Diosmetin inhibits cell proliferation and promotes apoptosis through STAT3/c-Myc signaling pathway in human osteosarcoma cells

BACKGROUND: Diosmetin is a bioflavonoid compound naturally abundant in citrus fruits. It is found to perform a variety of activities, while its antitumor property in osteosarcoma, a malignant tumor with unmet clinical treatment, remained unknown. METHODS: Colony formation assay, cell cycle analysis and apoptosis analysis were conducted respectively to observe the effect of diosmetin on cell proliferation and apoptosis in human osteosarcoma cells. Western blot and immunoprecipitation were used to detect the expression of apoptotic molecules and activation of STAT3/c-Myc pathway in Saos-2 and U2SO cells. RESULTS: Diosmetin significantly inhibited cell proliferation, induced cell cycle arrest at G2/M phase and promoted cell apoptosis in both Saos-2 and U2SO cells. Moreover, Diosmetin downregulated the expression of anti-apoptotic protein Bcl-xL while upregulated the levels of pro-apoptotic proteins including cleaved Caspase-3, cleaved-PARP and Bax. Furthermore, diosmetin dose-dependently inhibited STAT3 phosphorylation, reduced the expression of its downstream protein c-Myc and impeded the interaction between STAT3 molecules. CONCLUSIONS: These results suggest that diosmetin exerts anti-osteosarcoma effects by suppressing cell proliferation and inducing apoptosis via inhibiting the activation of STAT3/c-Myc signaling pathway, which provide the possibility for diosmetin to be a chemotherapeutic candidate for osteosarcoma.

Vitamina D, miR-21 y marcadores de apoptosis en pacientes con diabetes tipo I / Vitamin D, miR-21 and apoptosis markers in patients with type 1 diabetes

La diabetes Tipo 1 (DT1) es una compleja enfermedad autoinmune con una etiología aún desconocida. La vitamina D ha sido ampliamente estudiada debido a su potencial terapéutico en los potenciales nuevos casos de DT1. Por otra parte, los microARNs (miRs) han sido propuestos como posibles biomarcadores en diversos procesos biológicos como en la apoptosis e inflamación. El objetivo de este estudio fue evaluar el efecto de la suplementación con vitamina D sobre el perfil de expresión del miR-21 y marcadores de apoptosis tales como: BCL2, STAT3, TIPE2 y DAXX, en células mononucleares periféricas provenientes de pacientes con DT1 y sujetos controles. RESULTADOS: El perfil de expresión de miR-21 se encontró disminuido en los pacientes con DT1 en comparación con los controles. La expresión relativa de BCL2 se encontró aumentada en controles al comparar con pacientes DT1 en todas las condiciones experimentales. La expresión relativa de DAXX mostró un perfil de expresión diferencial al comparar pacientes con DT1 versus controles (p=0.006). CONCLUSIÓN: El estímulo con vitamina D parece tener un posible efecto regulador sobre los genes BCL2 y DAXX.

Type 1 diabetes (T1D) is a complex chronic autoimmune disease. Vitamin D has been one of the most studied therapeutic potential outbreaks related to T1D. Specific miRNAs have been proposed as potential biomarkers in several biological processes as apoptosis and inflammation. The aim of this study was to evaluate the effect of vitamin D on the expression profiles of miR-21 and apoptotic markers BCL2, STAT3, TIPE2 and DAXX, in PBMCs from T1D patients and control subjects. RESULTS: miR-21 expression was increased in controls regarding T1D patients. BCL2 was increased in controls compared to T1D patients in all experimental conditions. DAXX showed different expression patterns between T1D patients and controls (p=0.006). CONCLUSION: Vitamin D showed a possible regulation effect on apoptosis markers mainly through the regulation of BCL2 and DAXX

CXCL8(3-72) K11R/G31P protects against sepsis-induced acute kidney injury via NF-κB and JAK2/STAT3 pathway

BACKGROUND: Acute kidney injury (AKI), which is mainly caused by sepsis, has high morbidity and mortality rates. CXCL8(3-72) K11R/G31P (G31P) can exert therapeutic effect on inflammatory diseases and malignancies. We aimed to investigate the effect and mechanism of G31P on septic AKI. METHODS: An AKI mouse model was established, and kidney injury was assessed by histological analysis. The contents of serum creatinine (SCr) and blood urea nitrogen (BUN) were measured by commercial kits, whereas neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) were detected by enzyme-linked immunosorbent assay (ELISA) kits. The expressions of CXCL8 in serum and kidney tissues were determined using ELISA and immunohistochemical analysis, respectively. Apoptosis rate of renal tissue was detected by terminal deoxynucleotidyl transfer-mediated dUTP nick end labeling (TUNEL) analysis. The expressions of inflammatory cytokines were measured by quantitative real-time PCR and Western blot, respectively. The apoptosis-related proteins, JAK2, STAT3, NF-κB and IκB were determined by Western blot. RESULTS: G31P could reduce the levels of SCr, BUN, HGAL and KIM-1 and inhibit the renal tissue injury in AKI mice. G31P was also found to suppress the serum and nephric CXCL8 expressions and attenuated the apoptosis rate. The levels of inflammatory cytokines, pro-apoptotic proteins were decreased, while the anti-apoptotic proteins were increased by G31P in AKI mice. G31P also inhibited the activation of JAK2, STAT3 and NF-κB in AKI mice. CONCLUSION: These results suggest that G31P could protect renal function and attenuate the septic AKI. Our findings provide a potential target for the treatment of AKI.

Effect of lncRNA HULC knockdown on rat secreting pituitary adenoma GH3 cells

Pituitary adenoma is one of the most common tumors in the neuroendocrine system. This study investigated the effects of long non-coding RNAs (lncRNAs) highly up-regulated in liver cancer (HULC) on rat secreting pituitary adenoma GH3 cell viability, migration, invasion, apoptosis, and hormone secretion, as well as the underlying potential mechanisms. Cell transfection and qRT-PCR were used to change and measure the expression levels of HULC, miR-130b, and FOXM1. Cell viability, migration, invasion, and apoptosis were assessed using trypan blue staining assay, MTT assay, two-chamber transwell assay, Guava Nexin assay, and western blotting. The concentrations of prolactin (PRL) and growth hormone (GH) in culture supernatant of GH3 cells were assessed using ELISA. The targeting relationship between miR-130b and FOXM1 was verified using dual luciferase activity. Finally, the expression levels of key factors involved in PI3K/AKT/mTOR and JAK1/STAT3 pathways were evaluated using western blotting. We found that HULC was highly expressed in GH3 cells. Overexpression of HULC promoted GH3 cell viability, migration, invasion, PRL and GH secretion, as well as activated PI3K/AKT/mTOR and JAK1/STAT3 pathways. Knockdown of HULC had opposite effects and induced cell apoptosis. HULC negatively regulated the expression of miR-130b, and miR-130b participated in the effects of HULC on GH3 cells. FOXM1 was a target gene of miR-130b, which was involved in the regulation of GH3 cell viability, migration, invasion, and apoptosis, as well as PI3K/AKT/mTOR and JAK1/STAT3 pathways. In conclusion, HULC tumor-promoting roles in secreting pituitary adenoma might be via down-regulating miR-130b, up-regulating FOXM1, and activating PI3K/AKT/mTOR and JAK1/STAT3 pathways.

Rhein alleviates renal interstitial fibrosis by inhibiting tubular cell apoptosis in rats

BACKGROUND: Ureteral obstruction causes injury of the renal tissues and can irreversibly progress to renal fibrosis, with atrophy and apoptosis of tubular cells. The goal of the current study was to examine the effects of rhein on the apoptosis o renal tubular cells as well as renal fibrosis using a rodent model of unilateral ureteral obstruction (UUO). METHODS: UUO was induced through ureteral ligation, then animals received treatments with rhein or vehicle. The control rats only received sham operation. The renal tissue was harvested 1 week after surgery for assessment of kidney fibrosis. RESULTS: The expressions of collagen I and α-smooth muscle actin (α-SMA), as well as the severity of renal tubular apoptosis and fibrosis were time-dependently increased following UUO. Treatments with rhein partially inhibited such responses. Renal interstitial fibrosis was associated with STAT3 (signal transducer and activator of transcription 3) phosphorylation as well as altered expressions of Bax and Bcl2, both apoptosis-related proteins. Treatment with rhein also partly blocked these responses. CONCLUSION: These findings demonstrated that rhein mitigated apoptosis of renal tubular cell as well as renal fibrosis in a UUO rodent model. This curative effect is likely mediated via suppression of STAT3 phosphorylation.

Avaliação dos níveis de células CD44high/CD24low E TCD3+, e da expressão dos fatores de transcrição SOX2 e STAT3 no carcinoma escamoso de pênis / Evaluation of CD44highCD24low and TCD3 + cell levels, and expression of SOX2 and STAT3 transcription factors in penile squamous cell carcinoma

Introdução: O câncer de pênis (CaPe) tem uma alta incidência em alguns países subdesenvolvidos. O tratamento padrão é a remoção do tumor primário e a linfadenectomia inguinal, em alguns pacientes. O fator prognóstico mais importante é a doença linfonodal, porém os métodos de estadiamento, disponíveis na atualidade são pouco precisos, e a alta taxa de morbidade da linfadenectomia tem estimulado o estudo de biomarcadores preditivos de metástases em linfonodos, selecionando os pacientes que necessitam da linfadenectomia. Os marcadores STAT3 e CD44, CD24, e SOX2+ são conhecidos por serem considerados marcadores de diagnóstico e prognóstico em outros cânceres, mas sem estudos no câncer de pênis. Objetivo: Avaliar os níveis das células CD44high/CD24low E TCD3+, e a expressão dos fatores de transcrição SOX2 e STAT3 no carcinoma escamoso de pênis. Métodos: Estudo transversal realizado no Hospital de Câncer de Pernambuco (HCP) e Laboratório de Pesquisa Translacional do Instituto de Medicina Integral Prof. Fernando Figueira (IMIP), no período de março de 2015 a dezembro de 2017 em uma população de 38 pacientes com CaPe e controles. Foram realizadas análises de SOX2, STAT3, CD24 e CD44 no sangue e tecido tumoral por citometria de fluxo. Resultados: A mediana da idade foi de 61 anos (37 - 80 anos). A maioria dos pacientes apresentou tumor localizado na glande (76,3%), tipo histológico usual (71,0%), de grau 1 (86,8%), sem invasão vascular e perineural (86,8% e 68,4%), sem presença de metástases (86,8%). Com relação ao estadiamento, 55,3% dos pacientes apresentavam estágio pT2 e 34,2% pT1. Foram encontrados níveis elevados de CD44highCD24low, CD44highCD24lowpSTAT3+ e CD44highCD24lowSOX2+nos pacientes, quando comparados aos controles (p<0,05). Foram observados níveis percentuais reduzidos de leucócitos SOX2+ e TCD3+/SOX2+, e elevados leucócitos pSTAT3 nos pacientes, quando comparados aos dos controles saudáveis. Foram constatados níveis elevados de CD44highCD24low e de leucócitos SOX2+ no grupo de pacientes com invasão perineural, tamanho tumoral > 3 cm e em estádio pT2 (p<0,05). Foram encontradas: associação dos níveis de célulasCD44high/CD24low e no sangue periférico com o tamanho do tumor (p=0,04) e status de sobrevida (p=0,01) e de CD44high/CD24lowSOX2+ no sangue e tumor com status de sobrevida nos pacientes;expressão reduzida de pSTAT3 no sangue de pacientes com invasão perineural e elevados no tecido tumoral no grupo em estádio pT1;alta expressão de TCD3+ no sangue e tecido tumoral de pacientes com tumor ≤3cm;aumento de TCD3+SOX2+ no sangue de pacientes sem invasão perineural e em estádio pT1.Observou-se correlação de CD44highCD24lowpSTAT3+ (r=0,669; p=0,024), de pSTAT3 (r=0,487; p=0,018) e de TCD3+SOX2+ (r=0,404; p=0,029) entre o sangue e o tecido tumoral. Conclusão: As moléculas CD44, CD24 e SOX2 foram marcadores de doença avançada, sendo associados ao pior prognóstico no câncer de pênis, porém, p-STAT3 e TCD3+ foram associados a um prognóstico mais favorável neste estudo

Introduction: Penile cancer (CaPe) has a high incidence in some underdeveloped countries. The standard treatment is removal of the primary tumor and inguinal lymphadenectomy in some patients. At the moment, the most important prognostic factor is lymph node disease, but the staging methods currently available are not very accurate and the high morbidity rate of lymphadenectomy has prompted the study of biomarkers that may predict lymph node metastasis, and aid in the selection of patients who need lymphadenectomy. The markers STAT3 and CD44, CD24, SOX2 + are known to be markers of diagnosis and prognosis in other cancers, but there are no such studies on penile cancer. Aim: To evaluate the levels of CD44high/ CD24low and TCD3+ cells, and the expression of SOX2 and STAT3 transcription factors in squamous cell carcinoma of the penis. Methods: A cross-sectional study was conducted at the Hospital de Cancer de Pernambuco (HCP) and Translational Research Laboratory at the Prof. Fernando Figueira Institute of Integral Medicine (IMIP), from March 2015 to December 2017 of a population of 38 patients with CaPe and controls. Analyses of SOX2, STAT3, CD24 and CD44 were performed on blood and tumor tissue by flow cytometry. Results: The median age was 61 years (37-80 years). Most of the patients presented tumor localized in the glans (76.3%), histological type (71.0%), grade 1 (86.8%), without vascular and perineural invasion (86.8% and 68.4% %), without metastases (86.8%). Regarding staging, 55.3% of the patients had pT2 and 34.2% pT1. High levels of CD44highCD24low, CD44highCD24lowpSTAT3+and CD44highCD24low,SOX2+ were found in the patients compared to the controls (p <0.05). Reduced percentage levels of SOX2+ and TCD3+/ SOX2+ leukocytes and high pSTAT3 leukocytes were observed in the patients when compared to healthy controls. There were elevated levels of CD44highCD24low and SOX2+ leukocytes in the group of patients with perineural invasion, tumor size> 3 cm and at pT2 stage (p <0.05). Peripheral blood CD44highCD24low levels were found to be associated with the tumor size(p = 0.04) and survival status (p = 0.01) and CD44highCD24lowSOX2+ levels in the blood and tumor with survival status in patients. Reduced levels of pSTAT3 in the blood of patients with perineural invasion and elevated tumor tissue in the pT1 stage. There were elevated levels of TCD3 + in the blood and tumor tissue of patients with tumors of ≤3cm. Increased TCD3 + SOX2 + was found in the blood of patients without perineural and pT1 stage invasion. CD44highCD24lowpSTAT3 + (r = 0.669; p = 0.024), pSTAT3 (r = 0.487, p = 0.018) and TCD3 + SOX2 + (r = 0.404, p = 0.029) correlations were observed between blood and tumor tissue. Conclusion: CD44, CD24 and SOX2 molecules were markers of advanced disease, and were associated with the worst prognosis in penile cancer. However, p-STAT3 and TCD3 + were associated with a more favorable prognosis in this study

Differences in Hepatic Expression of Iron, Inflammation and Stress-Related Genes in Patients with Nonalcoholic Steatohepatitis

Abstract: Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide. We have previously shown that hepatic reticuloendothelial system (RES) iron deposition is associated with an advanced degree of nonalcoholic steatohepatitis (NASH) in humans. In this study, we aimed to determine differentially expressed genes related to iron overload, inflammation and oxidative stress pathways, with the goal of identifying factors associated with NASH progression. Seventy five patients with NAFLD were evaluated for their biochemical parameters and their liver tissue analyzed for NASH histological characteristics. Gene expression analysis of pathways related to iron homeostasis, inflammation and oxidative stress was performed using real-time PCR. Gene expression was compared between subjects based on disease status and presence of hepatic iron staining. We observed increased gene expression of hepcidin (HAMP) (2.3 fold, p = 0.027), transmembrane serine proteinase 6 (TMPRSS6) (8.4 fold, p = 0.003), signal transducer and activator of transcription 3 (STAT3) (5.5 fold, p = 0.004), proinflammatory cytokines; IL-1β (2.7 fold, p = 0.046) and TNF-α (3.8 fold, p = 0.001) in patients with NASH. TMPRSS6, a negative regulator of HAMP, is overexpressed in patients with NASH and HIF1α (hypoxia inducible factor-1) is downregulated. NAFLD patients with hepatic iron deposition exhibited higher hepcidin expression (3.1 fold, p = 0.04) but lower expression of cytokines. In conclusion, we observed elevated hepatic HAMP expression in patients with NASH and in NAFLD patients who had hepatic iron deposition, while proinflammatory cytokines displayed elevated expression only in patients with NASH, suggesting a regulatory role for hepcidin in NAFL to NASH transition and in mitigating inflammatory responses.

Activación de STAT3 por hipoxia en modelos in vitro de cáncer de cuello uterino y en células endoteliales / STAT3 activation by hypoxia in in vitro models of cervix cancer and endothelial cells

resumen Introducción: El microambiente tumoral influye en el comportamiento de las células cancerosas. Especialmente, el estímulo de agentes estresantes, como la hipoxia, se convierte en un factor crítico para la evolución y el tratamiento del cáncer. La reacción celular frente a diversos estímulos se manifiesta en la activación de vías de señalización como la JAK/STAT, una de las más importantes por sus efectos en la diferenciación y proliferación celular. Objetivo: Evaluar el estado de la vía JAK/STAT mediante la expresión o activación de la proteína STAT3 en células de cáncer de cuello uterino (HeLa) y en células endoteliales (EA.hy926) sometidas a hipoxia. Materiales y métodos: Las líneas celulares se sometieron a condiciones de hipoxia física (1 % de O2) o química (100 μM de deferoxamina, DFO) durante dos, seis y 24 horas. Mediante Western blot se determinó el cambio en la expresión y activación de STAT3, y mediante inmunofluorescencia indirecta su localización subcelular. Resultados:. La hipoxia se evidenció por la activación y translocación al núcleo del HIF-1. Ni la hipoxia física ni la química alteraron la expresión de STAT3, pero sí la activación, según se comprobó por su fosforilación y su translocación al núcleo en los dos modelos bajo estudio. Conclusiones: Se evidenció la importancia de la hipoxia como un estímulo que modifica la activación de la proteína STAT3 en las células HeLa y EA.hy926, lo cual la convierte en un elemento importante en el diseño de estrategias terapéuticas contra el cáncer.

Abstract Introduction: The biological behavior of cancer cells is influenced by the tumor microenvironment in which they develop. In this context, stressor stimuli such as hypoxia are considered critical for tumor development and therapeutic management. Cellular response to various stimuli is evidenced in the activation of intracellular signaling pathways such as JAK/STAT, which is one of the most important for its effects in differentiation and cell proliferation. Objective: To evaluate the condition of the JAK/STAT pathway through the expression/activation of the STAT3 protein in cervix cancer cells (HeLa) and endothelial cells (EA.hy926) subjected to ypoxia. Material and methods: Cell lines were subjected to physical (1% O2) or chemical (deferoxamine, DFO, 100 μM) hypoxia for 2, 6 and 24 hours. Changes in the expression and activation of STAT3, and its subcellular localization by indirect immunofluorescence, were determined by western blot. Results: Hypoxia was evidenced by the activation and translocation to the nucleus of HIF-1. Neither physical nor chemical hypoxia altered STAT3 expression, but it did affect its activation, as seen in its phosphorylation and translocation to the nucleus in the two models under study. Conclusions: The present study highlights the importance of hypoxia as a stimulus that modifies the activation of the STAT3 protein in HeLa and EA.hy926 cells, which makes it an important factor in the design of therapeutic strategies against cancer.

Hepato-protective effect of rutin via IL-6/STAT3 pathway in CCl4-induced hepatotoxicity in rats

BACKGROUND: Carbon tetrachloride (CCl4) induces hepatotoxicity in animal models, including the increased blood flow and cytokine accumulation that are characteristic of tissue inflammation. The present study investigates the hepato-protective effect of rutin on CCl4-induced hepatotoxicity in rats. RESULTS: Forty male Wistar rats were divided into four groups. Group I (control group) received 1 mL/kg of dimethyl sulfoxide intragastrically and 3 mL/kg olive oil intraperitoneally twice a week for 4 weeks. Group II received 70 mg/ kg rutin intragastrically. Groups III and IV received CCl4 (3 mL/kg, 30 % in olive oil) intraperitoneally twice a week for 4 weeks. Group IV received 70 mg/kg rutin intragastrically after 48 h of CCl4 treatment. Liver enzyme levels were determined in all studied groups. Expression of the following genes were monitored with real-time PCR: interleukin-6 (IL-6), dual-specificity protein kinase 5 (MEK5), Fas-associated death domain protein (FADD), epidermal growth factor (EGF), signal transducer and activator of transcription 3 (STAT3), Janus kinase (JAK), B-cell lymphoma 2 (Bcl2) and B-cell lymphoma-extra-large (Bcl-XL). The CCl4 groups showed significant increases in biochemical markers of hepatotoxicity and up-regulation of expression levels of IL-6, Bcl-XL, MEK5, FADD, EGF, STAT3 and JAK compared with the control group. However, CCl4 administration resulted in significant down-regulation of Bcl2 expression compared with the control group. Interestingly, rutin supplementation completely reversed the biochemical markers of hepatotoxicity and the gene expression alterations induced by CCl4. CONCLUSION: CCl4 administration causes alteration in expression of IL-6/STAT3 pathway genes, resulting in hepatotoxicity. Rutin protects against CCl4-induced hepatotoxicity by reversing these expression changes.

Transcriptional activity of the 5'-flanking region of the thyroid transcription factor-1 gene in human thyroid cell lines

Thyroid transcription factor-1 (TTF-1, NKX2-1) is a homeodomain-containing transcriptional factor that binds to and activates the promoters of thyroid and lung-specific genes, such as thyroglobulin, thyroid peroxidase, and thyroid stimulating hormone receptor. TTF-1 is known to play a key role in the development of the thyroid. However, the precise mechanism of TTF-1 gene transcription in human thyroid cells has not been studied. The expression of transcriptional activity in various lengths of the 5'-flanking region of the human TTF -1 gene was studied in TTF-1 positive and negative human thyroid cell lines. Increased transcriptional activity was observed in thyroid cell lines containing plasmids that coded for a sequence proximal to the transcription start site of exon 1 of the TTF-1 gene. However, we did not observe any difference in promoter activity in the region up to -2.6 kb from the proximal transcription start site of the TTF-1 gene between TTF-1 positive and negative cells. These results suggest that the proximal 5'-flanking region of the human TTF -1 gene does not contain sufficient cis-active regulatory information to direct gene expression in thyroid cells,and that other cis-or trans-acting factors participate in the thyroid specific gene expression of TTF-1.