Real-time Cell Analysis System Research Articles

The Intracellular Delivery Of Anti-HPV16 E7 scFvs Through Engineered Extracellular Vesicles Inhibits The Proliferation Of HPV-Infected Cells.

PurposeSingle-chain variable fragments (scFvs) are one of the smallest antigen-binding units having the invaluable advantage to be expressed by a unique short open reading frame (ORF). Despite their reduced size, spontaneous cell entry of scFvs remains inefficient, hence precluding the possibility to target intracellular antigens. Here, we describe an original strategy to deliver scFvs inside target cells through engineered extracellular vesicles (EVs). This approach relies on the properties of a Human Immunodeficiency Virus (HIV)-1 Nef mutant protein referred to as Nefmut. It is a previously characterized Nef allele lacking basically all functions of wt Nef, yet strongly accumulating in the EV lumen also when fused at its C-terminus with a foreign protein. To gain the proof-of-principle for the efficacy of the proposed strategy, the tumor-promoting Human Papilloma Virus (HPV)16-E7 protein was considered as a scFv-specific intracellular target. The oncogenic effect of HPV16-E7 relies on its binding to the tumor suppressor pRb protein leading to a dysregulated cell duplication. Interfering with this interaction means impairing the HPV16-E7-induced cell proliferation.MethodsThe Nefmut gene was fused in frame at its 3ʹ-terminus with the ORF coding for a previously characterized anti-HPV16-E7 scFv. Interaction between the Nefmut-fused anti-HPV16-E7 scFv and the HPV16-E7 protein was tested by both confocal microscope and co-immunoprecipitation analyses on co-transfected cells. The in cis anti-proliferative effect of the Nefmut/anti-HPV16-E7 scFv was assayed by transfecting HPV16-infected cells. The anti-proliferative effect of EVs engineered with Nefmut/anti-HPV16-E7 scFv on HPV16-E7-expressing cells was evaluated in two ways: i) through challenge with purified EVs by a Real-Time Cell Analysis system and ii) in transwell co-cultures by an MTS-based assay.ResultsThe Nefmut/anti-HPV16-E7 scFv chimeric product is efficiently uploaded in EVs, binds HPV16-E7, and inhibits the proliferation of HPV16-E7-expressing cells. Most important, challenge with cell-free EVs incorporating the Nefmut/anti-HPV16-E7 scFv led to the inhibition of proliferation of HPV16-E7-expressing cells. The proliferation of these cells was hindered also when they were co-cultured in transwells with cells producing EVs uploading Nefmut/anti-HPV16-E7 scFv.ConclusionOur data represent the proof-of-concept for the possibility to target intracellular antigens through EV-mediated delivery of scFvs. This finding could be relevant to design novel methods of intracellular therapeutic interventions.

Estimating Dynamic Cellular Morphological Properties via the Combination of the RTCA System and a Hough-Transform-Based Algorithm.

The xCELLigence real-time cell analysis (RTCA) system has the potential to detect cellular proliferation, migration, cytotoxicity, adherence, and remodeling. Although the RTCA system is widely recognized as a noninvasive and efficient tool for real-time monitoring of cellular fate, it cannot describe detailed cell morphological parameters, such as length and intensity. Transforming growth factor beta(TGF-β) induced the epithelial–mesenchymal transition (EMT), which produces significant changes in cellular morphology, so we used TGF-β to treat A549 epithelial cells in this study. We compared it with lipopolysaccharide (LPS) and cigarette smoke extract (CSE) as stimulators. We developed an efficient algorithm to quantify the morphological cell changes. This algorithm is comprised of three major parts: image preprocessing, Hough transform (HT), and post-processing. We used the RTCA system to record the A549 cell index. Western blot was used to confirm the EMT. The RTCA system showed that different stimulators produce different cell index curves. The algorithm determined the lengths of the detected lines of cells, and the results were similar to the RTCA system in the TGF-β group. The Western blot results show that TGF-β changed the EMT markers, but the other stimulator remained unchanged. Optics-based computer vision techniques can supply the requisite information for the RTCA system based on good correspondence between the results.

Famotidine has a neuroprotective effect on MK-801 induced toxicity via the Akt/GSK-3β/β-catenin signaling pathway in the SH-SY5Y cell line

Schizophrenia cannot be treated sufficiently with existing antipsychotic drugs. Taken into account that increased Glycogen Synthase Kinase 3 Beta (GSK-3β) activity is associated with schizophrenia pathophysiology and certain antipsychotics can be able to decrease GSK3β activity, inhibition of GSK-3β activity could be a novel approach for the treatment of schizophrenia. In the present study MK-801, a widely used chemical for the in vivo/in vitro modeling of schizophrenia was selected to evoke a detrimental effect on cellular survival via GSK3β and related proteins. A limited number of studies have reported the curative effects of famotidine, an antiulcer drug, in schizophrenic patients. To the best of our knowledge, no study investigated the molecular mechanism of the beneficial effect of famotidine in the patients. A recent study based on computerized drug modeling software (docking) indicated that famotidine might inhibit the GSK3β activity due to its chemical structure independent from histaminergic receptors. In this study, we aimed to investigate the effects of famotidine on the Akt/GSK-3β/β-catenin signaling pathway on SH-SY5Y neuroblastoma cells in the presence of MK-801. We investigated the effects of famotidine, olanzapine (an antipsychotic drug), and SB 415286 (specific GSK-3β inhibitor), on the basal cellular survival and MK-801 induced neuronal death beside of Akt/GSK-3β/β-catenin protein and gene expressions in SH-SY5Y cells. Cell viability, protein and gene expressions were determined by the real-time cell analysis (xCELLigence) system, western blotting and real-time polymerase chain reactions (Rt-PCR), respectively. Our findings suggested that MK-801 administration decreased cell survival probably via the increasing GSK-3β gene expression and activity in the SH-SY5Y cells. Pre-treatments with famotidine, olanzapine, and SB 415286 prevented MK-801 induced cell death via inhibitory effects on the MK-801 induced GSK-3β activity. Overall, the present results suggest that famotidine has a neuroprotective effect against MK-801 via modulation of the Akt/GSK-3β/β-catenin signaling pathway, an important mechanism in schizophrenia neurobiology.

Conditioned medium from asbestos-exposed fibroblasts affects proliferation and invasion of lung cancer cell lines.

The importance of the role of fibroblasts in cancer microenvironment is well-recognized. However, the relationship between fibroblasts and asbestos-induced lung cancer remains underexplored. To investigate the effect of the asbestos-related microenvironment on lung cancer progression, lung cancer cells (NCI-H358, Calu-3, and A549) were cultured in media derived from IMR-90 lung fibroblasts exposed to 50 mg/L asbestos (chrysotile, amosite, and crocidolite) for 24 h. The kinetics and migration of lung cancer cells in the presence of asbestos-exposed lung fibroblast media were monitored using a real-time cell analysis system. Proliferation and migration of A549 cells increased in the presence of media derived from asbestos-exposed lung fibroblasts than in the presence of media derived from normal lung fibroblasts. We observed no increase in proliferation and migration in lung cancer cells cultured in asbestos-exposed lung cancer cell medium. In contrast, increased proliferation and migration in lung cancer cells exposed to media from asbestos-exposed lung fibroblasts was observed for all types of asbestos. Media derived from lung fibroblasts exposed to other stressors, such as hydrogen peroxide and UV radiation didn’t show as similar effect as asbestos exposure. An enzyme-linked immunosorbent assay (ELISA)-based cytokine array identified interleukin (IL)-6 and IL-8, which show pleiotropic regulatory effects on lung cancer cells, to be specifically produced in higher amounts by the three types of asbestos-exposed lung fibroblasts than normal lung fibroblasts. Thus, the present study demonstrated that interaction of lung fibroblasts with asbestos may support the growth and metastasis of lung cancer cells and that chrysotile exposure can lead to lung cancer similar to that caused by amphibole asbestos (amosite and crocidolite).

Real-time cell analysis (RTCA) to measure killer cell activity against adherent tumor cells in vitro.

The enhancement of immune responses against tumor cells is a main focus of cancer immunotherapy. Immunotherapeutic approaches comprise a broad range of clinical applications including adjuvant therapies, check point inhibitors, cellular therapies, oncolytic viruses or targeted biologics such as bispecific antibodies. The usage of bispecific antibodies is one promising approach to enhance cytotoxicity and to selectively target effector cells to tumor-associated antigens. Here, we discuss the real-time cell analysis system as a suitable in vitro method to determine the interaction of tumor cell with effector cells alone or within a heterogeneous mixture of immune cells in peripheral blood or within tumor-infiltrating cells. The determination of cytotoxic effector cell activity using the real-time cell analyzer is highly useful to monitor the dynamic cellular interplay over extended periods of time.

Abstract 5264: Metabolic reprogramming with β-alanine to overcome chemotherapy resistance in pancreatic cancer

Abstract Background: Increased aerobic glycolysis (Warburg effect) leading to acidic microenvironment confers chemoresistance to weakly basic Gemcitabine, the standard treatment in Pancreatic ductal adenocarcinoma (PDAC). Metabolomic perturbations in two PDAC cell lines having differential sensitivity to Gemcitabine was investigated by profiling over 40 metabolites using 1H-NMR spectroscopy. Notably, a significant decrease in the concentration of β-alanine (βA) was observed in Gem resistant compared to Gem sensitive cell line, suggesting βA may have a role in chemotherapy response. We aimed to evaluate the role of βA supplementation in improving Gem efficacy in Gem resistant cell lines Methods: PDAC Panc-1 cell line extracts treated with Gem with and without βA supplementation were analysed via 1H-NMR spectroscopy. Aerobic glycolysis was quantified by measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) using the Seahorse XF analyser platform. Global mRNA profiling was done by Next Generation Sequencing. Proliferation, migration and cell cycle analysis was measured by Xcelligence real time cell analysis system and flow cytometry, respectively. PANC-1 and BxPC-3 tumor xenografts treated with Gemcitabine with and without βA for 4 weeks and the tumor volumes and weights were measured. Results: βA supplementation in Gem resistant cells results in: (1) decreased basal glycolytic rate, proton leak and ATP production; (2) increased pyruvate, decreased lactic acid and NAD+ concentrations with decreased LDH-A transcription; also increased carnosine levels with a more basic pH in the conditioned media; (3) enhanced sensitivity to Gem treatment, with a significant reduction in proliferation, cell migration, and increased apoptosis and relative hENt-1 mRNA levels and (5) xenograft tumor volumes and weights were significantly reduced upon βA supplementation. Conclusion: βA supplementation reprograms cell metabolism by normalizing energy production and reducing microenvironment acidification, thus enhancing chemo-sensitivity in Gem resistant PDAC cells and xenografts. βA can be potentially used as a co-therapeutic in PDAC. Note: This abstract was not presented at the meeting. Citation Format: Danny Yakoub, Smitha T. Totiger, Alexandra Moran, Sujit Suwal, Julio Pimentel, Lauren O'Donell, Jamie Walls, Nagaraj Nagathihalli, Nipun Merchant. Metabolic reprogramming with β-alanine to overcome chemotherapy resistance in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5264.

Modified CAR T cells targeting membrane-proximal epitope of mesothelin enhances the antitumor function against large solid tumor

Mesothelin (MSLN) is an attractive antigen for chimeric antigen receptor (CAR) T therapy and the epitope selection within MSLN is essential. In this study, we constructed two types of CARs targeting either region I of MSLN (meso1 CAR, also known as a membrane-distal region) or region III of MSLN (meso3 CAR, also known as a membrane-proximal region) using a modified piggyBac transposon system. We reported that, compared with meso1 CAR T cells, meso3 CAR T cells express higher levels of CD107α upon activation and produce increased levels of interleukin-2, TNF-α, and IFN-γ against multiple MSLN-expressing cancer cells in vitro. In a real-time cell analyzer system and a three-dimensional spheroid cancer cell model, we also demonstrated that meso3 CAR T cells display an enhanced killing effect compared with that of meso1 CAR T cells. More importantly, in a gastric cancer NSG mice model, meso3 CAR T cells mediated stronger antitumor responses than meso1 CAR T cells did. We further identified that meso3 CAR T cells can effectively inhibit the growth of large ovarian tumors in vivo. Collectively, our study provides evidences that meso3 CAR T-cell therapy performs as a better immunotherapy than meso1 CAR T-cell therapy in treating MSLN-positive solid tumors.

Ethyl Acetate Extract of Asclepias curassavica Induced Apoptosis in Human Cancer Cells via Activating p38 and JNK MAPK Signaling Pathways.

Background. Asclepias curassavica L. (Asclepiadaceae), as a traditional medicinal plant, is used as treatment for tumors in traditional Chinese and Indian medical practice. However, its underlying molecular mechanisms remain largely unresolved. The current study investigated its antitumor activity and the underlying molecular mechanisms. Method. Cell viability was detected by a real-time cell analysis system and MTT assay. Antitumor effect of ethyl acetate extract of Asclepias curassavica (EAAC) on NIC-H1975 tumors in vivo was assessed in BALB/c-nu/nu mouse. Apoptosis was measured using Hoechst33342 staining and Annexin V/PI-staining. Apoptosis-related proteins and MAPK signaling pathways were analyzed based on Western blot assay. Results. EAAC exhibited the highest cytotoxic activity in vitro than other polar parts. Meanwhile, EAAC could inhibit sensitive cell line NIC-H1975 proliferation in a concentration-dependent and time-dependent manner. Furthermore, EAAC had a significant inhibitory effect on NIC-H1975 tumor growth in BALB/c-nu/nu mouse. NIC-H1975 cells showed obvious apoptosis characteristics after EAAC treatment. Fas, caspase family members caspase 3, caspase 9, and caspase 8 showed dose-dependent induction by EAAC treatment, with increasing PARP cleavage. Additionally, EAAC significantly downregulated antiapoptotic proteins Bcl-2, XIAP, survivin, and Mcl-1 and upregulated proapoptosis proteins Bak, Bax, as well as activation of p38 and JNK MAPK signaling pathways. Moreover, inhibiting p38 and JNK MAPK by pharmacological inhibitors abrogated EAAC-induced apoptosis. Conclusion. Our data indicated that EAAC exerted potent antitumor effect both in vitro and in vivo by triggering the apoptotic pathway.

Ex Vivo Phenotyping and Potency Monitoring of CD19 CAR T Cells With a combined Flow Cytometry and Impedance-based Real-Time Cell Analysis Workflow

Abstract Cancer immunotherapy is increasingly utilized for cancer treatment. CARs (Chimeric antigen receptors) targeting CD19 have produced strong antitumor responses in hematologic malignancies, but tumor regression has seldom occurred when CARs targeting other antigens. The state of T-cell differentiation and T cell exhaustion influences the engraftment and persistence of CAR-expressing T cells following adoptive transfer. Here we report the use of a combined impendence-based Real-Time Cell Analysis (RTCA) and flow cytometry cell analysis workflow for ex vivo cytolytic potency monitoring of CD19 CAR T cell, as well as the examination of phenotypic and functional responses to antigen exposure over time. CD19 CAR T cell killing of HEK293 CD19 with different E:T ratio was monitored by RTCA system. Cytokine productions were measured by NovoCyte® Quanteon™ flow cytometer after 24 hrs. Co-incubation with HEK293 CD19, phenotypic and functional responses of CD19 CAR T cells towards exposure of different antigen levels were systematically examined over time. Both assays reveal that the CD19 CAR T cells show high dose-dependent killing activities against HEK293 CD19 but not against HEK293. Using the multi-color Flow Cytometry immunophenotyping, CD4 dim and CD8 dim populations are identified as effective killing cells which are enriched in central memory (CD45RA+CCR7−) cells while normal CD4+ or CD8+ T cells consist of predominant naïve-like (CD45RA+CCR7+) T cells after co-incubation with HEK293 CD19 cells. Exhaustion marker (PD-1, LAG-3, TIM-3) are overexpressed in normal CD4+ and CD8+ populations but not in CD4 dim and CD8 dim populations with CD19 antigen exposure. After antigen exposure, T cell activation levels change significantly.

Baicalein’in Farklı Meme Kanseri Hücre hatlarındaki Kinetik Etkilerinin Değerlendirilmesi

The aim of this study was to compare the antiproliferative effects of baicalein which is a flavonoide on MCF-7 and MDA-MB-231 cell line. The experiments were carried out with the evaluation of the parameters. Including cell viability, cell index, mitotic index, labelling index and apoptotic index. With the cell viability test, IC50 concentrations of baicalein for MCF-7 and MDA-MB-231 cells were determined as 10 µM and 30 µM, respectively and these concentrations were used in all experiments. The results showed that the IC50 concentrations decreased the values of cell viability, cell index, mitotic index and labelling index and increased the apoptotic index value for both cell types. These decreases and increases are statistically significant (p<0.01). In addition, curves of the graphs obtained from the xCelligence Real-Time Cell Analysis System showed that baicalein has cytoskeletal effects on MCF-7 cells and cytostatic effects on MDA-MB-231 cells. The results obtained from this study suggest that baicalein can be used in patients with luminal A and triple negative breast cancer in the clinic by supporting the future studies due to its antiproliferative effects on MCF-7 and MDA-MB-231 cells.

MicroRNA mimics that target the placental renin-angiotensin system inhibit trophoblast proliferation.

In early gestation, the human placental renin-angiotensin system (RAS) is upregulated and plays a role in placental development. Among other functions, signalling through the angiotensin II type 1 receptor (AT1R) initiates proliferation. Many microRNAs (miRNAs) targeting placental RAS mRNAs are downregulated at this time. We propose that in early gestation miRNAs that target the placental RAS are downregulated, allowing for the increased RAS expression and proliferation required for adequate placentation. HTR-8/SVneo cells (an immortalized human trophoblast cell line) were used to assess the effect of nine miRNA mimics (at 0.08, 0.16, 0.32 and 0.64 ng/μL) on trophoblast cell proliferation and predicted RAS target mRNAs. The effect of the miRNA mimics on the rate of cell proliferation was assessed using the xCELLigence real-time cell analysis system over 48 h. Levels of miRNAs and predicted RAS target mRNAs were determined by RT-PCR (qPCR, n = 9/group). Statistically different levels of expression were determined (P < 0.05). All nine miRNA mimics significantly affected the proliferation rates of HTR-8/SVneo cells. Five of the miRNA mimics (miR-181a-5p (predicted to target: renin (REN), angiotensin converting enzyme (ACE)), miR-378 (REN, ACE), miR-663 (REN), miR-483-3p (ACE, ACE2, angiotensinogen (AGT), angiotensin II type 1 receptor (AGTR1)) and miR-514 (AGT)) were associated with a dose-dependent reduction in cell proliferation. Seven of the mimics significantly decreased expression of at least one of their predicted target RAS mRNAs. Our study shows that miRNAs targeting placental RAS mRNAs play a role in controlling trophoblast proliferation. As placentation is largely a process of proliferation, changes in expression of these miRNAs may be partly responsible for the expression of the placental RAS, proliferation and placentation.

Fisetin induces autophagy in pancreatic cancer cells via endoplasmic reticulum stress- and mitochondrial stress-dependent pathways

Pancreatic cancer is one of the most aggressive tumors and patients have poor survival rates. Fisetin, a natural flavonoid, was recently reported to have antitumor effects in various cancer models. Autophagy is a conserved catabolic process that maintains cellular homoeostasis in response to stress, and together with apoptosis, determines cell fate. Herein, we examined the effect of fisetin on pancreatic cancer. We reveal that fisetin inhibits PANC-1 cell proliferation using a real-time cell analysis system. Moreover, the in vivo antitumor effect of fisetin was verified in pancreatic cancer using a luciferase-expressing murine xenograft pancreatic cancer model. We found that the AMPK/mTOR signaling pathway was enhanced after fisetin treatment; however, autophagy was not diminished by adding the AMPK inhibitor compound C. Thus, we hypothesized that an another autophagy regulating pathway existed. RNA-seq analysis revealed that the unfolded protein response pathway, which is activated by ER stress, was enriched. We also found that the stress-induced transcription factor p8 was increased in fisetin-treated PANC-1 cells, and that fisetin-induced autophagy was blocked by silencing p8. We revealed that p8-dependent autophagy was AMPK-independent, and that p8 regulated ATF6, ATF4, and PERK in response to ER stress via p53/PKC-α-mediated signaling. Furthermore, mitophagy was associated with Parkin and PINK1 in response to mitochondrial stress. Interestingly, ATF4 and ATF6 were increased in cells treated with fisetin and compound C. Moreover, inhibiting the AMPK/mTOR pathway with compound C may upregulate p8-dependent autophagy. Thus, there may be crosstalk between the AMPK/mTOR and p8-dependent pathways.

Limitations of Cross-Talk Between Osteosarcoma and Bone Marrow-Derived Mesenchymal Stem Cells

Objectives: Metastasis is a multi-step process which leads the tumor cells to escape from primer tumor region due to their need to gain malign phenotypes. While the effect of bone marrow-derived mesenchymal stem cells upon metastasis is not certain, some studies point out bone marrow-derived mesenchymal stem cells BM-MSCs to have this ability due to cell-cell interaction, released cytokines, and organization with the extracellular matrix in the micro-environment. Cross-talk via soluble factors also shifts the metastatic character.Patients and Methods: In this study, the effects of mesenchymal stem cells on tumor behavior by creating different microenvironments in 3-dimensional 3D in vitro cancer model is analyzed. The BM-MSCs and osteosarcoma cells were co-cultured via hanging-drop modeled 3D structure in normoxic and hypoxic conditions, and the cross-talk was modeled to measure their chemoattractant effects. The invasion and migration rates were measured with xCELLigence DP real-time cell analysis system. Mann Whitney U Test was used to compare independent samples. All P-values

Cytotoxic, apoptotic and cell migration inhibitory effects of atranorin on SPC212 mesothelioma cells

Objective: To investigate the effects of atranorin, a lichen secondary metabolite, on SPC212 malignant mesothelioma cells in vitro. Methods: SPC212 malignant mesothelioma cell line was used. 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide assay was used to evaluate cytotoxic effects of atranorin and cisplatin at 24, 48 and 72 h. Hematoxylin-eosin staining and 4’,6-diamidino-2-phenylindole, dihydrochloride staining were used for determining cell and nucleus morphology, respectively. Wound healing assay was used for investigating cell migration. The xCELLigence real-time cell analysis system was used for determining cell proliferation. Results: Atranorin at 5-450 μΜ decreased cell viability at 24, 48 and 72 h. IC50 values of atranorin were 300.94, 292.6 and 278.02 μM at 24, 48 and 72 h, respectively; meanwhile, the IC50 values of cisplatin were 128.00, 34.37 and 17.05 μM at 24, 48 and 72 h, respectively. Furthermore, atranorin disrupted cell and nuclear morphology with increasing concentrations. Atranorin significantly reduced cell migration by 38%, 37% and 35% at 300, 250 and 200 μM, respectively (P Conclusions: Atranorin has cytotoxic, antiproliferative, apoptotic and cell migration inhibitory effects on SPC212 malignant mesothelioma cancer cells.

Cytotoxicity of bracket identification dyes

To investigate the potential cytotoxicity of the bracket identification dyes commonly used in orthodontic fixed appliances. Six bracket brands representing the market in various aspects were selected. Ten sets of each bracket brand were acquired, and the identification dyes on them were scraped. They were tested for cytotoxicity at three different levels of concentration (2.5 mg/mL, 5 mg/mL, and 10 mg/mL), with the aid of a real-time cell analysis system. The results were compared within and between the groups. One-way analysis of variance and Tukey's post hoc test were used for statistical analysis. None of the six investigated dyes displayed cytotoxicity at the 2.5 mg/mL concentration. Of the investigated brands, three at 5 mg/mL and four at 10 mg/mL displayed cytotoxicity. Some of the identification dyes in this study did display cytotoxicity at the higher concentrations tested. Alternative methods for bracket identification should be considered.

Application of Real-Time Cell Electronic Analysis System in Modern Pharmaceutical Evaluation and Analysis.

Objective: We summarized the progress of the xCELLigence real-time cell analysis (RTCA) technology application in recent years for the sake of enriching and developing the application of RTCA in the field of Chinese medicine. Background: The RTCA system is an established electronic cellular biosensor. This system uses micro-electronic biosensor technology that is confirmed for real-time, label-free, dynamic and non-offensive monitoring of cell viability, migration, growth, spreading, and proliferation. Methods: We summarized the relevant experiments and literature of RTCA technology from the principles, characteristics, applications, especially from the latest application progress. Results and conclusion: RTCA is attracting more and more attention. Now it plays an important role in drug screening, toxicology, Chinese herbal medicine and so on. It has wide application prospects in the area of modern pharmaceutical evaluation and analysis.

The Effect of Flubendazole on Adhesion and Migration in SW480 and SW620 Colon Cancer Cells.

Colon cancer is the most common type of gastrointestinal cancer. Despite advances during the last two decades, the efficacy of colorectal cancer treatment is still insufficient and new anticancer agents are necessary. In our study, colon cancer cells derived from a primary tumor (SW480) and lymph node metastasis (SW620) from the same patient were used and compared. The effect of flubendazole (FLU) on cell adhesion and migration was monitored using the x-CELLigence Real-Time Cell Analysis system. Expressions of molecules involved in adhesion and migration were analyzed using RT-PCR and western blot. Furthermore, RNA silencing of nuclear factor-κB in SW620 cells was used to determine the involvement of the NF-κB p65 regulation pathway in FLU action. FLU significantly suppressed the adhesion of SW480 cells and reduced the expression of adhesion markers (ICAM-1, αE-catenin; β-catenin; integrin α5 and β1). Moreover, a significant anti-migratory potential of FLU was manifested in the SW620 cells. In addition, FLU suppressed the phosphorylation of NF-κB p65 and potentiated the suppression of several metastatic markers (ICAM-1, EpCAM, integrin α5, β1, α-tubulin) caused by NF-κB p65 silencing. FLU has a significant anti-migratory effect in intestinal cancer cell SW480 and its lymph node metastatic cells SW620. FLU decreases the expression of some proteins involved in metastatic processes and inhibits activation of NF-κB p65.

Chemical, biological and molecular modelling analyses to probe into the pharmacological potential of Antidesma madagascariense Lam.: A multifunctional agent for developing novel therapeutic formulations

Antidesma madagascariense Lam. (AM), an indigenous medicinal plant to the Mascarene Islands, is used for the treatment of several diseases. We endeavoured to validate its use via evaluating the kinetics of inhibition of crude aqueous extract (CAE) and crude methanol extract (CME) of AM against key metabolic enzymes (pancreatic lipase, cholesterol esterase [CEase], acetylcholinesterase [AChE], and urease). In vitro antiglycation, antioxidant, cytotoxicity using iCELLigence real time cell analysis system and WST-1 methods, were used. LC–ESI–MS/MS was employed to determine the phenolic composition of the extracts and interaction of selected compounds to the studied enzymes was determined using in silico docking. AChE was inhibited by the CME of AM and CEase by the CAE. Both extracts were active inhibitors of urease and pancreatic lipase. Hyperoside (271.97 μg/g extract), present in large amount in the CME, docked to the enzymatic pocket of urease and CEase. The extracts showed competitive and mixed inhibition of urease and pancreatic lipase, respectively. The antioxidant capacity of the CME (6.61 μg GAE/mg crude extract) was higher compared to CAE (2.20 μg GAE/mg crude extract). AM extracts were significantly (p < 0.05) less potent than aminoguanidine in preventing advanced glycation end products formation. Toxicological screening revealed that both extracts were non-toxic on HEK-293 cells. AM crude extracts at concentrations ranging from 78 to 312 μg/ml did not cause a visible change in cell morphology compared to control. This study supports the safe use of AM as a biomedicine for the management and/or treatment of common non-communicable diseases.

CLEC3B is downregulated and inhibits proliferation in clear cell renal cell carcinoma.

Dysregulation of C-Type Lectin Domain Family 3 Member B (CLEC3B) in serum or tumor tissues has been reported in patients with various cancer types. However, the expression and function of CLEC3B in clear cell renal cell carcinoma (ccRCC) remain unknown. To examine the function of CLEC3B in ccRCC, The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were examined to determine the expression of CLEC3B at the transcriptional level and it was demonstrated that CLEC3B mRNA was significantly downregulated in ccRCC compared with normal tissues (P<0.0001 and P=0.0392 in TCGA and GEO databases, respectively). The downregulation of CLEC3B was further validated at the protein level in 78.9% of ccRCCs by immunohistochemistry. To investigate the potential genetic mechanism for CLEC3B downregulation in ccRCC, copy number analysis was performed by profiling the copy number variation data from the TCGA project and it was revealed that the copy number loss of CLEC3B was prevalent in up to 88.1% of patients with ccRCC. CLEC3B genetic deletion was coupled with the well-known genetic loss of the von Hippel-Lindau tumor suppressor, which is a characteristic oncogenic event during ccRCC carcinogenesis. The downregulation of CLEC3B was associated with tumor progression and predicted unfavorable prognostic outcomes in the TCGA cohort. Real-time cell analyzer system technology revealed that CLEC3B inhibited the proliferation of ccRCC cell lines in vitro and that the mitogen-activated protein kinase pathway may contribute to this process. CLEC3B demonstrated substantial positive associations with proliferation inhibitors, but inverse associations with proliferation inducers and markers in two large ccRCC cohorts, suggesting that CLEC3B was able to identify ccRCCs with a lower proliferation capacity. In conclusion, the results of the present study propose that CLEC3B is a promising target for therapeutic intervention in ccRCC.

PO-179 Identification of C-Met and insulin receptor heterodimer as a mediator for hepatocellular carcinoma

IntroductionHepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths and obesity is predicted to be the leading cause for HCC in the following decades. Hyperglycemia and hyperinsulinemia are the two obesity-related factors associated with poor prognosis and survival. Hepatocyte growth factor (HGF) receptor, c-Met overactivation is common and results in higher proliferation and metastasis of HCC cells and drug resistance. In HCC patients, c-Met activation is often observed in the absence of HGF.Material and methodsCells were starved for 24 hours before hyperglycemic (HG, 25 mM) and/or hyperinsulinemic (HI, 100 nM) induction. Lysates were analysed by western blotting using the antibodies detecting activatory phosphorylations of c-Met, IR, Erk 1/2 and Akt. Receptor dimerization was studied by coimmunoprecipitation and colocalization using live-cell imaging. c-Met(SU11274) and IR(HMPA-(AM)3) inhibitors were used to reverse the effects of activations. Tumour cell metabolism was analysed using Seahorse Analyzer with cells expressing predominantly either IR isoform A or B. Proliferation and adhesion capacities were investigated using real time cell analysis system xCELLigence for 96 hours. Tumour cell survival in HG/HI blood circulation was simulated by circulation under shear stress for 4 hours in a fluidic system. Migration and invasion are studied using Boyden chamber.Results and discussions c-Met expression and activation was elevated when induced by high glucose/insulin. Also c-Met and IR were found to form a heterodimer upon HG/HI induction which explains a mechanism of c-Met activation even when HGF is absent in the tumour microenvironment. Both IR isoforms A/B were found to colocalize with c-Met and mediate the effect. In the presence of inhibitors, receptor activations and colocalizations were disrupted. c-Met inhibition resulted in a shift in tumour cell metabolism; diminished glycolysis and oxidative phosphorylation, which offers a role for rendering the cells more aggressive. Elevated adhesion, proliferation, migration, invasion and survival were observed upon induction which are controlled by Erk 1/2 and Akt activations, reversed by c-Met inhibition.ConclusionHere we identified that obesity-related prognostic factors, hyperglycemia and hyperinsulinemia, increase invasive and metastatic capacity of HCC cells through transactivation of c-Met by IR, which might also be a mechanism for drug resistance and offer an approach for targeted therapy.