Hydroxyurea modifies mesenchymal stromal cells functionality by senescence induction

Мезенхималне стромалне ћелије (МСЋ) су популација матичних ћелија које се одликују имунорегулаторним карактеристикама и диференцијационим потенцијалом ка остеобластима, хондроцитима и адипоцитима. МСЋ се могу изоловати из скоро свих адултних ткива а најчешће се добијају из костне сржи. Познато је да ови прогенитори који се налазе у циркулацији и костној сржи могу имати улогу како у стимулацији тако и у инхибицији пролиферације малигних ћелија и индукције профибротичног фенотипа. На карактеристике МСЋ могу утицати бројни фактори попут инфламаторних цитокина, реактвних кисеоничних врста (енг. reactive oxygen species, ROS), азот моноксида (енг. nitric oxide, NO) и индуктора стреса који се повезују и са настанком ћелијског старења, сенесценције. Хидроксиуреа (ХУ) је антинеопластични агенс који инхибира рибонуклеотид-редуктазу и користи се у терапији хематолошких малигнитета. Познато је да ХУ има цитостатски ефекат и проузрокује ДНК оштећење које може водити у превремену сенесценцију, али утицај ХУ на карактеристике МСЋ и интеракцију са ћелијама у околини посредством узгредног, bystander ефекта није до сада проучаван. Циљ ове студије био је да се анализира ефекат ХУ на карактеристике МСЋ, као што су морфологија, имунофенотип, диферецијациони капацитет, испољеност сенесцентног фенотипа, имуномодулаторне, профибротичне способности ових ћелија као и њихову улогу у туморогенези. Методе: МСЋ су успешно изоловане из периферне крви и костне сржи здравих донора а потом су окарактерисане према критеријумима Комитета за МСЋ Међународног друштва за ћелијску терапију. Генотоксични ефекат, индукција сенесцентних и профибротичних маркера под утицајем ХУ су анализирани имунохистохемијском и имунофлуоресцентном методом. Продукција унутарћелијског ROS (енг. reactive oxygen species) и NO (енг. nitric oxide) је анализирана употребом флуоресцентних реагенаса DCF и DAF. Анализа ћелијског циклуса и пролиферације, као и детекција површинских антигена је извршена методом проточне цитометрије. Утицај ХУ на активацију mTOR, MAPK, JAK/STAT и TGFbeta/SMAD сигналних путева процењен је методом имуноблота. Резултати: МСЋ су окарактерисане према способности адхерирања за пластику, експресији мезенхималних уз одсуство хематопоетских површинских антигена и диференцијационом потенцијалу ка остеобластима и адипоцитима...Mesenchymal stromal cells (MSC) are the population of stem cells with immunoregulatory features and significant differentiation potential towards osteoblast, chondrocytes and adipocytes. MSC have been isolated from abundant adult tissues, most frequently from bone marrow. As a circulating and the bone marrow microenvironment progenitors, those cells have a dual role as a promoters or inhibitors of both, malignant cell proliferation and profibrotic phenotype induction. Variation of MSC characteristics are associated with numerous factors such as inflammatory cytokines, reactive oxygen species (ROS), nitric oxide (NO) and stress inducers that could also induce senescence. Hydroxyurea (HU) is an antineoplastic agent that functions as the ribonucleotide reductase inhibitor and is mainly used in the treatment of hematological malignancies. As a DNA replication stress inducer HU may trigger a premature senescence-like cell phenotype, though its influence on MSC characteristics and repercussion on bystander cell proliferation has not elucidated yet. The aim of this study was to estimate the effect of HU on MSC morphology, immunophenotype, multilineage differentiation, senescencent phenotype, immunomodulatory and profibrotic activity as well as the roll of the HU treated MSC in tumorogenesis. Methods: MSC were successfully isolated from bone marrow and peripheral blood healthy donors. They were characterised in the presence and absence of HU, by criteria from MSC Committee of the International Society for Cellular Therapy. Genotoxic effect of HU, as well as the expression of senescence and profibrotic markers, were estimated by immunohistochemistry and immunofluorescence. Intracellular ROS and NO production was determined by fluorogenic molecules DCF and DAF. Cell cycle analysis, cell proliferation and surface markers detection was performed by flow cytometry. Influence of HU on activation of mTOR, MAPK, JAK/STAT and TGF/SMAD were determined by immunoblothing methods. Results: MSC were characterised by their plastic surface adhesion, expression of mesenchymal cell surface markers, lack of the expression of hematopoietic markers and the capacity to differentiate towards osteoblast and adipocytes. Examining the HU effect on MSC, we found that HU has the mild cytostatic effect and provokes cell cycle arrest in the S phase as the consequence of the DNA damage response evidenced by expression of gamaH2A.X and micronuclei..

Transforming growth factor-beta1 and myeloid-derived suppressor cells: A cancerous partnership

Transforming growth factor-beta1 (TGF-beta 1) plays a crucial role in tumor progression. It can inhibit early cancer stages but promotes tumor growth and development at the late stages of tumorigenesis. TGF-beta 1 has a potent immunosuppressive function within the tumor microenvironment that largely contributes to tumor cells' immune escape and reduction in cancer immunotherapy responses. Likewise, myeloid-derived suppressor cells (MDSCs) have been postulated as leading tumor promoters and a hallmark of cancer immune evasion mechanisms. This review attempts to analyze the prominent roles of both TGF-beta 1 and MDSCs and their interplay in cancer immunity. Furthermore, therapies against either TGF-beta 1 or MDSCs, and their potential synergistic combination with immunotherapies are discussed. Simultaneous TGF-beta 1 and MDSCs inhibition suggest a potential improvement in immunotherapy or subverted tumor immune resistance

Estramustine Phosphate Inhibits TGF-beta-Induced Mouse Macrophage Migration and Urokinase-Type Plasminogen Activator Production

Transforming growth factor-beta (TGF-beta) has been demonstrated as a key regulator of immune responses including monocyte/macrophage functions. TGF-beta regulates macrophage cell migration and polarization, as well as it is shown to modulate macrophage urokinase-type plasminogen activator (uPA) production, which also contributes to macrophage chemotaxis and migration toward damaged or inflamed tissues. Microtubule (MT) cytoskeleton dynamic plays a key role during the cell motility, and any interference on the MT network profoundly affects cell migration. In this study, by using estramustine phosphate (EP), which modifies MT stability, we analysed whether tubulin cytoskeleton contributes to TGF-beta-induced macrophage cell migration and uPA expression. We found out that, in the murine macrophage cell line RAW 264.7, EP at noncytotoxic concentrations inhibited cell migration and uPA expression induced by TGF-beta. Moreover, EP greatly reduced the capacity of TGF-beta to trigger the phosphorylation and activation of its downstream Smad3 effector. Furthermore, Smad3 activation seems to be critical for the increased cell motility. Thus, our data suggest that EP, by interfering with MT dynamics, inhibits TGF-beta-induced RAW 264.7 cell migration paralleled with reduction of uPA induction, in part by disabling Smad3 activation by TGF-beta

Long-Term Effects of Maternal Deprivation on the Volume of Dopaminergic Nuclei and Number of Dopaminergic Neurons in Substantia Nigra and Ventral Tegmental Area in Rats

Early life adversities leave long-lasting structural and functional consequences on the brain, which may persist later in life. Dopamine is a neurotransmitter that is extremely important in mood and motor control. The aim of this study was to investigate the effect of maternal deprivation during the ninth postnatal day on the volume of dopaminergic nuclei and the number of dopaminergic neurons in adolescence and adulthood. Maternally deprived and control Wistar rats were sacrificed on postnatal day 35 or 60, and the dopaminergic neurons were stained in coronal histological sections of ventral midbrain with the tyrosine hydroxylase antibody. The volume of dopaminergic nuclei and the number of dopaminergic neurons in the substantia nigra (SN) and ventral tegmental area (VTA) were analyzed in three representative coordinates. Maternal deprivation caused weight loss on postnatal day 21 (weaning) and corticosterone blood level elevation on postnatal days 35 and 60 in stressed compared to control rats. In maternally deprived animals, the volumes of SN and VTA were increased compared to the controls. This increase was accompanied by an elevation in the number of dopaminergic neurons in both nuclei. Altogether, based on somatic and corticosterone level measurements, maternal deprivation represents a substantial adversity, and the phenotype it causes in adulthood includes increased volume of the dopaminergic nuclei and number of dopaminergic neurons

Hydroxyurea-induced senescent peripheral blood mesenchymal stromal cells inhibit bystander cell proliferation of JAK2V617F-positive human erythroleukemia cells

Hydroxyurea (HU) is a nonalkylating antineoplastic agent used in the treatment of hematological malignancies. HU is a DNA replication stress inducer, and as such, it may induce a premature senescence-like cell phenotype; however, its repercussion on bystander cell proliferation has not been revealed so far. Our results indicate that HU strongly inhibited peripheral blood mesenchymal stromal cells (PBMSC) proliferation by cell cycle arrest in S phase, and that, consequently, PBMSC acquire senescence-related phenotypical changes. HU-treated PBMSC display increased senescence-associated beta-galactosidase levels and p16(INK4) expression, as well as DNA damage response and genotoxic effects, evidenced by expression of gamma H2A.X and micronuclei. Moreover, HU-induced PBMSC senescence is mediated by increased reactive oxygen species (ROS) levels, as demonstrated by the inhibition of senescence markers in the presence of ROS scavenger N-acetylcysteine and NADPH oxidase inhibitor Apocynin. To determine the HU-induced bystander effect, we used the JAK2V617F-positive human erythroleukemia 92.1.7 (HEL) cells. Co-culture with HU-induced senescent PBMSC (HU-S-PBMSC) strongly inhibited bystander HEL cell proliferation, and this effect is mediated by both ROS and transforming growth factor (TGF)-beta expression. Besides induction of premature senescence, HU educates PBMSC toward an inhibitory phenotype of HEL cell proliferation. Finally, our study contributes to the understanding of the role of HU-induced PBMSC senescence as a potential adjuvant in hematological malignancy therapies

Hydroxyurea modifies mesenchymal stromal cells functionality by senescence induction

Мезенхималне стромалне ћелије (МСЋ) су популација матичних ћелија које се одликују имунорегулаторним карактеристикама и диференцијационим потенцијалом ка остеобластима, хондроцитима и адипоцитима. МСЋ се могу изоловати из скоро свих адултних ткива а најчешће се добијају из костне сржи. Познато је да ови прогенитори који се налазе у циркулацији и костној сржи могу имати улогу како у стимулацији тако и у инхибицији пролиферације малигних ћелија и индукције профибротичног фенотипа. На карактеристике МСЋ могу утицати бројни фактори попут инфламаторних цитокина, реактвних кисеоничних врста (енг. reactive oxygen species, ROS), азот моноксида (енг. nitric oxide, NO) и индуктора стреса који се повезују и са настанком ћелијског старења, сенесценције. Хидроксиуреа (ХУ) је антинеопластични агенс који инхибира рибонуклеотид-редуктазу и користи се у терапији хематолошких малигнитета. Познато је да ХУ има цитостатски ефекат и проузрокује ДНК оштећење које може водити у превремену сенесценцију, али утицај ХУ на карактеристике МСЋ и интеракцију са ћелијама у околини посредством узгредног, bystander ефекта није до сада проучаван. Циљ ове студије био је да се анализира ефекат ХУ на карактеристике МСЋ, као што су морфологија, имунофенотип, диферецијациони капацитет, испољеност сенесцентног фенотипа, имуномодулаторне, профибротичне способности ових ћелија као и њихову улогу у туморогенези. Методе: МСЋ су успешно изоловане из периферне крви и костне сржи здравих донора а потом су окарактерисане према критеријумима Комитета за МСЋ Међународног друштва за ћелијску терапију. Генотоксични ефекат, индукција сенесцентних и профибротичних маркера под утицајем ХУ су анализирани имунохистохемијском и имунофлуоресцентном методом. Продукција унутарћелијског ROS (енг. reactive oxygen species) и NO (енг. nitric oxide) је анализирана употребом флуоресцентних реагенаса DCF и DAF. Анализа ћелијског циклуса и пролиферације, као и детекција површинских антигена је извршена методом проточне цитометрије. Утицај ХУ на активацију mTOR, MAPK, JAK/STAT и TGFbeta/SMAD сигналних путева процењен је методом имуноблота. Резултати: МСЋ су окарактерисане према способности адхерирања за пластику, експресији мезенхималних уз одсуство хематопоетских површинских антигена и диференцијационом потенцијалу ка остеобластима и адипоцитима...Mesenchymal stromal cells (MSC) are the population of stem cells with immunoregulatory features and significant differentiation potential towards osteoblast, chondrocytes and adipocytes. MSC have been isolated from abundant adult tissues, most frequently from bone marrow. As a circulating and the bone marrow microenvironment progenitors, those cells have a dual role as a promoters or inhibitors of both, malignant cell proliferation and profibrotic phenotype induction. Variation of MSC characteristics are associated with numerous factors such as inflammatory cytokines, reactive oxygen species (ROS), nitric oxide (NO) and stress inducers that could also induce senescence. Hydroxyurea (HU) is an antineoplastic agent that functions as the ribonucleotide reductase inhibitor and is mainly used in the treatment of hematological malignancies. As a DNA replication stress inducer HU may trigger a premature senescence-like cell phenotype, though its influence on MSC characteristics and repercussion on bystander cell proliferation has not elucidated yet. The aim of this study was to estimate the effect of HU on MSC morphology, immunophenotype, multilineage differentiation, senescencent phenotype, immunomodulatory and profibrotic activity as well as the roll of the HU treated MSC in tumorogenesis. Methods: MSC were successfully isolated from bone marrow and peripheral blood healthy donors. They were characterised in the presence and absence of HU, by criteria from MSC Committee of the International Society for Cellular Therapy. Genotoxic effect of HU, as well as the expression of senescence and profibrotic markers, were estimated by immunohistochemistry and immunofluorescence. Intracellular ROS and NO production was determined by fluorogenic molecules DCF and DAF. Cell cycle analysis, cell proliferation and surface markers detection was performed by flow cytometry. Influence of HU on activation of mTOR, MAPK, JAK/STAT and TGF/SMAD were determined by immunoblothing methods. Results: MSC were characterised by their plastic surface adhesion, expression of mesenchymal cell surface markers, lack of the expression of hematopoietic markers and the capacity to differentiate towards osteoblast and adipocytes. Examining the HU effect on MSC, we found that HU has the mild cytostatic effect and provokes cell cycle arrest in the S phase as the consequence of the DNA damage response evidenced by expression of gamaH2A.X and micronuclei..

Novel Patents Targeting Interleukin-17A; Implications in Cancer and Inflammation

Background: IL-17A is a founding member of the IL-17 family that has been implicated in the pathogenesis of inflammatory-associated diseases such as cancer and autoimmune disease. In cancer, IL-17A participates in many key events for tumor development, in part by affecting innate and adaptive immune system and also by direct modulation of many pro-tumor events. Moreover, IL-17A dysregulation at the site of inflammation is associated with rheumatoid arthritis, multiple sclerosis, psoriasis, among others. IL-17A has emerged as a topic of interest and is under profound investigation for its involvement in several types of inflammatory-associated diseases. Objective: This review aims to present an overview of the state of the art of IL-17A role in cancer and inflammation, as well as to describe recent patents targeting IL-17A with relevant clinical and biological properties for the prevention and treatment of cancer and inflammatory diseases. Methods: Relevant information was obtained by searching in PubMed using IL-17A or IL-17, cancer and inflammation as keywords, while relevant patents were obtained mainly from Google Patents. Results: Literature data indicated IL-17A as important biomolecule in the physiopathology of cancer and inflammatory diseases. Whereas, novel patents (2010 to 2017) targeting IL-17A are focused mainly on describing strategies to modulate IL-17A per se, co-modulation by bispecific antibodies to blocking IL-17A and important cytokines for IL-17A functions, upstream mechanisms and compounds to regulate IL-17A expression. Conclusion: The promising effects of patented agents against IL-17A may open new opportunities to therapeutic intervention targeting at different levels of involvement in the pathogenesis of cancer and inflammatory diseases

Paclitaxel inhibits transforming growth factor-beta-increased urokinase-type plasminogen activator expression through p38 MAPK and RAW 264.7 macrophage migration

Purpose: Transforming growth factor-beta (TGF-beta) induces alternative macrophage activation that favors tumor progression and immunosuppression. Meanwhile, paclitaxel (PTx) induces macrophage (M phi) polarization towards antitumor phenotype. TGF-beta also increases tumor stroma macrophage recruitment by mechanisms that include cell motility enhancement and extracellular matrix degradation. In this study, we aimed to determine whether PTx regulates macrophage migration and urokinase-type plasminogen activator (uPA) expression induced by TGF-beta. Methods: We used mouse macrophage RAW 264.7 cells treated with PTx and TGF-beta combinations. Proliferation was analyzed by MTT and cell cycle assays. Immunofluorescence was performed to determine tubulin cytoskeleton and Smad3 nuclear localization. Western blot and transcriptional luciferase reporters were used to measure signal transduction activation. Migration was determined by wound healing assay. uPA activity was determined by zymography assay. Results: PTx decreased RAW 264.7 cell proliferation by inducing G2/M cell cycle arrest and profoundly modified the tubulin cytoskeleton. Also, PTx inhibited TGF-beta-induced Smad3 activation. Furthermore, PTx decreased cell migration and uPA expression stimulated by TGF-beta. Remarkably, p38 MAPK mediated PTx inhibition of uPA activity induced by TGF-beta but it was not implicated on cell migration inhibition. Conclusions: PTx inhibits TGF-beta induction of mouse M phi migration and uPA expression, suggesting that PTx, as TGF-beta targeting therapy, may enhance MT anticancer action within tumors

Oxidative and nitrosative stress in myeloproliferative neoplasms: the impact on the AKT/mTOR signaling pathway

Purpose: A common feature of malignancies is increased reactive oxygen species (ROS) and reactive nitrogen species (RNS). We analyzed the influence of oxidative and nitrosative stress on the activation of AKT/mTOR signaling pathway in myeloproliferative neoplasms (MPN). Methods: Oxidative stress-induced gene expression in circulatory CD34(+) cells of MPN patients was studied by microarray analysis. Biomarkers of oxidative and nitrosative stress were determined using spectrophotometry in plasma and erythrocyte lysate. The levels of nitrotyrosine, inducible NO synthase (iNOS) and AKT/mTOR/p70S6K phosphorylation were determined by immunocytochemistry and immunoblotting in granulocytes of MPN patients. Results: Antioxidants superoxide dismutase 2 (SOD2) and glutathione peroxidase 1 (GPx1) gene expression were increased in circulatory CD34(+) cells, while SODI and GPx enzymes were reduced in the erythrocytes of MPN. Plasma malonyl-dialdehyde and protein carbonyl levels were elevated in MPN. The total antioxidant capacity in plasma and erythrocyte catalase (CT) activities was the most prominent in primary myelofibrosis (PMF) with JAK2V617F heterozygosity. The total nitrite/ nitrate (NOx) level was augmented in the plasma of PMF patients (p lt 0.001), while nitrotyrosine and iNOS were generally increased in the granulocytes of MPN patients. Activation of AKT/m TOR signaling was the most significant in PMF (p lt 0.01), but demonstrated JAK2V617F dependence and consequent p70S6K phosphorylation in the granulocytes of essential thrombocytemia (ET) and polycythemia vera (PV) patients. Hydrogen peroxide stimulated mTOR pathway, iNOS and nitrotyrosine quantities, the last one prevented by the antioxidant nacetyl-cysteine (NAC) in the granulocytes of MPN. Conclusion: Our study showed increased levels of oxidative and nitrosative stress parameters in MPN with JAK2V617F dependence. The ROS enhanced the constitutive activation of AKT/mTOR signaling and nitrosative parameters in MPN