distinctive strategy of this paper is based on the stringent environmental control and optimizing the excitation light dose by (i) using a low-power pulsed blue light-emitting diode with short pulse duration of 1.29 ms and (ii) adding a nontoxic fluorescent dye called carboxyfluorescein-diacetate-succinimidyl-ester (CFSE) to improve the fluorescence signals. To demonstrate the usefulness of the strategy, fluorescence signals and proliferation of dual-marked cells, during 5-h fluorescence imaging under pulsed excitation, were compared with those kept under continuous excitation and nonmarked reference cells. The results demonstrated 3% cell division and 2% apoptosis due to pulsed excitation compared to no division and 85% apoptosis under the continuous irradiation. Therefore, our strategy allows live cell imaging to be performed over longer time scales than with conventional continuous excitation.     

Influence of estradiol analogue on cell growth,morphology and death in esophageal carcinoma cells

2-Methoxyestradiol-bis-sulphamate is a bis-sulphamoylated derivative of the naturally occurring 17-beta-estradiol metabolite namely 2-methoxyestradiol. 2-Methoxyestradiol-bis-sulphamate is regarded as a potential anticancer drug with increased antiproliferative activity when compared to 2-methoxyestradiol. The aim of this pilot in vitro study was to determine the influence of 2-methoxyestradiol-bis-sulphamate on cell growth, morphology and possible induction of certain types of cell death in the SNO esophageal carcinoma cell line. A dose-dependent study (0.2-1.0μM) was conducted with an exposure time of 24 hours. Data revealed that 2-methoxyestradiol-bis-sulphamate reduced cell numbers statistically significantly to 74% after exposure to 0.4μM of the drug. Morphological studies including light microscopy demonstrated hallmarks of apoptosis, while fluorescent microscopy revealed both the presence of apoptosis and autophagy as types of cell death being induced in SNO cells after 24 hours of exposure to 0.4μM 2-methoxyestradiol-bis-sulphamate.