Proliferation In Presence Research Articles

Foxp3 inhibitory peptide encapsulated in a novel CD25-targeted nanoliposome promotes efficient tumor regression in mice.

P60, a Foxp3 inhibitory peptide, can hinder the regulatory T cell (Treg) activity and impair tumor proliferation. However, low systemic stability and poor specificity have led to daily dosing to achieve therapeutic effect. Therefore, this study aims to improve P60 stability and specific delivery through its encapsulation in liposomes targeting CD25, constitutively expressed in Tregs. P60 liposomes formulated with DSPE-PEG750 or DSPE-PEG2000 were incubated with DSPE-PEG2000-Maleimide micelles conjugated to Fab' fragments of anti-CD25 to develop two targeted formulations or immunoliposomes (IL): IL-P602000 (DSPE-PEG2000 only) and IL-P60750 (combining DSPE-PEG750 and DSPE-PEG2000). P60 encapsulation efficiency was 50%-60% irrespective of PEG chain length. Treg uptake was 2.5 and 14 times higher for IL-PEG750 compared with IL-PEG2000 and non-targeted liposomes, respectively, in in-vitro assays. In fact, IL-P60750 allowed CD8+ T cells ex-vivo proliferation in presence of Treg at doses 10-20 times lower than for free P60. Antitumor response of P60 and IL-P60750 in monotherapy and combined with anti-PD-1 was evaluated in MC38 and LLCOVA tumor bearing mice. In MC38 model, IL-P60750 monotherapy induced total tumor regression in 40% of mice reaching 100% for anti-PD-1 combination. This effect was associated with a significant increase in activated CD8+ T cells in tumors. Notably, IL-P60750 also inhibited human Treg in ex-vivo assay, showing the translational capability of this formulation. In conclusion, IL-P60750 formulated with different PEG chain lengths, has demonstrated antitumor efficacy by selective inhibition of Treg activity and enhances the effect of anti-PD1. Altogether, this novel IL represents a promising nanoplatform for cancer immunotherapies.

Seasonal Immune Rhythms of head kidney and spleen cells in the freshwater Teleost, Channa punctatus

Annual rhythms in immune function are the reflection of a crucial physiological strategy to deal with environmental stressors. The fish are pivotal animal models to study the annual rhythm and to understand the evolution of the vertebrate biological system. The current research was planned to assess the annual changes in the innate immune functions of immune cells in a teleost, Channa punctatus. Head kidney and splenic macrophage phagocytosis, superoxide generation, and nitrite release were evaluated to assess innate immunity. Cell-mediated immunity was measured through head kidney and splenic lymphocyte proliferation in presence of mitogens. The superoxide anion generation by the cells of head kidney and spleen was maximum in October. A bimodal pattern in nitrite production was observed with the first peak in November and the second in March. Cosinor analysis revealed a statistically significant annual rhythm in nitrite production. Similarly, phagocytosis and lymphocyte proliferation also showed statistically significant annual rhythms. It was concluded that animals maintain an optimum immune response in seasonally changing environments. Elevated immunity during certain times of the year might assist animals deal with seasonal environmental stressors. Further research may be focused upon measuring survival rate and reproductive success after season induced elevated immunity.

Topographical influence of electrospun basement membrane mimics on formation of cellular monolayer

Functional unit of many organs like lung, kidney, intestine, and eye have their endothelial and epithelial monolayers physically separated by a specialized extracellular matrix called the basement membrane. The intricate and complex topography of this matrix influences cell function, behavior and overall homeostasis. In vitro barrier function replication of such organs requires mimicking of these native features on an artificial scaffold system. Apart from chemical and mechanical features, the choice of nano-scale topography of the artificial scaffold is integral, however its influence on monolayer barrier formation is unclear. Though studies have reported improved single cell adhesion and proliferation in presence of pores or pitted topology, corresponding influence on confluent monolayer formation is not well reported. In this work, basement membrane mimic with secondary topographical cues is developed and its influence on single cells and their monolayers is investigated. We show that single cells cultured on fibers with secondary cues form stronger focal adhesions and undergo increased proliferation. Counterintuitively, absence of secondary cues promoted stronger cell–cell interaction in endothelial monolayers and promoted formation of integral tight barriers in alveolar epithelial monolayers. Overall, this work highlights the importance of choice of scaffold topology to develop basement barrier function in in vitro models.

Apatite-Graphene and Apatite-Graphene Oxide Nanocomposites: Hybrid Materials with Tailored Biological and Luminescent Properties

Apatite nanocomposites with graphene (G) or graphene oxide (GO) nanoflakes, as well as with related carbonaceous materials, present promising applications in hard tissue engineering, biomedicine, or drug delivery. Different methodologies have been explored in the last years to prepare apatite-based nanocomposites. Sitting drop vapour diffusion (SDVD) methodology induces the heterogeneous nucleation of biomimetic apatite on the reinforcement material, improving biological properties of the nanocomposites. In this work SDVD was used to prepare G-apatite and GO-apatite nanocomposites. Prior to the SDVD experiments, G flakes were obtained by sonication-assisted liquid-phase exfoliation (LPE) using L-Alanine (L-Aln) as dispersing biomolecule, while a commercial aqueous Graphene Oxide (GO) dispersion was used for the nucleation essays in presence of the same biomolecules. A parallel set of nucleation experiments was performed in presence of Tb3+ ions, to endow the nanocomposites of luminescent properties. Characterization by XRD, FTIR, and TEM demonstrated the heterogeneous nucleation of needle-shaped apatite nanocrystals on the surfaces of G and GO flakes. Fluorescence spectroscopy certified the presence of Tb3+ ions in the nanocomposites resulting in luminescent materials which can be used in imaging or theragnostic. Finally, in vitro tests with human mesenchymal stem cells revealed excellent cytocompatibility and cell proliferation in presence of the nanocomposites.

Evaluation of triazophos induced immunotoxicity of spleen and head kidney in fresh water teleost, Channa punctata

The utilization of pesticides has increased for destroying pests and protecting crops in the agriculture field. Triazophos is a commonly used organophosphorous insecticide that causes alterations in haematological and histological parameters in fish. The present study was designed to evaluate the effect of triazophos induced innate and cell mediated immunotoxicity in freshwater teleost, Channa punctata. Fishes were exposed to triazophos at concentrations 5 and 10% of LC50 value for 10 and 20 days. Splenic and head kidney macrophage phagocytosis, nitric oxide production and superoxide production were assayed to evaluate the innate immunity. Cell-mediated immunity was measured through splenic and head kidney lymphocyte proliferation in presence of T and B cell mitogens. Results of the present study revealed that macrophage phagocytosis was significantly reduced after in vivo triazophos treatment. Differential suppressive effect of triazophos was also observed where mitogen induced splenic and head kidney lymphocyte proliferations were reduced after 10 and 20 days treatment. Concentration dependent effect of triazophos was observed in in vivo studies where the production of reactive oxygen and nitrogen intermediates were suppressed. This study describes the first investigation of the effect of triazophos on immune functions and will help to determine appropriate ecotoxicity and immunotoxicity in freshwater teleosts.

Abstract LT011: Pharmacological blockade of platelet-CysLT1 receptor counteracts platelet protumoral action and prevents breast cancer cell metastasis to bone and lungs

Abstract Metastases are the main cause of death in cancer patients. However, few metastasis-specific targets have been exploited therapeutically. Blood platelets are well known to contribute to the poor survival of patients with advance cancers. Unfortunately, platelets are mandatory for human life due to their paramount function in hemostasis. In order to define new anti-metastasis therapies exhibiting a high specificity for the pathological tumor cell-platelet interactions without abrogating normal platelet functions we developed a high throughput assay that showed that pharmacological blockade of cysteinyl leukotriene receptor 1 (CysLT1) with two already FDA-approved drugs in asthma (Zarfirlukast and Montelukast), dose-dependently inhibited the mitogenic action of human platelets on human MDA-B02/Luc breast cancer cells. Both drugs had no impact on cell proliferation in presence of serum suggesting a specific alteration of platelet function. This effect was specific to CysLT1 as the CysLT2 inhibitor HAMI3379 remained inefficient even at high doses. All human breast cancer cells including MDA-B02/Luc cells used in this study did not express CysLT1 whereas platelets do suggesting that platelet-CysLT1 was the one contributing to the protumoral action of platelets. As described previously cysteinyl leukotriene D4 (LTD4), the strongest agonist of CysLT1, has no direct effect on platelet aggregation but instead potentiates the action of platelet agonists. We next injected MDA-B02/Luc cells into the tail artery of nude mice that received a daily treatment with Zafirlukast (0.4mg/kg per os). After 7 days bone marrow cells were collected and placed in culture in presence of puromycine for 14 day. After staining with crystal violet clones of tumor cells that colonized bone (TCB) were quantified. In these conditions Zafirlulast significantly inhibited by 90% the number of TCB. In order to confirm this anti-metastasis effect we used a syngenic animal model of spontaneous metastasis dissemination of murine 4T1 breast cancer cells after orthotopic xenograph in the mammary gland of BALB/c immunocompetent mice. Animals were treated with Zafirlukast (0.4mg/kg/day, i.p.), the common chemotherapeutic agent Paclitaxel (30mg/kg/2 days, i.p), or both drugs combined starting at day 3 post-tumor cell injections until sacrifice at day 12. Lungs were collected, minced, treated with collagenase and placed in culture for 14 days in presence of 6-Thioguanine. After staining with crystal violet clones of tumor cells that colonized the lungs (TCL) were quantified. In these conditions, the number of TCL was reduced by 88% with Zafirlulast compared with 60% with Paclitaxel. The combined treatment of Zafirlukast + Paclitaxel remarkably inhibited by 100% the number of TCL. In contrast, Zafirlukast alone or in combination with Paclitaxel had no significant effect on primary tumor growth. In conclusion we described for the first time that blockade of platelet-CysLT1 receptor altered the protumoral action of platelets and prevented breast cancer cell metastasis to bone and lungs. Citation Format: Lou Saier, Olivier Peyruchaud. Pharmacological blockade of platelet-CysLT1 receptor counteracts platelet protumoral action and prevents breast cancer cell metastasis to bone and lungs [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT011.

Efficient Expression, Purification and Functional Evaluation of Human Epidermal Growth Factor-Collagen Binding Domain Hybrid Protein on Growth Stimulation of Human Fibroblast Cells

Growth factors such as human epidermal growth factor (hEGF) have recently received high interest in regenerative medicine and pharmaceutical industries mainly due to their ability to restore tissues proliferation and improvement of their biological functions. In spite of various hEGF applications, its efficient expression in Escherichia coli could not yet reach an industrial reality mainly due to the lack of the ability of folding into the correct 3D structure because of three disulfide bonds in monomer hEGF. To address these challenges, here a fusion hEGF protein with a C terminus of collagen binding domain (CBD) along with intein protein with self-splicing property and ELP sequence was constructed by a three-step cloning procedure. This enabled us to purify recombinant hEGF without using chromatography columns. Following the confirmation of the construct by colony PCR, restriction enzymes analysis and sequencing, the 62[Formula: see text]kDa band of ELP-INTEIN-hEGF-CBD were observed on SDS-PAGE and confirmed by western blotting. Subsequently, the mitotic activity in Balb/c 3T3 cells proliferation in presence of recombinant hEGF-CBD compared with commercial hEGF using methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay which showed that our purified recombinant protein stimulates the cell proliferation similar to the commercial protein. Our strategy could be considered as a new feasible approach to produce hEGF in E. coli for pharmaceutical and clinical applications.

Sex steroids exert a suppressive effect on innate and cell mediated immune responses in fresh water teleost, Channa punctatus

The main objective of this study was to evaluate the potential role of two important sex steroids, crucial for reproductive success, on innate and cell mediated immune responses in a seasonally breeding, economically important fish, Channa punctatus. Intraperitoneal injections of testosterone and progesterone were given to different groups of fishes. Spleen and head kidney macrophages were isolated and studied for phagocytosis. Superoxide production and nitrite release by phagocytes were also investigated. Cell mediated immunity was measured by lymphocyte proliferation in presence of T and B cell mitogens. In vitro effect of steroids on mitogen induced lymphocyte proliferation was also analyzed. Results of the present investigation revealed the suppressive effects of testosterone and progesterone on immune responses of cells from spleen and head kidney. Concentration dependent effect of sex steroids were observed in vitro studies where phagocytosis and lymphocyte proliferation were suppressed. Immunosupression by these hormones may be the cost of reproduction and it is postulated that by suppressing immune responses, these steroids may, therefore, act as a physiological check regulating the relative amount of energy invested into either reproductive effort or immunocompetence.

PS1305 CHARACTERIZATION OF ENDOGENOUS CXCR4 INHIBITORY PEPTIDES TO TARGET WALDENSTRÖM'S MACROGLOBULINEMIA

Background:Waldenströḿs Macroglobulinemia (WM) is an indolent B‐cell lymphoma with elevated monoclonal IgM serum levels and bone marrow infiltration of lymphoplasmacytic cells. Whole‐genome sequencing has led to identification of two prevalent mutations affecting MYD88 and CXCR4 that allow the classification into 3 genotypes. It was shown that CXCR4 mutations contribute to malignant growth and are associated with Ibrutinib resistance in WM. So far, naturally occurring mechanisms counteracting CXCR4 driven proliferation in cancer are largely unknown. We screened a human blood‐derived peptide library in order to identify novel anti‐cancer peptides that target CXCR4. This approach succeeded in identifying an as‐yet‐unknown CXCR4 blocking peptide called EPI‐X4 (endogenous peptide inhibitor of CXCR4) that is generated naturally in the human body. The 16‐mer peptide is cleaved from its precursor albumin under acidic conditions and was shown to specifically interfere in the cross‐talk between CXCR4 positive cancer cells and their environment. Successful development of highly active and more stable EPI‐X4 derivatives (WSC02 and JM#21) could be of clinical relevance in the future.Aims:Our goal is to analyse the functional role of the naturally occurring EPI‐X4 and to test optimized EPI‐X4 derivatives as potential therapeutic tool in WM.Methods:We performed multiple cell line based functional assays such as apoptosis and migration as well as experiments with EPI‐X4 treated WM cells in vitro and in vivo using NSG mice. Overexpression of CXCR4 mutations was achieved via lentivirus‐mediated infection of WM cells. Whole transcriptome analysis based on next‐generation sequencing was carried out using the Illumina platform and validated via Real‐Time PCR. Proteomic analysis was done using highly sensitive tandem mass spectrometry.Results:We have investigated the effect of EPI‐X4 and its optimized derivatives on blocking the CXCR4 receptor, WM cell migration and inhibition of cell proliferation in presence or absence of different CXCR4 mutations. Efficient and dose depending blocking of CXCR4 by the peptides was documented by two different clones of CXCR4 antibodies, recognizing different epitopes. Both optimized forms of EPI‐X4 were able to abrogate growth and efficiently induced apoptosis in WM cells (up to 90% compared to the control). Migration of WM cells with or without activating CXCR4 mutations was inhibited by 50% for the naturally occurring EPI‐X4 and 95% for the optimized peptides compared to the control. Mice injected with WSC02 treated WM cells showed a prolonged survival of 26 days (median survival control 54,5 days versus 80,5 days for WSC02, respectively). To better understand the impact of EPI‐X4 on the transcriptome, we profiled EPI‐X4 treated cells at the mRNA level using RNA‐seq technique. By performing gene enrichment analysis, genes related to carbohydrate and lipid metabolism pathways as well as the RAF/RAS pathway, hypoxia signal cascades and p53 were enriched. Proteomic changes upon EPI‐X4 treatment on different WM cells are currently being analyzed.Summary/Conclusion:Collectively, our data indicate that naturally occurring mechanisms exist that counteract CXCR4 induced lymphoma growth and that this can be used to develop innovative treatment concepts which target CXCR4 mutations in WM.

Biocompatibility and Biological Efficiency of Inorganic Calcium Filled Bacterial Cellulose Based Hydrogel Scaffolds for Bone Bioengineering.

The principal focus of this work is the in-depth analysis of the biological efficiency of inorganic calcium-filled bacterial cellulose (BC) based hydrogel scaffolds for their future use in bone tissue engineering/bioengineering. Inorganic calcium was filled in the form of calcium phosphate (β-tri calcium phosphate (β-TCP) and hydroxyapatite (HA)) and calcium carbonate (CaCO3). The additional calcium, CaCO3 was incorporated following in vitro bio-mineralization. Cell viability study was performed with the extracts of BC based hydrogel scaffolds: BC-PVP, BC-CMC; BC-PVP-β-TCP/HA, BC-CMC-β-TCP/HA and BC-PVP-β-TCP/HA-CaCO3, BC-CMC-β-TCP/HA-CaCO3; respectively. The biocompatibility study was performed with two different cell lines, i.e., human fibroblasts, Lep-3 and mouse bone explant cells. Each hydrogel scaffold has facilitated notable growth and proliferation in presence of these two cell types. Nevertheless, the percentage of DNA strand breaks was higher when cells were treated with BC-CMC based scaffolds i.e., BC-CMC-β-TCP/HA and BC-CMC-β-TCP/HA-CaCO3. On the other hand, the apoptosis of human fibroblasts, Lep-3 was insignificant in BC-PVP-β-TCP/HA. The scanning electron microscopy confirmed the efficient adhesion and growth of Lep-3 cells throughout the surface of BC-PVP and BC-PVP-β-TCP/HA. Hence, among all inorganic calcium filled hydrogel scaffolds, ‘BC-PVP-β-TCP/HA’ was recommended as an efficient tissue engineering scaffold which could facilitate the musculoskeletal (i.e., bone tissue) engineering/bioengineering.

Characterization and Cytotoxicity Evaluation of a Marine Sponge Biosilica.

Bone fractures characterize an important event in the medical healthcare, being related to traumas, aging, and diseases. In critical conditions, such as extensive bone loss and osteoporosis, the tissue restoration may be compromised and culminate in a non-union consolidation. In this context, the osteogenic properties of biomaterials with a natural origin have gained prominence. Particularly, marine sponges are promising organisms that can be exploited as biomaterials for bone grafts. Thus, the objectives of this study were to study the physicochemical and morphological properties of biosilica (BS) from sponges by using scanning electron microscopy, Fourier-transform infrared, X-ray diffraction (SEM, FTIR and XRD respectively), mineralization, and pH. In addition, tests on an osteoblast precursor cell line (MC3T3-E1) were performed to investigate its cytotoxicity and proliferation in presence of BS. Bioglass (BG) was used as gold standard material for comparison purposes. Sponge BS was obtained, and this fact was proven by SEM, FTIR, and XRD analysis. Calcium assay showed a progressive release of this ion from day 7 and a more balanced pH for BS was maintained compared to BG. Cytotoxicity assay indicated that BS had a positive influence on MC3T3-E1 cells viability and qRT-PCR showed that this material stimulated Runx2 and BMP4 gene expressions. Taken together, the results indicate a potential use of sponge biosilica for tissue engineering applications.

Ethyl Acetate Extract of Licorice Root (Glycyrrhiza glabra) Enhances Proliferation and Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells.

Glycyrrhiza glabra (G. glabra) has been used as a flavoring and sweetener agent, in addition to its therapeutic properties. It is rich in phytoestrogen and may prevent osteoporosis caused by estrogen deficiency; however, there is no evidence for its effects on proliferation and osteogenesis in mesenchymal stem cells. So, we were encouraged to investigate whether the ethyl acetate extract of licorice root as a source of phytoestrogen can act similar to estrogen in cell culture. Furthermore, the analysis of the licorice extract (LE) based on HPLC-DAD-ESI-MS indicated that LE comprises phytoestrogen compounds, such as glabridin and glabrene. In this study, the effects of LE on proliferation of human bone-marrow mesenchymal stem cells (hBM-MSCs) were investigated using MTT assay. In addition, its effects on the osteogenesis were evaluated using alkaline phosphatase activity (ALP), calcium deposition, and bone specific gene expression such as ALP, osteocalcin, Runx2, and BMP-2. The quantitative gene expression was studied by real-time RT-PCR. Our results showed a significant increase in proliferation in presence of LE in concentration 10-50 µg/mL. The differentiation of hBM-MSCs increased in doses of LE (10-25 µg/mL) compared to the control group. The effects of LE were similar to those of 17β-estradiol (E2) (10-8 M) and were abolished by ICI 182,780 an antagonist of estrogen receptor (ER) (10-7), indicating that the stimulatory effects of LE occur through estrogen receptor-mediated mechanism . Taking these into account, LE may be a potential candidate for prevention of osteoporosis in menopausal women.

HBV Facilitated Hepatocellular Carcinoma Cells Proliferation by Up-Regulating Angiogenin Expression Through IL-6

Background/Aims: Patients with hepatitis B virus (HBV) infection are at a high risk of developing hepatocellular carcinoma (HCC). In this study, we aim to investigate the roles of HBV on angiogenin (ANG), as well as the effects on cell proliferation in presence of ANG down-regulation. Methods: Serum ANG was determined by ELISA. The expression of ANG mRNA and protein in HCC cell lines with or without HBV/HBx were determined. Western blot and ELISA were conducted to determine the effects of HBV/HBx on IL-6 expression. The role of IL-6 on ANG was evaluated by IL-6 recombinant protein or IL-6 neutralizing antibody. Immunofluorescence staining was used to detect the nuclear translocation of ANG. MTT was performed to evaluate the relative inhibition ratio. Result: In vivo experiments showed elevation of serum ANG in patients infected with HBV. In vitro experiments showed HBV and HBx contributed to the transcription and translation of ANG. ANG expression showed increase after IL-6 stimulation, and ANG protein decreased in the presence of IL-6 blocking with its antibody. HBV promoted nuclear translocation of ANG. Inhibiting ANG expression or blocking of nuclear transfer of ANG attenuated the 45S rRNA synthesis and cell proliferation. Conclusion: HBV and HBx protein can increase the level of ANG through IL-6. HBV and HBx contributed to the nuclear translocation of ANG. Cell proliferation was inhibited after inhibiting the expression or nuclear transfer of ANG.

Effects of system benzylaminopurine-adenine sulphate in combination with naphthalene acetic on in vitro regeneration and proliferation of pineapple (Ananas comosus (L.) Mill var. comosus)

In vitro micropropagation is now used in the production of healthy and homogenous pineapple planting materials. In order to increase the in vitro proliferation of pineapple, the synergetic effect of adenine sulphate (AdS) and benzylaminopurine (BAP) in presence or not of naphthalene acetic acid (NAA) was evaluated. Median axillary buds of crowns bursted 6 to 8 weeks old were cultured on Murashige and Skoog medium supplemented with BAP (0, 1 and 2 mg/l), NAA (0 and 0.5 mg/l) with or without AdS (40 mg/l). Five buds per treatment with four replicates were used. The addition of BAP to the medium stimulated regeneration (65 to 82%) and multiplication (3.04 to 6.13 plantlets/explant) which were further enhanced by the addition of NAA (98%; 6.42 plantlets/explant). AdS highly stimulated proliferation in presence of 2 mg/l BAP and NAA (14.96 plantlets/explant), plantlet mass (1.143g) but limited plantlet growth in height which was more induced in the absence of regulators (4.88 cm) and with AdS alone (5.63 cm). Thus, BAP is necessary for the proliferation of pineapple and the addition of 0.5 mg/l NAA to 2 mg/l BAP and 40 mg/l AdS optimizes the multiplication and growth parameters of pineapple plantlets. These results allow the mass propagation of pineapple’s homogeneous healthy planting materials.

PD-1 Blockade Promotes Emerging Checkpoint Inhibitors in Enhancing T Cell Responses to Allogeneic Dendritic Cells

Immune checkpoint inhibitors, which target coinhibitory T cell molecules to promote anticancer immune responses, are on the rise to become a new pillar of cancer therapy. However, current immune checkpoint-based therapies are successful only in a subset of patients and acquired resistances pose additional challenges. Finding new targets and combining checkpoint inhibitors might help to overcome these limitations. In this study, human T cells stimulated with allogeneic dendritic cells (DCs) were used to compare immune checkpoint inhibitors targeting TIM-3, BTLA, LAG-3, CTLA-4, and TIGIT alone or in combination with a PD-1 antibody. We found that PD-1 blockade bears a unique potency to enhance T cell proliferation and cytokine production. Other checkpoint inhibitors failed to significantly augment T cell responses when used alone. However, antibodies to TIM-3, BTLA, LAG-3, and CTLA-4 enhanced T cell proliferation in presence of a PD-1 antibody. Upregulation of coinhibitory T cell receptors upon PD-1 blockade was identified as a potential mechanism for synergistic effects between checkpoint inhibitors. Donor-specific variation in response to immune checkpoint inhibitors was attributed to the T cells rather than DCs. Additionally, we analyzed the regulation of checkpoint molecules and their ligands on T cells and allogeneic DCs in coculture, which suggested a PD-1 blockade-dependent crosstalk between T cells and APC. Our results indicate that several immune checkpoint inhibitors have the capacity to enhance T cell responses when combined with PD-1 blockade. Additional in vitro studies on human T cells will be useful to identify antibody combinations with the potential to augment T cell responses in cancer patients.

Quercetin induces apoptosis and necroptosis in MCF-7 breast cancer cells.

This study investigated the quercetin (Que) effects on growth of MCF-7 human cancer breast cell line and its cellular death mechanism. Quercetin has been found to be very efficacious against many different types of cancer cells. However, the study is not sufficiently powered to demonastrate anticancer mechanisms. MCF-7cells were treated by 50 µM/ ml of Que for 48 hours. MCF-7 cells were also pretreated with 10 Μm ZVAD (apoptosis inhibitor) or 3 mM Nec-1 (necroptosis inhibitor) for evaluation of cell death induced by apoptosis or necroptosis. MTT and clonogenicity assays revealed that the Que induced a significant increase in cell viability and proliferation in presence of Nec-1 in comparison to the presence of ZVAD (p < 0.05). Que also increased apoptosis as revealed by DAPI staining and morphology evaluations. Following Que treatment Bcl-2 expression was significantly decreased while Bax expression was significantly increased. Que in presence of Nec-1 decreased expression of Bax gene, reduced apoptotic index, increased cell viability and proliferation of MCF-7 cells in comparison to absence of Nec-1. MCF-7 cells showed a significantly increased expression of RIPK1 and RIPK3 in response to Que plus ZVAD in comparison to absence of ZVAD. Our results revealed that the high Que toxicity for breast cancer cells depends on multiple cell death pathways, which involve mainly necroptosis (Fig. 6, Ref. 21).

Abstract B18: Estrogen deficiency causes reawakening of dormant disseminated breast cancer cells via angiopoietin-1/Tie2 signaling

Abstract Introduction: Compared to premenopausal women, postmenopausal women with breast cancer are at increased risks of bone metastasis. When cancer cells migrate into bone marrow (BM), they stay dormant in stable microvasculature but proliferate in unstable microvasculature. Angiopoietin-1(ANGPT1)/Tie2 signaling regulates microvascular stability. ANGPT-1 stabilizes microvasculature, while ANGPT-2 destabilizes it and promotes neovascularization. We hypothesized that postmenopausal estrogen deficiency induces reawakening of the dormant tumor cells in BM through modulation of angiogenesis. We examined the effects of estrogen depletion on the proliferative status of breast cancer cells (BCCs) in a BM microvasculature model, and quantified the changes of ANGPT1 and ANGPT2 expressions in the model before and after estrogen depletion. Materials and Methods: Bone marrow microvasculature model was generated by using bone marrow mesenchymal stem cells (MSCs) and endothelial cells (ECs). To deplete estrogen in the model, charcoal-stripped serum-containing culture medium with or without 17β-estradiol (E2) were used. ANGPT1 and ANGPT2 protein levels in the model were determined by western blot. Estrogen receptor(ER)-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cell lines were used. Fluorescence-tagged tumor cells were seeded in the BM microvasculature models with or without estrogen and their proliferation were monitored by microscope and microscope fluorescence reader. Recombinant human ANGPT1 (RhANGPT1) and Tie2-specific blocking antibody were used for ANGPT1/Tie2 signaling modulation. Results: ANGPT2 expression was significantly increased in BM microvasculature model exposed to prolonged estrogen depletion. ANGPT1 expression was not changed. BCCs in the estrogen-depleted BM microvasculature model grew significantly faster than those in the model supplemented with E2. Blocking ANGPT1/Tie2 signaling in the model by Tie2-specific blocking antibody enhanced BCC proliferation in presence of estrogen, whereas RhANGPT1-treatment inhibited BCC proliferation in estrogen-depleted BM microvasculature model. In further, RhANGPT1 protected BCCs from doxorubicin-induced apoptosis. RhANGPT1 inhibited proliferation of Tie2-expressing MDA-MB-231 cells in absence of BM MSCs or ECs, suggesting direct interaction of ANGPT1 and Tie2-expressing tumor cells. Conclusions: With in vitro disseminated breast cancer cell model, we confirmed that estrogen deficiency modified bone marrow microvasculature to more metastasis-prone environment. We identified the ANGPT1/Tie2 signaling as a key regulator of angiogenic tumor cell dormancy. The results suggest that postmenopausal estrogen deficiency may lead to reawakening of dormant BCCs by altering ANGPT1 and ANGPT2 levels in BM microvasculature. Angiogenic dormancy in association with estrogen implies a new strategy against metastatic recurrence in postmenopausal women with remote breast cancer history. Citation Format: Hyun Ho Han, Nam Hoon Cho, Suki Kang, Joo Hyun Lee, Ji Eun Kim. Estrogen deficiency causes reawakening of dormant disseminated breast cancer cells via angiopoietin-1/Tie2 signaling. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B18.

HEDGEHOG/GLI-E2F1 axis modulates iASPP expression and function and regulates melanoma cell growth.

HEDGEHOG (HH) signaling is a key regulator of tissue development and its aberrant activation is involved in several cancer types, including melanoma. We and others have shown a reciprocal cross talk between HH signaling and p53, whose function is often impaired in melanoma. Here we present evidence that both GLI1 and GLI2, the final effectors of HH signaling, regulate the transcription factor E2F1 in melanoma cells, by binding to a functional non-canonical GLI consensus sequence. Consistently, we find a significant correlation between E2F1 and PATCHED1 (PTCH1), GLI1 and GLI2 expression in human melanomas. Functionally, we find that E2F1 is a crucial mediator of HH signaling and it is required for melanoma cell proliferation and xenograft growth induced by activation of the HH pathway. Interestingly, we present evidence that the HH/GLI-E2F1 axis positively modulates the inhibitor of apoptosis-stimulating protein of p53 (iASPP) at multiple levels. HH activation induces iASPP expression through E2F1, which directly binds to iASPP promoter. HH pathway also contributes to iASPP function, by the induction of Cyclin B1 and by the E2F1-dependent regulation of CDK1, which are both involved in iASPP activation. Our data show that activation of HH signaling enhances proliferation in presence of E2F1 and promotes apoptosis in its absence or upon CDK1 inhibition, suggesting that E2F1/iASPP dictates the outcome of HH signaling in melanoma. Together, these findings identify a novel HH/GLI-E2F1-iASPP axis that regulates melanoma cell growth and survival, providing an additional mechanism through which HH signaling restrains p53 proapoptotic function.

A2B-mediated endothelial cell proliferation is associated with decoupling of the NO and VEGF signaling pathway during pre-eclampsia.

A2B-mediated endothelial cell proliferation is associated with decoupling of the NO and VEGF signaling pathway during pre-eclampsia.

Differential suppressive effects of testosterone on immune function in fresh water snake, Natrix piscator: an in vitro study.

Reptiles represent the crucial phylogenetic group as they were the ancestors of both birds and mammals hence very important to study. The objectives of the present study were to investigate the potential roles of testosterone in the innate immune responses and splenic lymphocyte proliferation in fresh water snake, Natrix piscator. Animals were mildly anesthetized and spleens were taken out to study the splenic macrophage phagocytosis, super oxide production and nitrite release using in vitro testosterone. Splenic lymphocytes were isolated by density gradient centrifugation and were studied for mitogen induced proliferation in presence of in vitro testosterone. Testosterone suppressed the phagocytosis and nitrite release in a concentration dependent manner. Biphasic suppressive effect of testosterone was observed in superoxide production as judged by reduction of nitroblue tetrazolium salt where salt reduction was suppressed at lower and higher concentrations of testosterone. Mitogen induced splenic lymphocyte proliferation was also suppressed by testosterone. By suppressing immune responses, testosterone may, therefore, act as a physiological mechanism regulating the relative amount of energy invested into either reproductive effort or immunocompetence.