Real-time Cell Analysis System Research Articles

Abstract 1123: TRAF2 and NCK-interacting protein kinase (TNIK) regulates cancer stemness and adipogenesis of osteosarcoma cells

Abstract [Introduction] Osteosarcoma (OS) is a rare malignant bone tumor and affects predominantly adolescents and young adults. There is no molecular targeted therapy for OS and breakthrough intervention has long been awaited for decades. Disturbances in differentiation towards mature osteoblast from mesenchymal stem cell are related to OS pathogenesis. Wnt signaling is a key component in managing this differentiation process. We previously reported that TNIK was a positive regulator of Wnt signaling. To develop the first molecular targeted therapy for OS patients, we focused on Wnt pathway-targeted therapy in OS and initiated this study to demonstrate TNIK as a novel therapeutic target for OS treatment. [Methods] Eleven different osteosarcoma cell lines were used in the present study. Twenty clinical samples were obtained at biopsy and surgical resection from ten OS patients. TNIK expression was assessed by western blotting, immunofluorescence and immunohistochemical staining. The generation of the novel TNIK inhibitor was previously described (Masuda et al., 2016). Cells were cultured with TNIK inhibitor or siTNIK. Cell viability was evaluated by real-time cell analysis system or ATP quantitation assay. The effect of TNIK inhibitor in vivo was also assessed by a mouse xenograft model. RNA-seq was performed using the RNA extracted from vehicle or TNIK inhibitor-treated OS cells. Cancer stem cell (CSC)-like properties were assessed by soft-agar colony-formation assay, limiting dilution assay, ALDH activities and the expression of CSC markers. Adipogenic phenotypes were qualitatively and quantitatively analyzed by Oil red O staining and the expression of adipogenesis markers. [Results] TNIK was identified in all cell lines and most of cases. Immunohistochemistry of OS tissue microarray also revealed that 52 out of 55 OS samples were positive for TNIK protein. TNIK inhibitor suppressed OS cell proliferation both in vitro and in vivo. SiTNIK also inhibited OS cell growth. Global gene expression analysis revealed a significant decrease in the expression of the genes relating to Wnt signaling pathway and maintenance of stem cell pluripotency. Soft-agar colony-formation assay and limiting dilution assay revealed that TNIK inhibitor decreased sphere formation activities in OS cells. Accordingly, TNIK inhibitor significantly suppressed ALDH activities and the expression of the proteins that are involved in the maintenance of CSCs, including NANOG, SOX2, OCT4A and MYC. Concomitantly, TNIK inhibitor drove adipogenic transdifferentiation of OS cells with PPARG activation. TNIK knock-down U2OS cells created by shRNAs showed nearly identical results. [Conclusion] We present that TNIK inhibition promotes adipogenic transdifferentiation of OS cells and eliminates CSCs. Our study thus suggests the feasibility of TNIK as a target for OS treatment. Citation Format: Toru Hirozane, Mari Masuda, Naoko Goto, Teppei Sugano, Naofumi Asano, Eisuke Kobayashi, Keisuke Horiuchi, Hideo Morioka, Akira Kawai, Masaaki Sawa, Morio Matsumoto, Masaya Nakamura, Tesshi Yamada. TRAF2 and NCK-interacting protein kinase (TNIK) regulates cancer stemness and adipogenesis of osteosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1123.

Abstract 1760: The hexavalent CD40 agonist HERA-CD40L augments multi-level crosstalk between T cells and antigen-presenting cells

Abstract Introduction: HERA-CD40L is a novel hexavalent CD40 agonist engineered with the HERA-Technology developed by Apogenix. We have previously shown that the natural binding mode via the receptor/ligand binding domains and the high clustering capacity for the cognate receptor clearly distinguish HERA-CD40L from other, e.g. antibody-based, CD40-targeting compounds. Here, we report on the effects of HERA-CD40L on crosstalk between T cells and antigen presenting cells (APC) and the functional consequences in vitro. Materials & Methods: Biological activity of CD40 agonists was analyzed using co-cultures of primary T cells with B cells or monocytes/macrophages. All primary cells were isolated by negative selection using magnetic sorting from healthy donor buffy coats. Expression of CD markers upon CD40 ligation on B cells and monocytes was analyzed by flow cytometry (FC). Monitoring of T cell-induced killing of tumor cells primed in direct co-cultures with APC was done on a real-time cell analysis system (xCELLigence). For analysis of phagocytosis, we developed an FC-based assay employing primary monocytes/macrophages and Jurkat A3 cells. Results: Treatment of primary B cells and monocytes with HERA-CD40L induced expression of co-stimulatory molecules, like CD86, and promotes M1 maturation of naïve (M0) monocytes. In vitro, treatment of alternatively activated M2 macrophages with HERA-CD40L induced an M2 to M1 phenotype switch (re-programming) which concurs with CD16 downregulation and a dose-dependent decrease of phagocytic activity of re-programmed macrophages compared to M0 or M2 macrophages. Primary B cells and M1 macrophages enhanced the proliferation and cytotoxic activity of naïve T cells in direct co-cultures in the presence of HERA-CD40L. The activating effect on T cells required direct cell-cell contact with APC and was not observed in indirect co-cultures. Functionally, neutralization of either MHC-I or CD80/CD86 in direct co-cultures inhibited full activation of the T cells in vitro as shown by kill assays with various tumor cell lines. Conclusion: The hexavalent CD40 agonist HERA-CD40L produced by the Apogenix HERA-Technology is a potent immune-regulator acting on B cells and myeloid cells. HERA-CD40L promotes activation of B cells, maturation of APC and induces an M2 to M1 phenotype switch which inhibits tolerance-inducing phagocytic activity of the repolarized macrophages in vitro. In response to CD40 ligation on APC, an efficient anti-tumor response is conferred to primary T cells through cell-cell interactions via MHC-I and CD80/CD86. Citation Format: Christian Merz, Jaromir Sykora, Viola Marschall, David M. Richards, Meinolf Thiemann, Harald Fricke, Oliver Hill, Christian Gieffers. The hexavalent CD40 agonist HERA-CD40L augments multi-level crosstalk between T cells and antigen-presenting cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1760.

Synthesis of Biotinylated 2-methoxystypandrone and Identification of JAK2 and IKK as its Targets.

2-Methoxystypandrone (2-MS), isolated from the roots of Polygonum cuspidatum, is a potent dual inhibitor of the STAT3 and NF-κB pathways. To investigate the molecular targets and mechanisms of 2-MS. A biotin-conjugated 2-MS analog, named 2-MS-Biotin, was designed and synthesized. The effects of 2-MS-Biotin on the STAT3 and NF-κB pathways were examined by Western blotting. The cytotoxicity of 2- MS-Biotin was evaluated using real-time cell analysis system. Proteins directly bound to 2-MS-Biotin were pulled down through streptavidin agarose beads and were detected using Western blotting. 2-MS-Biotin retained the inhibition activities of the parent compound 2-MS on the STAT3 and NF-κB pathways as well as on cancer cell growth. Also, JAK2 and IKK proteins can be effectively pulled down by 2- MS-Biotin. Using 2-MS-Biotin as a tool, both JAK2 and IKK were identified as the targets of 2-MS.

The Effect of Folic Acid- and Caffeic Acid-Functionalized SPION on Different Cancer Cell Lines

In this study, caffeic acid (CFA)- and folic acid (FA)-conjugated superparamagnetic iron oxide bio-nanocomposite (SPION@PEG-FA-@CFA) was prepared and its cytotoxic and apoptotic effects against murine fibroblast (L929), glioblastoma (U87), adenocarcinoma, human breast cancer (MCF-7), epithelioid cervix carcinoma (HeLa), and lung carcinoma (A549) have been determined. A real-time cell analyzer system (RTCA) for cytotoxicity, annexin assay and TUNEL, caspase 3/7 expression level, and caspase 3/7 activities for apoptosis were used. The RTCA showed that the cytotoxic effects of the bio-nanocomposite were higher in U87, MCF-7, and HeLa cells (which are folic acid-positive cells (FAR (+))). TUNEL, annexin, and caspase assay results indicated that the bio-nanocomposite caused apoptotic and necrotic impacts on cells. Necrotic and apoptotic cell ratios in FAR-positive cells (L929 and A549) were lesser than those in U87, MCF-7, and HeLa cells (FAR (+)). Therefore, the obtained results showed that the bio-nanocomposite can be used as a nano-medicine for biomedical application (cancer treatments). Moreover, this work can also be useful for designing other functionalized materials which could act as biomaterial. This study will be also informative for the researchers to design novel materials for biomedical applications.

Spine-bellied sea snake (Hydrophis curtus) venom shows greater skeletal myotoxicity compared with cardiac myotoxicity

For the first time the impedance-based xCELLigence real-time cell analysis system was used to measure the myotoxicity of sea snake venom. With a focus on the spine-bellied sea snake (Hydrophis curtus), the venom of four sea snake species and three terrestrial snake species were compared for myotoxicity against a human skeletal muscle cell line (HSkMC). Hydrophis curtus venom was also tested on a human cardiac muscle cell line (HCM). Surprisingly, all four sea snake venoms tested on HSkMC produced an initial 100–280% rise in xCELLigence cell index that peaked within the first two hours before falling. The cell index rise of H. curtus venom was correlated with the WST-1 cell proliferation assay, which demonstrated an increase in mitochondrial metabolism. The myotoxicity of H. curtus was 4.7–8.2 fold less potent than the other sea snakes tested, the Australian beaked sea snake (Hydrophis zweifeli), the elegant sea snake (Hydrophis elegans) and the olive sea snake (Aipysurus laevis). If our cell-based results translate to H. curtus envenomations, this implies that H. curtus would be less myotoxic than the other three. Yet the myotoxicity of H. curtus venom to cardiac muscle cells was nine times weaker than for skeletal muscle cells, providing evidence that the venom has a selective effect on skeletal muscle cells. This evidence, combined with the slow-acting nature of the venom, supports a digestive role for sea snake myotoxins.

Oleanolic Acid, a Novel Endothelin A Receptor Antagonist, Alleviated High Glucose-Induced Cardiomyocytes Injury.

Endothelin-1 (ET-1) and its receptor endothelin A receptor (ET[Formula: see text] have been shown to be upregulated in a high glucose environment, which increase the incidence of diabetes-related heart failure. Our previous study demonstrated that oleanolic acid (OA), a natural compound found in Chinese herbs had ET-1 antagonistic effects. We aimed to verify whether OA could ameliorate diabetes mellitus (DM)-induced injury in cardiomyocytes by reducing the antagonistic effects of the ET-1 pathway. For the induction of high glucose-related injury in cardiomyocytes, neonatal rat ventricular cardiomyocytes (NRVMs) were subjected to culture medium containing 25[Formula: see text]mM of glucose. Natriuretic peptide B (BNP), mitochondrial membrane potential (MMP) and cell surface area were measured to evaluate the severity of NRVMs injury. mRNA expression of ET-1 and ETA was determined using quantitative PCR. Moreover, a Ca[Formula: see text] influx assay was used to evaluate potential ETA antagonistic effects. Molecular docking of OA and ETA was performed using the Sulflex-Dock program. Human induced pluripotent stem cell (iPS-C)-derived cardiomyocytes and real time cell analysis system (RTCA) were used to verify the effect of OA on the ET-1 pathway. High glucose levels increased the expression of BNP at both mRNA and protein levels in cardiomyocytes. Moreover, cell surface area and MMP were also elevated in a high glucose environment. High glucose-induced injury in NRVMs was not reversible by hypoglycemic therapy. In addition, ETA was upregulated by high glucose treatment and levels could not be reduced by hypoglycemic treatment. The Ca[Formula: see text] influx assay on ETA/HEK293 cells showed that OA had a partial ETA antagonistic effect. Molecular docking approaches showed that OA was docked into the active site of ETA. Furthermore, functionality tests based on iPS-C and RTCA demonstrated that treatment with OA could reverse ET-1-induced alternation of beating rates and amplitude. Thus, OA could reverse high glucose-induced BNP upregulation, and increased both the cell area and MMP in NRVMs. High glucose-induced irreversible ETA upregulation is a major reason of continuous diabetes-related injury in cardiomyocytes. Treatment with OA had a protective effect on high glucose-induced injury in cardiomyocytes through a partial ETA antagonistic role.

Functional analysis of ESM1 by siRNA knockdown in primary and metastatic head and neck cancer cells

Genetic factors play a large role in cancer, and thus, there is a great desire to understand the effects of different genes in cancer and to also develop gene therapy for better treatments. Therefore, the development of alternative diagnosis and therapy modalities is of utmost importance. The aim of our study was to illuminate the role of ESM1 (endothelial cell-specific molecule-1, also known as Endocan) in proliferation and migration of head and neck cancer, thus helping to pave the way for new treatment modalities and predictive biomarkers. ESM1 expression was shown with immunofluorescence assay using confocal laser scanning microscope in primary and metastatic head and neck cancer cells. ESM1 expression was knocked down by RNA interference in head and neck cancer cells. Knockdown efficiency was evaluated by quantitative real-time RT-PCR and Western blot. Cell proliferation and migration assays were performed by xCELLigence real-time cell analysis system. Immunofluorescence assay showed nuclear localization and high expression of ESM1 in primary and metastatic head and neck cancer cells. ESM1 mRNA and protein levels were significantly decreased in ESM1-knockdown cells compared to control. ESM1-knockdown cells showed reduced proliferation and migration activity when compared to control cells. These findings suggest that ESM1 has roles on proliferation and migration of head and neck cancer cells.

A novel lnc-PCF promotes the proliferation of TGF-\u03b21-activated epithelial cells by targeting miR-344a-5p to regulate map3k11 in pulmonary fibrosis

Emerging evidence suggests that microRNA (miRNA) and long noncoding RNA (lncRNA) play important roles in disease development. However, the mechanism underlying mRNA interaction with miRNA and lncRNA in idiopathic pulmonary fibrosis (IPF) remains unknown. This study presents a novel lnc-PCF that promotes the proliferation of TGF-β1-activated epithelial cells through the regulation of map3k11 by directly targeting miR-344a-5p during pulmonary fibrogenesis. Bioinformatics and in vitro translation assay were performed to confirm whether or not lnc-PCF is an actual lncRNA. RNA fluorescent in situ hybridization (FISH) and nucleocytoplasmic separation showed that lnc-PCF is mainly expressed in the cytoplasm. Knockdown and knockin of lnc-PCF indicated that lnc-PCF could promote fibrogenesis by regulating the proliferation of epithelial cells activated by TGF-β1 according to the results of xCELLigence real-time cell analysis system, flow cytometry, and western blot analysis. Computational analysis and a dual-luciferase reporter system were used to identify the target gene of miR-344a-5p, whereas RNA pull down, anti-AGO2 RNA immunoprecipitation, and rescue experiments were conducted to confirm the identity of this direct target. Further experiments verified that lnc-PCF promotes the proliferation of activated epithelial cells that were dependent on miR-344a-5p, which exerted its regulatory functions through its target gene map3k11. Finally, adenovirus packaging sh-lnc-PCF was sprayed into rat lung tissues to evaluate the therapeutic effect of lnc-PCF. These findings revealed that lnc-PCF can accelerate pulmonary fibrogenesis by directly targeting miR-344a-5p to regulate map3k11, which may be a potential therapeutic target in IPF.

ICELLigence real-time cell analysis system for examining the cytotoxicity of drugs to cancer cell lines.

The recently developed iCELLigence™ real-time cell analyzer (RTCA) can be used for the label-free real-time monitoring of cancer cell proliferation, viability, invasion and cytotoxicity. The RTCA system uses 16-well microtiter plates with a gold microelectrode biosensor array that measures impedance when cells adhere to the microelectrodes causing an alternating current. By measuring the electric field generated in this process, the RTCA system can be used for the analysis of cell proliferation, viability, morphology and migration. The present review aimed to summarize the working method of the RTCA system, in addition to discussing the research performed using the system for various applications, including cancer drug discovery via measuring cytotoxicity.

Luteolin-Loaded Spion as a Drug Carrier for Cancer Cell In Vitro

The aim of this study is to determine the cytotoxic and apoptotic effects of newly synthesized folic acid (FA) and luteolin (carboxylated luteolin (CL)) conjugated superparamagnetic nanoparticle (SPION@FA-PEG@CL) on folate receptor positive (FAR (+)) and negative (FAR (−)) cell lines in vitro. Cytotoxicity analysis by real-time cell analyzer system (RTCA) and apoptosis by TUNEL and Annexin assay, Caspase 3/7 activities, and Caspase 3/7 expression level were determined. According to RTCA results, SPION@FA-PEG@CL nanodrug showed higher cytotoxicity against U87, MCF-7, HeLa cell lines in comparison with L929 and A549 cell lines. TUNEL and Annexin assays indicated that SPION@FA-PEG@CL increased the apoptotic and necrotic cell ratio in FAR (+) cell. In L929 and A549 cells, are FAR (−) cell lines apoptotic and necrotic cell ratio was lower than FAR (+) cell lines (U87, MCF-7 and HeLa). Caspase 3 and 7 analyses also showed that SPION@FA-PEG@CL nanodrug have apoptotic and necrotic impact on cell lines, they were higher in FAR (+) cells than FAR (−) cells. According to results, synthesized SPION@FA-PEG@CL can be a novel nanodrug delivery system for in cancer chemotherapy.

Abstract 1688: HERA-CD40L: A novel hexavalent CD40 agonist with superior biological activity

Abstract Introduction: Targeted therapeutics for cancer treatment are mostly developed as antibodies, however, the natural signaling complexes of the members of the TNF superfamily and their receptors consist of clusters of trimers. Consequently, most of these bivalent agonistic antibodies depend on Fc receptor mediated crosslinking for biological activity. The HERA-Technology developed by Apogenix generates hexavalent fusion proteins targeting the TNF-receptor superfamily with high clustering capacity for the cognate receptor, which overcomes this disadvantage of antibody-based drugs. Here we compared the efficacy of different CD40 agonist formats, including the novel HERA-CD40L and the functional consequences of differential receptor clustering. Materials & Methods: Biological activity of CD40 agonists was compared using an engineered reporter cell line and by flow cytometric analysis of CD40-induced IκBα degradation in Ramos B cells. T lymphocytes and monocytes were isolated from buffy coats and expression of CD markers upon CD40 ligation was analysed by multicolor flow cytometry (MC-FC). Secretion of cytokines in response to CD40 ligation was determined by ELISA. Monitoring of T cell-induced killing of tumor cells in direct co-cultures employed a real-time cell analysis system (xCELLigence). Results: Direct comparison of bivalent CD40 antibodies with trivalent CD40L and the hexavalent HERA-CD40L in two independent bioactivity assay formats demonstrated that only the hexavalent agonist was fully active without additional crosslinking. In contrast to HERA-CD40L, neither the bivalent agonistic CD40 antibody nor the trivalent CD40L were able to upregulate expression of activation markers on B cells or to induce secretion of proinflammatory cytokines such as IL-12 and TNFα by PBMCs. In vitro generated M2-macrophages acquired an M1 phenotype and enhanced proliferation of naïve CD4+ T cells in direct co-culture. Similarly, direct co-culture of CD4+ T cells with Ramos B cells in the presence of HERA-CD40L induced cytotoxic activity of CD4+ cells against tumor cells. The activating effect was dependent on cell-cell contacts and was not observed in indirect co-cultures. Importantly, only the hexavalent HERA-CD40L showed full biological activity without additional crosslinking. Conclusion: The hexavalent CD40 agonist HERA-CD40L produced by Apogenix HERA-Technology platform triggers CD40 signaling on B cells and cells of the monocytic lineage, leads to direct cytolytic activation and proliferation of CD4+ T cells and shifts the M1/M2 balance towards proinflammatory conditions. Unlike bivalent CD40 antibodies or trivalent CD40L_ based agonists, the hexavalent HERA-CD40L forms highly clustered signaling complexes and thus exhibits superior biological activity over other agonistic formats without the need for Fc receptor mediated crosslinking. Citation Format: Christian Merz, Jaromir Sykora, Meinolf Thiemann, Viola Marschall, Karl H. Heinonen, Harald Fricke, Christian Gieffers, Oliver Hill. HERA-CD40L: A novel hexavalent CD40 agonist with superior biological activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1688. doi:10.1158/1538-7445.AM2017-1688

Toll-like receptor 5 agonist CBLB502 induces radioprotective effects in vitro.

CBLB502 derived from Salmonella flagellin is a novel agonist of Toll-like receptor 5 (TLR5). It has been shown that CBLB502 can exert high radioprotective efficacy on mice and primates from both GI and hematopoietic syndromes during whole-body irradiation with low toxicity and immunogenicity. However, no effective system has been used to investigate the protective effect of CBLB502 against irradiation and the related mechanism in vitro. In this study, we investigated the radioprotective properties of CBLB502 in HEK293-N-T cells constitutively expressing human TLR5 and NF-κB-dependent luciferase. HEK293-N-T cells were treated with different doses of CBLB502 prior to 60Co-γ ray irradiation. After irradiation, cell viability was real-time measured for 4 days by using the real-time cell analysis system. We found that CBLB502 was capable of efficiently maintaining the survival rate of irradiated HEK293-N-T cells. Then apoptotic cell death and cell cycle were detected by flow cytometry. The results showed that CBLB502 pre-treatment could reduce the apoptosis and promote the recovery of irradiated HEK293-N-T cells from G2-phase arrest in a dose-dependent manner. Our data indicated that CBLB502 has a direct radioprotective effect in vitro via anti-apoptosis and promotes cell cycle recovery. The method developed here could be an effective in vitro system to screen other TLR5-target radioprotectants like CBLB502.

Dexmedetomidine attenuates hypoxia/reoxygenation injury in primary neonatal rat cardiomyocytes.

Systemic administration of dexmedetomidine provides cardioprotection against ischemia/reperfusion (I/R) injury; however, the direct effects of dexmedetomidine on cardiomyocytes have not been clarified. The present study investigated the effects of dexmedetomidine on primary neonatal rat cardiomyocytes under hypoxic/reoxygenation (H/R) conditions. In order to simulate in vivo I/R injury, primary neonatal rat cardiomyocytes were cultured under hypoxic conditions for 1 h and subsequently reoxygenated for 24 h. The effects of preconditioning with dexmedetomidine 2 h before hypoxia and postconditioning during reoxygenation were also examined. Cellular viability and activity were analyzed by monitoring the dynamic response profile of living cells using a real-time cell analyzer system. A special scaled index, defined as the normalized cell index (NCI), was used to minimize the influence of inter-experimental variations. The dose-effect curve was generated from the area under the time-course curve values of NCI. H/R exposure markedly decreased cell viability and activity. Furthermore, no cytotoxicity was associated with a clinically relevant concentration of dexmedetomidine. Preconditioning with dexmedetomidine concentration-dependently ameliorated the reductions in NCI in cardiomyocytes following H/R injury. Additionally, postconditioning with dexmedetomidine improved the reductions in NCI at concentrations between 3 and 200 nM. Finally, the effect of 3–40 nM dexmedetomidine postconditioning was greater than preconditioning. These results indicated that preconditioning and postconditioning with dexmedetomidine attenuated H/R injury in primary neonatal rat cardiomyocytes at the cellular level.

Atractylenolide I stimulates intestinal epithelial repair through polyamine-mediated Ca2+ signaling pathway

BackgroundAn impairment of the integrity of the mucosal epithelial barrier can be observed in the course of various gastrointestinal diseases. The migration and proliferation of the intestinal epithelial (IEC-6) cells are essential repair modalities to the healing of mucosal ulcers and wounds. Atractylenolide I (AT-I), one of the major bioactive components in the rhizome of Atractylodes macrocephala Koidz. (AMR), possesses multiple pharmacological activities. This study was designed to investigate the therapeutic effects and the underlying molecular mechanisms of AT-I on gastrointestinal mucosal injury. MethodsScratch method with a gel-loading microtip was used to detect IEC-6 cell migration. The real-time cell analyzer (RTCA) system was adopted to evaluate IEC-6 cell proliferation. Intracellular polyamines content was determined using high performance liquid chromatography (HPLC). Flow cytometry was used to measure cytosolic free Ca2+ concentration ([Ca2+]c). mRNA and protein expression of TRPC1 and PLC-γ1 were determined by real-time PCR and Western blotting assay respectively. ResultsTreatment of IEC-6 cells with AT-I promoted cell migration and proliferation, increased polyamines content, raised cytosolic free Ca2+ concentration ([Ca2+]c), and enhanced TRPC1 and PLC-γ1 mRNA and protein expression. Depletion of cellular polyamines by DL-a-difluoromethylornithine (DFMO, an inhibitor of polyamine synthesis) suppressed cell migration and proliferation, decreased polyamines content, and reduced [Ca2+]c, which was paralleled by a decrease in TRPC1 and PLC-γ1 mRNA and protein expression in IEC-6 cells. AT-I reversed the effects of DFMO on polyamines content, [Ca2+]c, TRPC1 and PLC-γ1 mRNA and protein expression, and restored IEC-6 cell migration and proliferation to near normal levels. ConclusionOur data demonstrate that AT-I stimulates intestinal epithelial cell migration and proliferation via the polyamine-mediated Ca2+ signaling pathway. Therefore, AT-I may have the potential to be further developed as a promising therapeutic agent to treat diseases associated with gastrointestinal mucosal injury, such as inflammatory bowel disease and peptic ulcer.

Abstract B48: Structure to function: comparison of CD40 agonist formats reveals superior immune-modulating properties of hexavalent scCD40L-RBD-Fc fusion protein APG1233

Abstract Introduction: Initiation and perpetuation of anti-tumor responses of the immune system are in the focus of current approaches to fight cancer. Manipulation of the tumor microenvironment, e.g. shifting the M1/M2 ratio towards M1 is one goal to support immunologic cancer treatment. Here we examined and compared the functional effects of bivalent agonistic anti-CD40 antibodies, homo-trimeric trivalent CD40-Ligand and the novel hexavalent CD40-agonist APG1233 on the maturation and differentiation of primary monocytes, B cells and T cell mediated tumor cell killing. Materials and Methods: Following treatment with various CD40 agonists, cytokine secretion by monocytes, PBMCs and T cells from blood samples was assessed by ELISA. Monocytes isolated from healthy-donor blood samples were differentiated in vitro into either M1- or M2-type macrophages, or dendritic cells (DC) which was confirmed by multicolor flow-cytometry (MC-FC). Subsequently, we analyzed the respective M1- and M2-type macrophages and DCs regarding their ability to induce proliferation in a direct allogenic co-culture system with naïve CD4-positive T cells by a flow cytometry-based CFSE-assay. Macrophage plasticity, e.g. re-polarisation of M2-like to M1-like macrophages upon exposure to CD40 agonists was assessed by MC-FC. A real-time cell analysis system (Roche xCelligence RTCA DP) was used to attest T cell activation by CD40 stimulated B cells, resulting in killing of tumor cells in direct co-cultures. Results: Stimulation of CD40 on PBMCs, T cells and monocytes increased secretion of cytokines (e.g. IL-12, TNFαa, CCL4) dependent on the agonist format and moreover was strictly dependent on Fc-crosslinking when using agonistic anti-CD40 mAb. In vitro, when the hexavalent APG1233 was added to the cytokine cocktail during the in vitro differentiation process, the appearance of M1-type macrophages was substantially increased. Moreover, M2-macrophages underwent conversion and acquired M1-type surface markers after exposure to APG1233. Finally, in direct co-culture of the in vitro differentiated cell populations with naïve CD4+ T cells, M1-macrophages induced strong lymphocyte proliferation, while the induction by monocytes and M2-macrophages was low. On a functional level, T lymphocytes co-cultured with M1 or DC acquired direct cytotoxic activity against tumor cells in a real-time cell analysis assay. Similarly, induction of cytolytic activity of purified T cells in vitro required the presence of both CD40 expressing B cells and APG1233. Conclusion: Stimulation of CD40 on immune cells triggers development of anti-tumor responses, but efficacy of various agonist formats dramatically varies. Compared to trimeric CD40L formats and agonistic anti-CD40 mAbs, the novel hexavalent CD40 agonist APG1233 emerging from Apogenix HERA Technology platform excels as a strong inducer of B cell activation, M1-type macrophage differentiation and M2->M1 conversion. M1-macrophages generated in vitro are functional and enhance proliferation of naïve CD4+ T cells. CD40 stimulation on a CD40 expressing B cell line enhances its ability to activate T cells and trigger an anti-tumor response. Citation Format: Christian Merz, Jaromir Sykora, Thamara Beyer, Stefanie Knorn, Harald Fricke, Christian Gieffers, Oliver Hill, David M. Richards. Structure to function: comparison of CD40 agonist formats reveals superior immune-modulating properties of hexavalent scCD40L-RBD-Fc fusion protein APG1233. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B48.

Traditionally Used Lathyrus Species: Phytochemical Composition, Antioxidant Activity, Enzyme Inhibitory Properties, Cytotoxic Effects, and in silico Studies of L. czeczottianus and L. nissolia.

Members of the genus Lathyrus are used as food and as traditional medicines. In order to find new sources of biologically-active compounds, chemical and biological profiles of two Lathyrus species (L. czeczottianus and L. nissolia) were investigated. Chemical profiles were evaluated by HPLC-ESI-MSn, as well as by their total phenolic and flavonoid contents. In addition, antioxidant, enzyme inhibitory, and cytotoxic effects were also investigated. Antioxidant properties were tested by using different assays (DPPH, ABTS, CUPRAC, FRAP, phosphomolybdenum, and metal chelation). Cholinesterases (AChE and BChE), tyrosinase, α-amylase, and α-glucosidase were used to evaluate enzyme inhibitory effects. Moreover, vitexin (apigenin-8-C-glucoside) and 5-O-caffeoylquinic acid were further subjected to molecular docking experiments to provide insights about their interactions at molecular level with the tested enzymes. In vitro cytotoxic effects were examined against human embryonic kidney cells (HEK293) by using iCELLigence real time cell analysis system. Generally, L. czeczottianus exhibited stronger antioxidant properties than L. nissolia. However, L. nissolia had remarkable enzyme inhibitory effects against cholinesterase, amylase and glucosidase. HPLC-ESI-MSn analysis revealed that flavonoids were major components in these extracts. On the basis of these results, Lathyrus extracts were rich in biologically active components; thus, these species could be utilized to design new phytopharmaceutical and nutraceutical formulations.

Multiple pharmacological targets, cytotoxicity, and phytochemical profile of Aphloia theiformis (Vahl.) Benn.

Aphloia theiformis (Vahl.) Benn. (AT) is traditionally used in Sub-Saharan African countries including Mauritius as a biomedicine for the management of several diseases. However, there is a dearth of experimental studies to validate these claims. We endeavoured to evaluate the inhibitory effects of crude aqueous extract as traditionally used together with the crude methanol extracts of AT leaves on urease, angiotensin (I) converting enzyme (ACE), acetylcholinesterase (AChE), cholesterol esterase (CEase), glycogen phosphorylase a (GPa), and glycation in vitro. The crude extract showing potent activity against the studied enzymes was further partitioned using different solvents of increasing polarity. The enzyme inhibitory and antiglycation activities of each fraction was assessed. Kinetic of inhibition of the active crude extract/fractions on the aforementioned enzymes was consequently determined using Lineweaver-Burk plots. An ultra-high performance liquid chromatography (UHPLC-UV/MS) system was used to establish the phytochemical profile of AT. The real time cell analysis system (iCELLigence™) was used to monitor any cellular cytotoxicity of AT. Crude methanolextract (CME) was a potent inhibitor of the studied enzymes, with IC50 ranging from 696.22 to 19.73μg/mL. CME (82.5%) significantly (p<0.05) inhibited glycation and was comparable to aminoguanidine (81.5%). Ethyl acetate and n-butanol fractions of CME showed non-competitive, competitive, and uncompetitive mode of inhibition against ACE, CEase, and AChE respectively. Mangiferin, a xanthone glucoside was present in CME, ethyl acetate, and n-butanol fractions. Active extract/fractions were found to be non-cytotoxic (IC50>20μg/mL) according to the U.S National Cancer Institute plant screening program. This study has established baseline data that tend to justify the traditional use of AT and open new avenues for future biomedicine development.

Translationally controlled tumour protein TCTP is induced early in human colorectal tumours and contributes to the resistance of HCT116 colon cancer cells to 5-FU and oxaliplatin

BackgroundTranslationally controlled tumour protein TCTP is an anti-apoptotic protein frequently overexpressed in cancers, where high levels are often associated with poor patient outcome. TCTP may be involved in protecting cancer cells against the cytotoxic action of anti-cancer drugs. Here we study the early increase of TCTP levels in human colorectal cancer (CRC) and the regulation of TCTP expression in HCT116 colon cancer cells, in response to treatment with the anti-cancer drugs 5-FU and oxaliplatin.MethodsUsing immunohistochemistry, we assessed TCTP levels in surgical samples from adenomas and adenocarcinomas of the colon, compared to normal colon tissue. We also studied the regulation of TCTP in HCT116 colon cancer cells in response to 5-FU and oxaliplatin by western blotting. TCTP mRNA levels were assessed by RT-qPCR. We used mTOR kinase inhibitors to demonstrate mTOR-dependent translational regulation of TCTP under these conditions. Employing the Real-Time Cell Analysis (RTCA) System and the MTS assay, we investigated the effect of TCTP-knockdown on the sensitivity of HCT116 cells to the anti-cancer drugs 5-FU and oxaliplatin.Results1. TCTP levels are significantly increased in colon adenomas and adenocarcinomas, compared to normal colon tissue. 2. TCTP protein levels are about 4-fold upregulated in HCT116 colon cancer cells, in response to 5-FU and oxaliplatin treatment, whereas TCTP mRNA levels are down regulated. 3. mTOR kinase inhibitors prevented the up-regulation of TCTP protein, indicating that TCTP is translationally regulated through the mTOR complex 1 signalling pathway under these conditions. 4. Using two cellular assay systems, we demonstrated that TCTP-knockdown sensitises HCT116 cells to the cytotoxicity caused by 5-FU and oxaliplatin.ConclusionsOur results demonstrate that TCTP levels increase significantly in the early stages of CRC development. In colon cancer cells, expression of this protein is largely upregulated during treatment with the DNA-damaging anti-cancer drugs 5-FU and oxaliplatin, as part of the cellular stress response. TCTP may thus contribute to the development of anti-cancer drug resistance. These findings indicate that TCTP might be suitable as a biomarker and that combinatorial treatment using 5-FU/oxaliplatin, together with mTOR kinase inhibitors, could be a route to preventing the development of resistance to these drugs.

Lung cells support osteosarcoma cell migration and survival

BackgroundOsteosarcoma (OS) is the most common primary bone tumor, with a propensity to metastasize to the lungs. Five-year survival for metastatic OS is below 30%, and has not improved for several decades despite the introduction of multi-agent chemotherapy. Understanding OS cell migration to the lungs requires an evaluation of the lung microenvironment. Here we utilized an in vitro lung cell and OS cell co-culture model to explore the interactions between OS and lung cells, hypothesizing that lung cells would promote OS cell migration and survival. The impact of a novel anti-OS chemotherapy on OS migration and survival in the lung microenvironment was also examined.MethodsThree human OS cell lines (SJSA-1, Saos-2, U-2) and two human lung cell lines (HULEC-5a, MRC-5) were cultured according to American Type Culture Collection recommendations. Human lung cell lines were cultured in growth medium for 72 h to create conditioned media. OS proliferation was evaluated in lung co-culture and conditioned media microenvironment, with a murine fibroblast cell line (NIH-3 T3) in fresh growth medium as controls. Migration and invasion were measured using a real-time cell analysis system. Real-time PCR was utilized to probe for Aldehyde Dehydrogenase (ALDH1) expression. Osteosarcoma cells were also transduced with a lentivirus encoding for GFP to permit morphologic analysis with fluorescence microscopy. The anti-OS efficacy of Disulfiram, an ALDH-inhibitor previously shown to inhibit OS cell proliferation and metastasis in vitro, was evaluated in each microenvironment.ResultsLung-cell conditioned medium promoted osteosarcoma cell migration, with a significantly higher attractive effect on all three osteosarcoma cell lines compared to basic growth medium, 10% serum containing medium, and NIH-3 T3 conditioned medium (p <0.05). Lung cell conditioned medium induced cell morphologic changes, as demonstrated with GFP-labeled cells. OS cells cultured in lung cell conditioned medium had increased alkaline phosphatase staining.ConclusionsLung endothelial HULEC-5a cells are attractants for OS cell migration, proliferation, and survival. The SJSA-1 osteosarcoma cell line demonstrated greater metastatic potential than Saos-2 and U-2 cells. ALDH appears to be involved in the interaction between lung and OS cells, and ALP may be a valuable biomarker for monitoring functional OS changes during metastasis.

Evaluation of Antiangiogenic and Antimetastatic Effects of Penicillium chrysogenum Secondary Metabolites

Development of resistance to standard therapy and toxic side effects are the major challenges encountered in chemotherapy of colorectal cancer. Increasing evidence has shown the ability of natural products to overcome various challenging disorders. Since the development of antibiotics, fungi have been shown to be a wide useful source of lead compounds for the development of new pharmaceuticals. Fungal secondary metabolites from extreme environments are remarkable due to their diverse components, which makes them interesting candidates for drug discovery. In order to discover novel active antimetastatic and antiangiogenic compounds, secondary metabolites of halotolerant Penicillium chrysogenum-1(hPc-1) isolated from Tuz Lake in Turkey and ethylacetate extract of hPc-1 was prepared from the isolate of hPc-1 culture medium. Firstly, antioxidant activity of the extract was determined. Then cytotoxicity and cell proliferation were investigated by WST-1 assay and real-time cell analysis system-DP on human umbilical vein endothelial cells and colorectal carcinoma cells, respectively. Antiangiogenic effects on human umbilical vein endothelial cells were evaluated with cell migration and invasion assays by using real-time cell analysis system. Also mRNA expression levels of metastasis and angiogenesis related genes, VEGFA, VEGFB, EGFR, COX-10, ANGPT-1 and IL-8 were determined on both cell lines. Results indicated that hPc-1 extract exhibited significant antimetastatic and antiangiogenic effects. According to the real-time cell analysis system results, IC50 concentrations of hPc-1 extract on Caco2 and human umbilical vein endothelial cells were calculated as 55.2 and 37.89 µg/ml, respectively for 72 h. These results indicated that the secondary metabolites of hPc-1 extract could have the potential as a preventive and therapeutic agent in metastatic cancer processes.