Tumor Cells In Animals Research Articles

Galectins: a potential pharmacological target

Aim. To consider the use of galectin-1 and galectin-3 inhibitors as potential pharmacological targets in antitumor and antifibrotic therapy.The lecture includes the analysis of experimental research and review articles presented in the PubMed database. A brief description of the structure of galectins is given. Their generally accepted classification and features of the structure of the carbohydrate recognition domain in galectin-1 and galectin-3 are presented. The main part of the lecture describes the results of research on the development of carbohydrate-based (β-galactoside derivatives or analogues) and non-carbohydrate-based (peptide-based, carboxamide derivatives) inhibitors capable of interacting with galectin-1 and galectin-3.The results of experiments performed on animal models and tumor cell cultures demonstrate that the antitumor effect of galectin antagonists is realized through the suppression of proliferation and metastasis, activation of tumor cell apoptosis, and modulation of the antitumor immune response. Antagonists of galectin-1 and galectin-3 potentiate the effect of antitumor drugs and have an antifibrotic effect. Some of the compounds discussed in the lecture are undergoing clinical trials. The data presented in the lecture open up opportunities for the development and synthesis of new molecules of potential galectin-1 and 3 inhibitors.

Molecular-oriented therapy of lung tumors in animals with BRAF mutations – possibilities and limitations of the method

Lung cancer (LC) is malignant epithelial tumors that differ in origin, histological structure, clinical course, and treatment results. Lung cancer ranks first in terms of male morbidity in Russia, and in terms of mortality in the world. In animals, LC is diagnosed much less frequently, but with an increase in the number of animals kept in cities, the incidence of LC increases, since companion animals are exposed to the same risk factors as humans. We conducted a retrospective analysis of the medical records of 43 animals (28 cats and 15 dogs) admitted to the VEGA veterinary clinics for treatment in the period from 2020 to 2023. The criterion for the inclusion of animals in the study was a tumor process in the thoracic cavity and lungs, diagnosed by instrumental and laboratory methods. High-dose intravenous chemotherapy, which is used in humans, is ineffective in the treatment of LC in animals, therefore, it is necessary to search, develop and introduce into clinical practice targeted drugs that will be effective in animals. Since the most widely used group of targeted drugs in human medicine are low-molecular kinase inhibitors that act on the corresponding molecular target in the tumor, the development of molecular genetic methods available for practical use by veterinary oncologists for the study of tumor cells in animals is relevant in veterinary medicine. The choice of treatment methods and prognosis depend on the stage of the disease, therefore, the issue of introducing biomarkers of various types and stages of oncological processes into veterinary practice deserves research. Also, to improve the effectiveness of treatment, it is recommended to conduct research and develop a lung cancer staging scale for cats.

Dual Co-Stimulation with CD28 and ICOS Enhances T Cell Activation and Optimizes CAR-T Cell Antitumor Activity

Background: The Chimeric antigen receptor (CAR) T cell therapy for hematological malignancies has revolutionized anticancer therapy. CAR-T cell expansion and persistence are strikingly associated with the intrinsic programmes of T cells. However, the current process for CAR-T cells manufacturing resulted in high frequencies of exhausted and senescent CAR-T cells with reduced mitochondrial mass. T cell receptor (TCR) signaling cooperates with co-stimulatory signaling to regulate T cells activation and differentiation. Considering that ICOS has a well-defined role in T cell differentiation, especially with regard to the incorporation of ICOS into CAR to boost CAR-T cells antitumor activity, we therefore hypothesize that TCR activation in synergy with dual CD28 and ICOS co-stimulation rapidly triggers T cell activation and may be a potential approach to optimize CAR-T cells antitumor activity. Methods: In this study, we established a culture system using plate-coated CD3 in combination with CD28 and ICOS mAbs to simultaneously stimulate T cells, and further studied whether dual CD28 and ICOS co-stimulation could reprogram the transcriptional, epigenetic and proteomic states of T cells and optimize CAR-T cells antitumor activity. In vitro CAR-T cell phenotype and cytotoxicity were detected via flow cytometry, co-culture killing assay and degranulation assay. NSG mice were inoculated with Raji cells to establish xenograft mouse model, and the tumor load and survival time were dynamically monitored after treatment with CAR-T cells. Results: As expected, dual co-stimulation potently enhanced T cells activation and expansion via reprogramming their transcriptome, epigenome and proteome. Mechanistically, dual CD28 and ICOS co-stimulation rapidly upregulated the pathways such as ribosome, citrate cycle (TCA cycle) and oxidative phosphorylation (OXPHOS) to drive T cells exiting from quiescence. Moreover, metabolic pathways are rewired in CD28 and ICOS-stimulated T cells to guarantee sufficient energy for cell proliferation. Meaningfully, dual co-stimulation confers CAR-T cells with high lentiviral transfection efficiency, possibly correlating with highly open chromatin accessibility in CD28 and ICOS-stimulated T cells. CAR-T cells manufactured with dual CD28 and ICOS co-stimulation demonstrated significantly enhanced OXPHOS and mitochondrial biogenesis, resulting in a superior expansion and being more effective at killing tumor cells in immunodeficient animals. Conclusions: Collectively, our data indicated that activating CD28 and ICOS signaling during CAR-T cells preparation is a potential strategy for optimizing CAR-T cells antitumor activity.

Potential Effects of Geraniol on Cancer and Inflammation-Related Diseases: A Review of the Recent Research Findings.

Geraniol (GNL), a natural monoterpene, is found in many essential oils of fruits, vegetables, and herbs, including lavender, citronella, lemongrass, and other medicinal and aromatic plants. GNL is commonly used by the cosmetic and food industries and has shown a wide spectrum of pharmacological activities including anti-inflammatory, anticancer, antimicrobial, antioxidant, and neuroprotective activities. It represents a potential anti-inflammatory agent and a promising cancer chemopreventive agent, as it has been found to be effective against a broad range of cancers, including colon, prostate, breast, lung, skin, kidney, liver, and pancreatic cancer. Moreover, GNL scavenges free radicals and preserves the activity of antioxidant enzymes. In addition, GNL induces apoptosis and cell cycle arrest, modulates multiple molecular targets, including p53 and STAT3, activates caspases, and modulates inflammation via transcriptional regulation. In the present study, different modes of action are described for GNL's activity against cancer and inflammatory diseases. This compound protects various antioxidant enzymes, such as catalase, glutathione-S-transferase, and glutathione peroxidase. Experiments using allergic encephalomyelitis, diabetes, asthma, and carcinogenesis models showed that GNL treatment had beneficial effects with low toxicity. GNL has been shown to be effective in animal models and tumor cell lines, but there have not been any clinical studies carried out for it. The aim of the present review is to provide updated data on the potential effects of GNL on cancer and inflammation, and to enhance our understanding of molecular targets, involved pathways, and the possible use of GNL for clinical studies and therapeutic purposes in the treatment of cancer and inflammation-related diseases.

Fluorescence Labeling of Circulating Tumor Cells with a Folate Receptor-Targeted Molecular Probe for Diffuse In Vivo Flow Cytometry.

We recently developed a new instrument called "diffuse in vivo flow cytometry" (DiFC) for enumeration of rare fluorescently labeled circulating tumor cells (CTCs) in small animals without drawing blood samples. Until now, we have used cell lines that express fluorescent proteins or were pre-labeled with a fluorescent dye ex vivo. In this work, we investigated the use of a folate receptor (FR)-targeted fluorescence molecular probe for in vivo labeling of FR+ CTCs for DiFC. We used EC-17, a FITC-folic acid conjugate that has been used in clinical trials for fluorescence-guided surgery. We studied the affinity of EC-17 for FR+ L1210A and KB cancer cells. We also tested FR- MM.1S cells. We tested the labeling specificity in cells in culture in vitro and in whole blood. We also studied the detectability of labeled cells in mice in vivo with DiFC. EC-17 showed a high affinity for FR+ L1210A and KB cells in vitro. In whole blood, 85.4% of L1210A and 80.9% of KB cells were labeled above non-specific background with EC-17, and negligible binding to FR- MM.1S cells was observed. In addition, EC-17-labeled CTCs were readily detectable in circulation in mice with DiFC. This work demonstrates the feasibility of labeling CTCs with a cell-surface receptor-targeted probe for DiFC, greatly expanding the potential utility of the method for pre-clinical animal models. Because DiFC uses diffuse light, this method could be also used to enumerate CTCs in larger animal models and potentially even in humans.

Abstract 684: A semi-mechanistic platform model to capture individual animal responses to checkpoint inhibitors in a syngeneic mouse model

Abstract Syngeneic mouse models have been widely employed in preclinical discovery of checkpoint inhibitors as they enable study of drug impact on the intact immune system (Lechner, 2013; Murphy, 2015). However, the interpretation of such studies remains challenging partly due to the large variability in individual animal responses to drug treatment. In this work, we describe the generation of a model platform that captures essential aspects of the pharmacokinetics, cellular and tumor growth effects of murine surrogates of two checkpoint therapeutic antibodies, anti-PD1 and anti-CTLA4, in the CT26 syngeneic tumor model. The model describes individual animal responses with regard to drug exposure, key intra-tumoral cell kinetics and tumor volume changes and provides biologically plausible explanations for the observed differences between good and poor responders to treatment with anti-PD1 or anti-CTLA4. We used the model to predict the antibody dose-response relationships for individual animals and to identify dose thresholds above which complete tumor elimination can be achieved in good responders. In contrast, our models predict that poor responders would not achieve complete response even with much higher drug doses. The parameters in our model that impact the response in poor responders are not drug-related. This finding suggests that immune-cell related barriers have to be crossed in order to achieve a therapeutic response in these animals - possibly via combination therapy. In addition, we identified the net tumor cell doubling rate, one potential parameter that contributes to individual variability in response to treatment, as the most sensitive biological parameter determining tumor volume changes upon treatment with anti-PD1 or anti-CTLA4. Measuring individual animal tumor cell growth characteristics may help with the experimental design and qualification of animals for studies (in addition to absolute tumor volume), and thereby reduce inter-animal variability and enhance the interpretability of study results, especially in combination with a model such as the one presented here. This model platform can be adapted to capture and compare checkpoint drug effects in different syngeneic tumor models. Moreover, it can be expanded to add additional drug mechanisms and can serve as a tool to inform the experimental design of mouse studies. Citation Format: Alison M. Betts, Lin Lin, Carissa L. Young, Wenlian Qiao, Jatin Narula, Peter O'Brien, Derek Bartlett, Andrea Hooper, Jason Williams, John M. Burke, Joshua Apgar, Fei Hua, Lore Gruenbaum. A semi-mechanistic platform model to capture individual animal responses to checkpoint inhibitors in a syngeneic mouse model [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 684.

BI1071, a Novel Nur77 Modulator, Induces Apoptosis of Cancer Cells by Activating the Nur77-Bcl-2 Apoptotic Pathway.

Nur77 (also called TR3 or NGFI-B), an orphan member of the nuclear receptor superfamily, induces apoptosis by translocating to mitochondria where it interacts with Bcl-2 to convert Bcl-2 from an antiapoptotic to a pro-apoptotic molecule. Nur77 posttranslational modification such as phosphorylation has been shown to induce Nur77 translocation from the nucleus to mitochondria. However, small molecules that can bind directly to Nur77 to trigger its mitochondrial localization and Bcl-2 interaction remain to be explored. Here, we report our identification and characterization of DIM-C-pPhCF3 +MeSO3 - (BI1071), an oxidized product derived from indole-3-carbinol metabolite, as a modulator of the Nur77-Bcl-2 apoptotic pathway. BI1071 binds Nur77 with high affinity, promotes Nur77 mitochondrial targeting and interaction with Bcl-2, and effectively induces apoptosis of cancer cells in a Nur77- and Bcl-2-dependent manner. Studies with animal model showed that BI1071 potently inhibited the growth of tumor cells in animals through its induction of apoptosis. Our results identify BI1071 as a novel Nur77-binding modulator of the Nur77-Bcl-2 apoptotic pathway, which may serve as a promising lead for treating cancers with overexpression of Bcl-2.

Thermally Abused Frying Oil Potentiates Metastasis to Lung in a Murine Model of Late-Stage Breast Cancer.

Deep-frying is a popular form of food preparation used globally and throughout in the United States. Each time dietary oils are heated to deep-frying temperatures, they undergo chemical alterations that result in a new matrix of lipid structures. These lipid products include triglyceride dimers, polymers, oxidized triglycerides, and cyclic monomers, which raises nutritional concerns about associations between these lipid products and heightened health risks. Reports of associations between thermally abused frying oil and deleterious health outcomes currently exist, yet there is little information concerning the effects of thermally abused frying oil consumption and the progression of breast cancer. This study used a late-stage breast cancer murine model and in vivo bioluminescent imaging to monitor progression of metastasis of 4T1 tumor cells in animals consuming fresh soybean oil (SBO) and a thermally abused frying oil (TAFO). Bioluminescent and histologic examinations demonstrated that TAFO consumption resulted in a marked increase of metastatic lung tumor formation compared to SBO consumption. Further, in animals consuming the TAFO treatment diet, metastatic tumors in the lung displayed a 1.4-fold increase in the Ki-67 marker of cellular proliferation and RNA-sequencing analysis of the hepatic tissue revealed a dietary-induced modulation of gene expression in the liver.

Cysteine supplementation mitigates the toxicity associated with antitumor therapy of Ehrlich’s ascites fluid adsorbed over protein a containing Staphylococcus aureus cowan I†

Introduction:Previously, we have reported the amelioration of ad-AF induced hepatotoxicity with the exogenous supplementation of glutathione (GSH) without compromising the anti-tumor effect of ad-AF in ascites tumor model of mice with transplantable Ehrlich’s Ascites Tumor cells. Cellular uptake of glutathione (GSH) has its own limitations, therefore exogenous supplementation of L-cysteine (Cys) was tried to reduce the toxicity of ad-AF by providing –SH contents without compromising the anti-tumor property of adsorbed ascites fluid (ad-AF).Results:A significant increase in mean survival time (MST) of tumor bearing mice from 18.1 days to 32.9 days with exogenous supplementation of Cys was observed. Cys supplementation did not alter decline in body-weight gain, tumor cell counts as well as decrease in the viability of tumor cells in ascites tumor bearing animals. Similarly, Cys has been helpful to restore the hepatic –SH contents upto the levels of –SH content in tumor control group. The exogenous supplementation of Cys along with ad-AF has been helpful to restore the decline in the activities of phase-I and enhanced levels of glutathione-S-transferase (GST). The changes in the activities of different enzymes of phase-I and phase-II indicate the reduction in toxic insult induced by the therapeutic material (ad-AF). However, ad-AF treatment could not prevent tumor bearers from natural death due to tumor progression but significantly reduced the rate of tumor progression.Conclusions:Our study suggests that exogenous supplementation of Cys alongwith ad-AF could have a potential to be developed as a modality for the treatment of ascites tumor at least at experimental level.

EXPERIMENTAL STRATEGY FOR IN VITRO EVALUATION OF CYTOTOXIC / ANTITUMOR PROPERTIES OF NEW METAL COMPLEXES WITH DIFFERENT LIGANDS

PURPOSE. The aim of this study was to evaluate cytotoxic / antitumor properties of newly synthesized metal [Zn(II), Cu(II), Co(II), Ni(II), Zn(II)/Ag(I), Zn(II)/Au(I)] complexes with various ligands (Schiff bases, non-steroidal anti-inflammatory drugs, bile acids) and to introduce an optimized strategy for such investigations in our research activity. MATERIALS AND METHODS. Human and animal tumor and non-tumor cells were used as model systems. Short-term (3 – 96h) and long-term (> 2 weeks) experiments were carried out using cytotoxicity assays, cytological / immunocytochemical, biochemical and molecular-biological methods to assess the influence of the compounds on cell viability and proliferation and their ability to induce apoptosis/necrosis and/or autophagy. RESULTS. The examined metal complexes express cytotoxic activity that is time- and concentration-dependent and are more active than the corresponding ligands tested alone. Zn(II)/Au(I) and Zn(II)/Ag(I) complexes with Salen are found to be the most promising cytotoxic agents being effective also in multidrug resistant cancer cells. Their cytotoxic activity is higher than those of cisplatin, oxaliplatin and epirubicin. CONCLUSIONS. A complex approach, based on a wide range of cell cultures and methods with different molecular / cellular targets and mechanisms of action was optimized and successfully applied for the assessment of cytotoxicity of new metal complexes.

Surface Molecular Markers of Cancer Stem Cells: Computation Analysis of Full-Text Scientific Articles.

The data on cancer stem cell surface molecular markers of 27 most common cancer diseases were analyzed using natural language processing and data mining techniques. As a source, 8933 full-text open-access English-language scientific articles available on the Internet were used. Text mining was based on searching for three entities within one sentence, namely a tumor name, a phrase "cancer stem cells" or its synonym, and a name of differentiation cluster molecule. As a result, a list of surface molecular markers was formed that included markers most frequently mentioned in the context of certain tumor diseases and used in studies of human and animal tumor cells. Based on similarity of the associated markers, the tumors were divided into five groups.

Abstract 2792: The combination of immune checkpoint blockers with the anti-ICOS KY1044 antibody results in a strong tumor response

Abstract The last few years have shown a strong paradigm shift in cancer therapies with the approval of antibodies targeting immune checkpoints (CTLA-4, PD-1 and PD-L1). These immune checkpoint blockers (ICBs) are associated with a strong and long-lasting response in patients suffering from different malignancies. However, there is still a high proportion of patients showing intrinsic or acquired resistance to ICBs. Although the mechanisms associated with the lack of immune response are multiple, the presence of highly anti-inflammatory regulatory T cells (TRegs) in the tumour microenvironment (TME) is known to negatively affect the response to these therapies. Similarly, high TReg levels in the TME have been associated with poor prognosis in several cancers. Together, this highlights the potential of targeting and depleting TRegs to enhance anti-tumour responses. The Inducible T-cell costimulator (ICOS/CD278) is related to the CD28 superfamily and is induced when T cells get activated. ICOS expression levels vary in different immune cell subtypes and in different tissues. In preclinical mouse tumour models, TRegs (CD4+/FOXP3+) constitutively express ICOS on their surface and the expression of ICOS on TRegs is significantly higher than that on effector T cells (TEffs). In addition, ICOS expression on TRegs is higher in the TME than in the blood or spleen, which makes it a strong candidate for preferential depletion of TRegs in tumours. By immunizing Kymice™ in which the endogenous Icos gene has been knocked out, we identified a novel, fully human antibody called KY1044 that cross-reacts with mouse ICOS. KY1044 is an anti-ICOS subclass G1 kappa monoclonal antibody that selectively binds to dimeric ICOS (Fc fusion) with an affinity of less than 2nM. Using in vitro and in vivo approaches we demonstrate that KY1044 has a dual mechanism of action: (1) it promotes the preferential depletion of intratumoural ICOShigh TRegs resulting in an increase in the TEff:TReg ratio in the TME; and (2) it stimulates ICOSLow TEff cells. Using the mouse effector enabled version of KY1044 (mIgG2a) we confirm, using syngeneic models, a strong anti-tumour efficacy as monotherapy or in combination with surrogates of ICBs. We also demonstrated a tumour antigen specific immunity, as highlighted by the rejection of the original tumour cells in animals cured of the disease and re-challenged by the same cell line. Noteworthy, Pharmacodynamic studies demonstrate long-term depletion of TRegs and a significant increase in the TEff:TReg ratio in response to KY1044. In summary, our data demonstrate that targeting ICOS with KY1044 is a valid approach for manipulating the immune system and for inducing a strong anti-tumour response in several indications. The data presented here also warrant the assessment of KY1044 in cancer patients in a clinical trial. Citation Format: Richard C. Sainson, Matthew McCourt, Anil Thotakura, Nahida Parveen, Miha Kismac, Gwenoline Borhis, Joana Carvalho, Tracey Myers, Robert Rowlands, Hanif Ali, Hannah Craig, Vivian Wong, Qi Liang, Volker Germaschewski. The combination of immune checkpoint blockers with the anti-ICOS KY1044 antibody results in a strong tumor response [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 2792.

Abstract 2442: Detection of tissue hypoxia with EF5

Abstract Hypoxia, a condition of oxygen deprivation, is a salient feature of the tumor microenvironment. Hypoxia tumors are frequently resistant to conventional cancer therapies and are correlated with increased migratory and metastatic behavior. A number of detection modalities exist for the detection of hypoxia; however, only EF5 (2-[2-nitro-1H-imidazol-1-yl]-N-(2,2,3,3,3-pentafluoropropyl) acetamide) provides detailed information on the distribution of hypoxia in tissues at both the physiologic and pathologic levels. EF5 is a compound that, when injected into animal tissues, selectively binds to hypoxic cells and forms adducts. These adducts are detected by a mouse monoclonal antibody, clone ELK3-51, conjugated to fluorescent dyes including Cyanine 3, Alexa Fluor 488 and Cyanine 5. The use of EF5 and its specific fluorescent monoclonal antibody to selectively detect regions of hypoxia in mouse tissues is described. BALB/c mice between 7 to 10 weeks old and weighing between 18 to 30 g were used in the study. Mouse #1 was not treated with EF5 and its spleen was harvested as a negative control. Mouse #2 was injected with 10 mM EF5 (typically 1% of animal weight in mL) and allowed to run aerobically for 4 hours before euthanization. Mouse #3 was injected with EF5, euthanized after 10 minutes and kept warm at 37°C for 40 minutes to mimic the anaerobic hypoxic condition before the spleen was harvested (hypoxic control). Examination of spleen samples indicated highly specific binding of the EF5 adducts only in the anaerobic hypoxic mouse spleen. The use of EF5 with fluorescent conjugated antibodies provides a sensitive and quantitative method for the detection and measurement of tissue hypoxia in animal and human tumor tissues and cells. Citation Format: Kevin Su, Amy Noble, Nick Asbrock, Cameron Koch, Vi Chu. Detection of tissue hypoxia with EF5 [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 2442.

Comparative study of the effects of artesunate and garlic extract on tomato crown gall disease

Crown gall disease causes great economic losses worldwide by reducing crop yields and increases susceptibility to opportunistic pathogens. The anti-malarial Artesunate which is a derivative of artemisinin from Artemisia annua leaf and Garlic extracts known for their inhibitory and cytotoxic effects on the proliferation of human and animal tumor cells were investigated on rapidly dividing plant cells in Agrobacterium tumefaciens -induced crown gall tumor of tomato ( Solanum lycopersicum ) to determine their effects. Twenty four (24) tomato seedlings grown from seeds and divided into 4 treatment groups A, B, C and D were used for this study. Group A and B were inoculated with A. tumefaciens and treated with Artesunate and Garlic extract respectively for 3 weeks. Tomato seedlings of group C were the healthy seedlings that were not inoculated with A. tumefaciens and group D contains the seedlings that were inoculated with A. tumefaciens but not treated. Within two weeks of inoculation with A. tumefaciens , some of the diseased seedlings developed obvious galls (tumors) compared to the Artesunate and Garlic treated seedlings and the healthy seedlings. It was observed that Artesunate and Garlic extracts showed significant inhibition ( P ≤ 0.05) against gall (tumor) development. Artesunate and Garlic treated seedlings had better growth compared to the diseased seedlings. However, Garlic extract was observed to be more effective than Artesunate at inhibiting gall (tumor). This therefore confirmed the efficacy of garlic extracts and synthetic artesunate against crown gall disease of tomato. Keywords: Artesunate, Garlic, Crown gall tumor and Agrobacterium tumefaciens

Impact of neutron radiation on the viability of tumor cells cultured in the presence of boron-10 isotope

To investigate the impact of a neutron beam formed with the accelerator-based epithermal neutron source designed at the G.I. Budker Institute of Nuclear Physics (INP) on the viability of human and animal tumor cells cultured in the presence of boron-10 isotope. Human U251 and T98G glioma cells and Chinese hamster CHO-K1 and V-79 cells were incubated at various concentrations in the culture medium containing 10B-enriched L-boronophenylalanine. The cells were irradiated with a neuron beam using the accelerator-based epithermal neuron source. A clonogenic assay was used to evaluate the viability of the irradiated cells. The absorbed doses obtained from elastic scattering of fast neutrons by substance nuclei and the doses obtained from boron neutron capture were calculated using the NMS code. The absorbed doses of gamma-radiation were measured with a mixed radiation dosimeter. The viability of boron-containing and intact human U251 and T98G cell lines and Chinese hamster CHO-K1 and V-79 cells was analyzed after neutron beam radiation. Irradiation of all four cell lines were cultured in the presence of 10B was shown to reduce their colony-forming capacity compared with the control. Elevated boron levels in the culture medium resulted in a significant decrease in the proportion of survived cells. Radiation had the most pronounced impact on the proliferative capacity of the human U251 glioma cell lines. The cultures of human tumor cells and mammalian cells demonstrated that the neutron beam formed with the accelerator-based epithermal neutron source designed at the INP, was effective in reducing the viability of tumor cells in the presence of 10B.

Вплив супернатанта прогеніторних нейроклітин на цитотоксичну функцію лімфоцитів у щурів з гліомою

The impact of rat neurogenic progenitor cells supernatant (RPNS) on the cytotoxic function of lymphocytes in rats under conditions of physiological norm and experimentally modeled tumor (brain glioma strain 101.8) was studied. The research was carried out in animals with inoculated tumor without RPNS injection and with different regimes of RPNS injection (thrice repeated from 5th to 10th day after glioma inoculation as well as 1 week and 1 month before tumor inoculation). Comparison groups included rats without glioma who triple injected with RPNS; and intact animals (control). RPNS was received from suspension of neurogenic progenitor cells (NPC) of rat brain on 14th day of gestation and injected intraperitoneally (0,12 mg per animal). Cytotoxic function of lymphocytes of experimental rats was evaluated in MTT-colorimetric test with allogeneic glioma cells. RPNS administration increased the cytotoxic activity of lymphocytes in vitro tests with allogeneic tumor cells in intact animals (to 37-38%) as well as in rats with glioma (to 11-22%). Under the RPNS influence the life expectancy and median survival of tumor-bearing animals increased (an average of 3-4 days). RPNS input modes such as triple injection from 5th to 10th day after glioma inoculation and 1 week before inoculation were the most effective. Thus, indirect tumor-inhibiting effect under intraperitoneal. RPNS administration in rats with glioma is demonstrated, which is obviously due to increased efficiency of cytotoxic function of immune cells of animals with inoculated tumor under the influence of the factors produced by NPC.

Viability and proliferation of L929, tumour and hybridoma cells in the culture media containing sericin protein as a supplement or serum substitute.

Cell cultures often require the addition of animal serum and other supplements. In this study, silk sericin, a bioactive protein, recovered from the waste of silk floss production was hydrolysed into three pepsin-degraded sericin peptides with different ranges of molecular mass. Normal animal cells, tumour cells and hybridoma cells were cultured systematically in FBS culture media containing sericin as a supplement or serum substitute. The culture test and microscopic observation of L929 cells showed that the smaller molecular weight of the degraded sericin is most suitable for cell culture. The cell culture results showed that with the degradation of sericin, for normal mouse fibroblast L929 cells, addition of 0.75 % sericin into FBS culture medium yields cell viability that is superior to FBS culture medium alone. When all serum was replaced by sericin, cell viability in the sericin medium could reach about one half of that in FBS medium. When in a medium containing a mixture of FBS: sericin (6:4, v/v), the cell culture effect is about 80 %. For the cultures of four tumour and one hybridoma cells, regardless of the molecular weight range, these degraded sericin peptides could substitute all serum in FBS media. The cell viability and proliferation of these tumour and hybridoma cells are equivalent or superior to that in FBS medium. In other words, cell viability and proliferation of these tumour and hybridoma cells in sericin media are more preferable to serum media. The mechanism of the sericin protein to promote cell growth and proliferation will be further investigated later.

Anti-carcinogenic actions of glycoprotein conjugated with isoflavones from submerged-liquid culture of Agaricus blazei mycelia through reciprocal expression of Bcl-2 and Bax proteins

Glycoproteins isolated from fruit bodies and mycelial cultures of mushrooms exhibit anti-carcinogenic actions in human cancer cells and animal tumor cells by induction of apoptosis. Here, we report that isoflavone-conjugated glycoproteins (designate Gluvone), exhibit strong anti-carcinogenic effects on human breast cancer MCF-7 cells by induction of apoptosis. Gluvone with 9.4 kDa of molecular weight was isolated from submerged-liquid culture of Agaricus blazei mycelia (ABM) in soy flake-containing liquid medium. MCF-7 cells were incubated with various amounts of Gluvone (0~250 μM) for a period of 6 days. Gluvone exhibited anti-proliferative actions in a dose-dependent manner and 62% growth inhibition at 200 μM for 4 days relative to control. Hoechst 33258 staining analysis revealed that Gluvone induced formation of apoptotic bodies. Gluvone was associated with down-regulation of anti-apoptotic Bcl-2 protein expression as well as up-regulation of pro-apoptotic Bax protein expression. Gluvone treatment induced proteolytic activation of caspase-9 and caspase-3 through cytochrome c release from mitochondria to cytosol as well as concomitant degradation of poly (ADP-ribose) polymerase (PARP). In addition, Gluvone induced activation of caspase-8. Taken all together, these results indicate that the anti-proliferative effect of Gluvone is associated with induction of apoptotic cell death through the mitochondrial dysfunction pathway mediated by enhancement of Bax protein expression and suppression of Bcl-2 protein expression.

TM-01 * IDENTIFICATION OF GLUCOSE-6-PHOSPHATASE ALPHA AS A KEY METABOLIC REGULATOR FOR GLIOBLASTOMA CELL INVASION

Glioblastoma (GBM) remains the most aggressive primary brain cancer in adults. Similar to other cancers, GBM cells undergo metabolic reprogramming to promote proliferation and survival. Glycolytic inhibition is widely used to target such reprogramming. However, the stability of glycolytic inhibition in GBM remains unclear. In this study, we found that the expression of glucose-6-phosphatase-α (G6PC) was elevated in GBM when compared to normal brain ) (p < 0.001). Human-derived brain tumor initiating cells (BTICs) utilize this enzyme to counteract glycolytic inhibition induced by 2-Deoxy-D-glucose (2DG) and sustain malignant progression. Down-regulation of G6PC renders the majority of these cells unable to survive glycolytic inhibition, and promotes glycogen accumulation through the activation of glycogen synthase (GYS1) and inhibition of glycogen phosphorylase (PYGL). Moreover, BTICs that survive G6PC knockdown are less aggressive (reduced migration, invasion, proliferation, and increased astrocytic differentiation) (p < 0.001). Interestingly, after performing immunohistochemistry staining for G6PC in brain sections derived from animals implanted with G6PC knockdown cells for a survival study, we found a complete rescue of its expression in the tumor cells of these animals. These results explain the absence of survival differences observed between G6PC knockdown and wild type mice. Furthermore, these findings also suggest a very important role for G6PC in cell survival, since only the cells that carried G6PC where able to survive and invade in our animal model. Collectively, our findings establish G6PC as a key enzyme with pro-malignant functional consequences that has not been previously reported in GBM and identify it as a potential therapeutic target.

Abstract 2664: An anti-Jagged-1/-2 Probody demonstrates inhibition of Jagged-dependent Notch signaling and is activated in multiple types of tumors

Abstract Probodies represent a unique class of antibody therapeutics that specifically target therapeutic activity to diseased tissues, such as cancer. A Probody is a fully recombinant protein that contains a masking peptide that blocks the antibody's antigen-binding site and is linked to the light chain of the antibody by a protease-cleavable peptide substrate. Probodies are designed to be inactive in circulation and in healthy tissues but activated within the tumor microenvironment. The Probody™ approach is designed to widen the therapeutic index of antibodies to promising therapeutic targets that may be limited by significant toxicity. One such promising set of targets are components of the Notch pathway, which is involved in both oncogenic and healthy tissue signaling. We have developed a fully human IgG1 monoclonal antibody that binds and inhibits the activity of both human and mouse Jagged-1 and Jagged-2, two key ligands in the Notch signaling pathway. A Probody produced from this antibody induced dose-dependent inhibition of Notch signaling, resulting in anti-tumor activity in in vivo tumor models without significant toxicity. To further explore the clinical relevance of the anti-Jagged Probody for the treatment of cancer, we evaluated (i) the heterogeneity of Jagged expression in multiple tumor types, (ii) the activation of the Jagged Probody in tumor biopsies, and (iii) the blockade of Notch signaling using an intracellular pharmacodynamic biomarker. Patient tumor tissues were screened for Jagged-1/-2 IHC across lung, pancreatic and breast cancer_ indications where activation of the Notch pathway is implicated. Overall, more than 75% of patient tumors demonstrated moderate to high Jagged-1/-2 expression. We also developed and validated a technique termed IHZ™ analysis that enables the measurement of proteolytic Probody activation and binding to Jagged ligands ex vivo in human tumor sections. We evaluated 120 patients across the three tumor types and observed ∼99% of samples that were positive for antibody binding were also positive by IHZ analysis, signifying sufficient proteolytic activity to activate the Probody. Finally, we evaluated the accumulation of the Notch Intracellular Domain (NICD), a biomarker of Notch signaling, in the nucleus of tumor cells in animals treated with antibody. Notably, our data showed that Notch signaling blockade as indicated by a decrease in the accumulation of NICD correlated with dose-response in in vivo models. Taken together, our data suggest that the anti-Jagged-1/-2 Probody may have wide therapeutic utility as a tumor-specific inhibitor of Jagged-dependent Notch signaling in oncology. Citation Format: Olga Vasiljeva, Elizabeth Menendez, Jason Sagert, James W. West, Jennifer Richardson, Luc Desnoyers, Shouchun Liu, Judi Ford, Krishna Plou, Henry Lowman. An anti-Jagged-1/-2 Probody demonstrates inhibition of Jagged-dependent Notch signaling and is activated in multiple types of tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2664. doi:10.1158/1538-7445.AM2014-2664