B1 mRNA Levels Research Articles

New 2-oxoindole derivatives as multiple PDGFRα/ß and VEGFR-2 tyrosine kinase inhibitors

Two new series of N-aryl acetamides 6a-o and benzyloxy benzylidenes 9a-p based 2-oxoindole derivatives were designed as potent antiproliferative multiple kinase inhibitors. The results of one-dose NCI antiproliferative screening for compounds 6a-o and 9a-p elucidated that the most promising antiproliferative scaffolds were 6f and 9f, which underwent five-dose testing. Notably, the amido congener 6f was the most potent derivative towards pancreatic ductal adenocarcinoma MDA-PATC53 and PL45 cell lines (IC50 = 1.73 µM and 2.40 µM, respectively), and the benzyloxy derivative 9f was the next potent one with IC50 values of 2.85 µM and 2.96 µM, respectively. Both compounds 6f and 9f demonstrated a favorable safety profile when tested against normal prostate epithelial cells (RWPE-1). Additionally, compound 6f displayed exceptional selectivity as a multiple kinase inhibitor, particularly targeting PDGFRα, PDGFRβ, and VEGFR-2 kinases, with IC50 values of 7.41 nM, 6.18 nM, and 7.49 nM, respectively. In contrast, the reference compound Sunitinib exhibited IC50 values of 43.88 nM, 2.13 nM, and 78.46 nM against the same kinases. The derivative 9f followed closely, with IC50 values of 9.9 nM, 6.62 nM, and 22.21 nM for the respective kinases. Both 6f and 9f disrupt the G2/M cell cycle transition by upregulating p21 and reducing CDK1 and cyclin B1 mRNA levels. The interplay between targeted kinases and these cell cycle regulators underpins the G2/M cell cycle arrest induced by our compounds. Also, compounds 6f and 9f fundamentally resulted in entering MDA-PATC53 cells into the early stage of apoptosis with good percentages compared to the positive control Sunitinib. The in silico molecular-docking outcomes of scaffolds 6a-o and 9a-p in VEGFR-2, PDGFRα, and PDGFRβ active sites depicted their ability to adopt essential binding interactions like the reference Sunitinib. Our designed analogs, specifically 6f and 9f, possess promising antiproliferative and kinase inhibitory properties, making them potential candidates for further therapeutic development.

Combination of Quercetin or/and siRNA-loaded DDAB-mPEG-PCL hybrid nanoparticles reverse resistance to Regorafenib in colon cancer cells

BackgroundColorectal cancer (CRC) is the second leading cause of cancer death. Although Regorafenib showed survival benefits in patients with CRC, reports imply the recurrence of malignant phenotype resulting from chemotherapy. Evidence demonstrated that a5β1 integrin plays an important role in the Regorafenib treatment, which, may be led to resistance. In this study, the effects of /siRNA or/ and Quercetin loaded DDAB-mPEG-PCLnanoparticles could reverse this resistance phenotype in colon cancer cells in vitro.MethodsRegorafenib-resistant Ls-180 colon cancer cell line was developed by long-term exposure to Regorafenib. Quercetin and Regorafenib were separately encapsulated into mPEG-PCL micelles through the nano-precipitation method and characterized by DLS. Optimized doses of Quercetin and Regorafenib were used for combination therapy of resistant cells followed cytotoxicity study using MTT. Gene expression levels of the β1 subunit of integrin were determined by the real-time method of RT-PCR.ResultsDeveloped Regorafenib resistant LS-180 showed to have Regorafenib IC50 of 38.96 ± 1.72 µM whereas IC50 in non-resistant cells were 8.51 ± 0.29 µM, which meaningful was lower statistically compared to that of a resistant one. The β1 mRNA level of whole α5β1 integrin was significantly higher in the resistant cells compared to those of non-resistant ones. Gene expression levels in each siRNA-loaded nanoparticle and Quercetin-loaded one were lower than that in mock experiments. Finally, when these two types of nanoparticles were used to treat resistant cells, gene expression decrease of integrin indicated a greater effect that could be capable of reverse resistancy.ConclusionResults of this study demonstrated another confirmation of involving integrins in cancer resistance following chemotherapy using Regorafenib. Also, it indicated how using siRNA targeting integrin could enhance the plant derivatives like Quercetin effects to reverse resistance in vitro.

Abstract P029: Variable Expression Of Integrin Subunits In The Male And Female Kidneys Suggests A Key Factor Responsible For The Higher Prevalence Of Chronic Kidney Diseases In Women

Clinical and epidemiological studies have highlighted the sex differences in the incidence of chronic kidney diseases. However, the underlying pathological mechanisms contributing to the higher prevalence of chronic kidney disease in women are not well known. The cell’s reciprocal communication to the basement membrane is one of the important factors in maintaining kidney functions and injury responses. The cell-basement membrane communication is mediated by cell surface receptors. Integrins are heterodimeric cell surface receptors that consist of noncovalently associated α and β subunits and are the principal receptor that transduces signaling between the cell and basement membrane. This study investigated the sex difference in the expression pattern of integrin α2, α5, and integrin β1. The kidney cortex from 2 months old C57BL/6 mice (both male and female) was investigated for the expression pattern of integrin using q-RTPCR and immunoblotting. Immunoblotting shows a 35% decrease in integrin β1 protein in females (1.13±0.15) compared to males (1.73±0.36). This was further confirmed by the fold change in mRNA expression using q-RTPCR that shows significantly lower integrin β1 mRNA levels in females compared to males 1.097 ± 0.08 (male) and 0.693± 0.11(female). The expression of collagen receptor, integrin α2 did not show a significant difference in expression between males and females in immunoblotting (0.23±0.14 vs 0.78±0.35) and q-RTPCR (1.07±0.46 vs 1.36±0.47). The receptor for fibronectin, integrin α5 also did not alter between males and females by immunoblotting (1.00±0.28 vs 0.98±0.17) and q-RTPCR (1.05±0.55 vs 1.04±0.38). The cell-specific expression in kidneys is progressing using immune histochemistry and primary cell cultures. Integrin β1 KO mice developed higher fibrosis than WT mice when subjected to injury. This observed lower integrin β1 expression could be an important factor in the modification of cell microenvironment and contributes to the higher chronic kidney disease progression in women that require further investigation.

Estradiol and 1α,25(OH)2 vitamin D3 share plasma membrane downstream signal transduction through calcium influx and genomic activation in immature rat testis

The aim of this study was to investigate the rapid response pathway and gene and protein expression profiles of the rat testis in response to estradiol (E2) and 1α,25(OH)2 vitamin D3 (1,25-D3), to understand how they mediate their effects on the first spermatogenic wave. To do this, we compared the effects of 1,25-D3 and E2 on 45calcium(Ca2+) uptake and the involvement of estrogen receptors (ESR) in their rapid responses. Additionally, we studied the downstream signal transduction effects of 1,25-D3 and E2 on cyclin A1/B1 and cellular cycle protein expression. As previously observed for 1,25-D3, E2 also increased 45Ca2+ uptake in immature rat testes via voltage-dependent Ca2+ channels, Ca2+-dependent chloride channels and via the activation of protein kinase C, protein kinase A and mitogen-activated protein kinase kinase (MEK). Elevated aromatase expression by testes was observed in the presence of 1,25-D3 and both hormones decreased ESR mRNA expression. Furthermore, 1,25-D3 and E2 diminished cyclin A1 mRNA expression, but E2 did not affect cyclin B1 mRNA levels. Consistent with these findings, the immunocontent of cyclin A1 and B1 in the testes was also increased by 1,25-D3 and E2. 1,25-D3 increased expressions of the p16 and p53 proteins, supporting the anti-proliferative and pro-apoptotic properties of 1,25-D3, while E2 also augmented p16. Data indicate that both hormones trigger rapid responses at the plasma membrane that may control the expression of gene and proteins related to cell cycle regulation, and thereby modulate spermatogenesis.

Tissue specificity of senescent cell accumulation during physiologic and accelerated aging of mice.

Senescent cells accumulate with age in vertebrates and promote aging largely through their senescence‐associated secretory phenotype (SASP). Many types of stress induce senescence, including genotoxic stress. ERCC1‐XPF is a DNA repair endonuclease required for multiple DNA repair mechanisms that protect the nuclear genome. Humans or mice with reduced expression of this enzyme age rapidly due to increased levels of spontaneous, genotoxic stress. Here, we asked whether this corresponds to an increased level of senescent cells. p16Ink4a and p21Cip1 mRNA were increased ~15‐fold in peripheral lymphocytes from 4‐ to 5‐month‐old Ercc1 −/∆ and 2.5‐year‐old wild‐type (WT) mice, suggesting that these animals exhibit a similar biological age. p16Ink4a and p21Cip1 mRNA were elevated in 10 of 13 tissues analyzed from 4‐ to 5‐month‐old Ercc1 −/∆ mice, indicating where endogenous DNA damage drives senescence in vivo. Aged WT mice had similar increases of p16Ink4a and p21Cip1 mRNA in the same 10 tissues as the mutant mice. Senescence‐associated β–galactosidase activity and p21Cip1 protein also were increased in tissues of the progeroid and aged mice, while Lamin B1 mRNA and protein levels were diminished. In Ercc1 −/Δ mice with a p16Ink4a luciferase reporter, bioluminescence rose steadily with age, particularly in lung, thymus, and pancreas. These data illustrate where senescence occurs with natural and accelerated aging in mice and the relative extent of senescence among tissues. Interestingly, senescence was greater in male mice until the end of life. The similarities between Ercc1 −/∆ and aged WT mice support the conclusion that the DNA repair‐deficient mice accurately model the age‐related accumulation of senescent cells, albeit six‐times faster.

Optimal dietary curcumin improved growth performance, and modulated innate immunity, antioxidant capacity and related genes expression of NF-κB and Nrf2 signaling pathways in grass carp (Ctenopharyngodon idella) after infection with Aeromonas hydrophila

This study investigated the effects of dietary curcumin on growth performance, non-specific immunity, antioxidant capacity and related genes expression of NF-κB and Nrf2 signaling pathways in grass carp (Ctenopharyngodon idella). A total of 525 juvenile grass carps with mean initial body weight of (5.30 ± 0.10) g were randomly distributed into five groups with three replicates each, fed five diets containing graded levels of curcumin (0, 196.11, 393.67, 591.46 and 788.52 mg/kg diet) for 60 days. After feeding trial, fifteen fish per tank were challenged with Aeromonas hydrophila and the mortalities were recorded for 7 days. The results showed that optimal dietary curcumin (393.67 mg/kg diet) improved the weight gain (WG) and specific growth rate (SGR) of juvenile grass carp, reduced feed conversion ratio (FCR) and the mortalities after challenge (P < 0.05). Moreover, optimal dietary curcumin increased the activities of lysozyme (LYZ) and acid phosphatase (ACP), and complement 3 (C3) and C4 levels, decreased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in serum of grass carp after injection with A. hydrophila (P < 0.05). Meanwhile, optimal dietary curcumin up-regulated the mRNA levels of LYZ, C3 and antimicrobial peptides [hepcidin, liver-expressed antimicrobial peptide-2 (LEAP-2), β-defensin], and anti-inflammatory cytokines of interleukin-10 (IL-10) and transforming growth factor β1 (TGF-β1), and inhibitor of κBα (IκBα), whereas down-regulated pro-inflammatory cytokines of tumor necrosis factor-α (TNF-α), IL-1β, IL-6 and IL-8, and nuclear factor kappa B p65 (NF-κB p65), IκB kinases (IKKα, IKKβ and IKKγ) mRNA levels in the liver and blood of grass carp after injection with A. hydrophila (P < 0.05). In addition, optimal dietary curcumin increased the reduced glutathione (GSH) content and activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST) and glutathione reductase (GR), reduced reactive oxygen species (ROS) and malondialdehyde (MDA) levels in the liver of grass carp after injection with A. hydrophila (P < 0.05). Meanwhile, optimal dietary curcumin up-regulated the mRNA levels of these antioxidant enzymes and nuclear factor erythroid 2-related factor 2 (Nrf2), whereas down-regulated Kelch-like ECH-associated protein (Keap) 1a and Keap 1b mRNA levels (P < 0.05) in the liver and blood of grass carp after injection with A. hydrophila. Thus, optimal dietary curcumin supplementation could promote growth of juvenile grass carp, reduce FCR, and enhance disease resistance, innate immunity and antioxidant capacity of fish, attenuating inflammatory response. However, dietary excessive curcumin had negative effect on fish. Based on second-order regression analysis between dietary curcumin contents and weight gain, the optimum requirement of dietary curcumin in juvenile grass carp was determined to be 438.20 mg/kg diet.

Analysis of Lamin B1, Vimentin and Anti-Ku86 as Prospective Biomarkers of Hepatocellular Carcinoma in Patients with Hepatitis C Virus Infection.

Hepatocellular carcinoma (HCC) is the fifth most common malignancy and most frequently develops in patients with cirrhosis. Surveillance strategies are recommended in high-risk groups because early detection of small lesions improves the likelihood of curative treatment. This study investigated the prospective clinical significance of serum levels of anti-Ku86 and plasma levels of lamin B1and vimentin as early markers of HCC. We recruited 74 patients at Assiut University Hospital-37 with HCC and 37 with chronic liver disease (liver cirrhosis patients)-and 36 age- and sex-matched healthy controls. Lamin B1 and vimentin mRNA expression levels were evaluated by reverse transcription-PCR and serum levels of anti-Ku86 were measured by enzyme-linked immunosorbent assay. Compared with liver disease patients and controls, HCC patients showed higher levels of lamin B1 mRNA (sensitivity, 96%; specificity, 65%), vimentin mRNA (sensitivity, 94%; specificity, 92%), and anti-Ku86 (sensitivity, 94%; specificity, 80%). LaminB1 levels were significantly higher in patients with a tumor size < 2 cm than in patients with tumors 2-5 cm and >5cm in size. Lamin B1 had significant positive correlations with alpha-fetoprotein (AFP) (P=0.034) and anti-Ku86 (P=0.002). Receiver operating characteristic curves for differentiating HCCfrom liver cirrhosis revealed a higher area under the curve(AUC).for vimentin than for AFP, lamin B1, and anti-Ku86 for the diagnosis of HCC (P<0.001). Circulating levels of anti-Ku86, lamin B1,and vimentin might be potential surrogate markers of HCC, either alone or in combination with AFP. However, independent and discriminative serological biomarkers with higher sensitivity and specificity are still needed for the early detection of HCC.

Antifibrotic effect of pirfenidone on orbital fibroblasts in patients with thyroid-associated ophthalmopathy and its mechanisms

Objective: To investigate the effects of pirfenidone on orbital fibroblasts (OFs) from patients with thyroid-associated ophthalmopathy (TAO) and its underlying mechanisms. Methods: OFs from patients with TAO were isolated and cultured in DMEM. Cells were divided into four groups and treated with 0, 250, 500 and 1 000 μg/ml pirfenidone for 24, 48 or 72 hours, respectively. Cell proliferation was detected by tetramethyl azo salt (MTT) assay, and cell viability was determined by trypan blue. Transforming growth factor (TGF) β1 mRNA level was determined by real-time fluorescence quantitative PCR (RT-qPCR). Type Ⅰ and type Ⅲ collagen secreted from cultured cells were measured by enzyme-linked immuno sorbent assay (ELISA). Results: (1) The primary cultured OFs had typical fibroblast spindle-like morphology. (2) MTT assay showed that pirfenidone treatment significantly inhibited the proliferation of OFs in a dose-dependent manner (P<0.05) with the proliferation rates of pirfenidone treated groups of -15.31%, -24.92%, -48.53% from 250, 500, 1 000 μg/ml after 72 h, respectively, in which the inhibition effect of 1 000 μg/ml pirfenidone was significantly different from the other two treated groups (P<0.05). There were no significant differences in the inhibitory effect of the same concentration group among different time points at 24 h, 48 h and 72 h (P>0.05). Trypan blue showed that the survival rate of OFs in different concentrations of pirfenidone from 0,250, 500, 1 000 μg/ml at 72 h were 78.37%, 79.21%, 78.24% and 76.28%, respectively. There were no significant differences between each drug treated and the control group (P>0.05). (3) RT-qPCR results showed that the mRNA expression levels of TGFβ1 at 250, 500, 1 000 μg/ml pirfenidone treated groups at 72 h were 0.760±0.010, 0.440±0.006, and 0.290±0.002, respectively. Compared with the control group (0.950±0.014), the differences were statistically significant (all P<0.05). Moreover, TGFβ1 mRNA expression level in 1 000 μg/ml pirfenidone treated group was significantly lower than those in the other two treated groups (all P<0.05). The secretion of type Ⅰ collagen (0.633±0.006, 0.527±0.003 and 0.402±0.008) and type Ⅲ collagen (0.511±0.003, 0.439±0.007 and 0.223±0.006) in 250, 500 and 1 000 μg/ml pirfenidone treated groups at 72 h were significantly lower than those in the control group (0.794±0.005, 0.527±0.007, all P<0.05). Type Ⅰ and type Ⅲ collagen secretion in 1 000 μg/ml pirfenidone treated group were significantly lower than those in the other two groups (P<0.05). Conclusions: Pirfenidone inhibits the cell proliferation, TGFβ1 expression and collagen secretion of OFs, which may contribute to the anti-fibrotic effect of pirfenidone.

MicroRNA-144 inhibits cell proliferation, migration and invasion in human hepatocellular carcinoma by targeting CCNB1

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignancies with a high morbidity and mortality worldwide. MicroRNAs are key regulators of HCC genesis. However, the regulatory role and underlying mechanisms of microRNA in HCC is still limited.MethodsCyclin B1 (CCNB1) mRNA levels were examined in non-tumor and liver cancer of The Cancer Genome Atlas (TCGA) cohort. CCNB1 was knockdown to evaluate the HCC cell proliferation, migration and invasion. MicroRNA-144 targeting CCNB1 was identified with TargetScan analysis and confirmed with reporter assay. Overexpression of MicroRNA-144 was achieved using microRNA mimics and function of microRNA-144 was tested in vitro HCC cell line proliferation and in vivo tumor formation experiments.ResultsHere, we found that the high level expression of CCNB1 was closely associated with poor prognosis in HCC patients. Knockdown of CCNB1 by RNA interference significantly inhibited cell proliferation, migration and invasion in HCC. Furthermore, we found that miR-144 directly targeted CCNB1 and inhibited CCNB1 expression. Moreover, in vivo experiments of subcutaneous tumor formation further demonstrated that miR-144 delayed tumor formation by negative regulation of CCNB1.ConclusionTherefore, we conclude that microRNA-144/CCNB1 axis plays an important role in human HCC. Therapies targeting microRNA-144 could potentially improve HCC treatment.

Effect of Exogenous Zinc on MsrB1 Expression and Protein Oxidation in Human Lens Epithelial Cells.

Aging has been related to zinc deficiency, resulting in protein oxidation and age-related decline of methionine sulfoxide reductase (Msr) activity. This study was designed to investigate the levels of methionine sulfoxide reductase B1 (MsrB1) mRNA and oxidized proteins in human lens epithelial (hLE) cells after treatment with exogenous zinc. The role of exogenous zinc in regulation of MsrB1 gene expression and protein oxidation in hLE cells was studied by MTT assay, oxidized protein measurement kit, and real-time PCR. The results showed that hLE cell viability was significantly decreased by MsrB1 gene knockdown or peroxynitrite (ONOO-) treatment, while it was significantly increased after treatment with exogenous zinc (P < 0.05). Protein carbonyl content in hLE cell by MsrB1 gene knockdown or ONOO- treatment was significantly decreased after treatment with ZnSO4 (P < 0.01). And exogenous zinc could increase the level of MsrB1 in hLE cell under normal (P < 0.001) and oxidative stress (P < 0.01) conditions. In conclusion, exogenous zinc could protect hLE cells against MsrB1 gene knockdown or ONOO--induced cell death by upregulation of MsrB1 involved in the elimination of reactive oxygen species (ROS) and oxidized proteins.

Using Lamin B1 mRNA for the early diagnosis of hepatocellular carcinoma: a cross-sectional diagnostic accuracy study

Background : Hepatocellular carcinoma (HCC) is vital medical issue in Egypt. It accounts for 70.48% of all liver tumors among Egyptians. The aim of this study was to determine the diagnostic role of plasma levels of mRNA of lamin B1 by RT-qPCR as an early marker of HCC. Methods: This study was conducted at the Clinical Pathology Department in collaboration with the Department of Tropical Medicine and Infectious Diseases at Ain Shams University Hospitals. It included 30 patients with primary HCC and viral cirrhosis (all were hepatitis C virus-positive) (Group I), in addition to 10 patients with chronic liver diseases (Group II) and 10 healthy age- and sex-matched subjects (Group III). Group I was further classified according to the Barcelona-Clinic Liver Cancer Staging System. Serum α-fetoprotein (AFP) chemiluminescent-immunoassays and RT-qPCR analysis of plasma lamin B1 mRNA levels were performed for all participants. Results: AFP and lamin B1 significantly elevated in patients with HCC compared to those in the other studied groups. AFP and lamin B1 status could discriminate group I from group II and III. A significant increase was found among the three Barcelona stages with regards to AFP and lamin B1 levels. A significant decrease was found between group II and stage 0, A and B with regards to AFP and lamin B1. Lamin B1 and AFP could both differentiate HCC patients with one tumor nodule (T1) from those with two or more tumor nodules (T2&Tm), as well as between those with tumor sizes >3 cm and ≤3 cm. Conclusion: Measurement of lamin B1 mRNA is recommended in patients with chronic liver disease with normal serum AFP, especially in known cirrhotic patients that deteriorate rapidly without any apparent etiology.

Using Lamin B1 mRNA for the early diagnosis of hepatocellular carcinoma: a cross-sectional diagnostic accuracy study.

Background: Hepatocellular carcinoma (HCC) is vital medical issue in Egypt. It accounts for 70.48% of all liver tumors among Egyptians. The aim of this study was to determine the diagnostic role of plasma levels of mRNA of lamin B1 by RT-qPCR as an early marker of HCC. Methods: This study was conducted at the Clinical Pathology Department in collaboration with the Department of Tropical Medicine and Infectious Diseases at Ain Shams University Hospitals. It included 30 patients with primary HCC and viral cirrhosis (all were hepatitis C virus-positive) (Group I), in addition to 10 patients with chronic liver diseases (Group II) and 10 healthy age- and sex-matched subjects (Group III). Group I was further classified according to the Barcelona-Clinic Liver Cancer Staging System. Serum α-fetoprotein (AFP) chemiluminescent-immunoassays and RT-qPCR analysis of plasma lamin B1 mRNA levels were performed for all participants. Results: AFP and lamin B1 significantly elevated in patients with HCC compared to those in the other studied groups. AFP and lamin B1 status could discriminate group I from group II and III. A significant increase was found among the three Barcelona stages with regards to AFP and lamin B1 levels. A significant decrease was found between group II and stage 0, A and B with regards to AFP and lamin B1. Lamin B1 and AFP could both differentiate HCC patients with one tumor nodule (T1) from those with two or more tumor nodules (T2&Tm), as well as between those with tumor sizes >3 cm and ≤3 cm. Conclusion: Measurement of lamin B1 mRNA is recommended in patients with chronic liver disease with normal serum AFP, especially in known cirrhotic patients that deteriorate rapidly without any apparent etiology.

Deficiency of NOD1 Improves the β-Adrenergic Modulation of Ca2+ Handling in a Mouse Model of Heart Failure.

Heart failure (HF) is a complex syndrome characterized by cardiac dysfunction, Ca2+ mishandling, and chronic activation of the innate immune system. Reduced cardiac output in HF leads to compensatory mechanisms via activation of the adrenergic nervous system. In turn, chronic adrenergic overstimulation induces pro-arrhythmic events, increasing the rate of sudden death in failing patients. Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) is an innate immune modulator that plays a key role in HF progression. NOD1 deficiency in mice prevents Ca2+ mishandling in HF under basal conditions, but its role during β-adrenergic stimulation remains unknown. Here, we evaluated whether NOD1 regulates the β-adrenergic modulation of Ca2+ signaling in HF. Ca2+ dynamics were examined before and after isoproterenol perfusion in cardiomyocytes isolated from healthy and from post-myocardial infarction (PMI) wild-type (WT) and Nod1-/- mice. Isoproterenol administration induced similar effects on intracellular [Ca2+]i transients, cell contraction, and sarcoplasmic reticulum (SR)-Ca2+ load in healthy WT and Nod1-/- cells. However, compared with WT-PMI cells, isoproterenol exposure induced a significant increase in the [Ca2+]i transients and cell contraction parameters in Nod1-/--PMI cells, which mainly due to an increase in SR-Ca2+ load. NOD1 deficiency also prevented the increase in diastolic Ca2+ leak (Ca2+ waves) induced by isoproterenol in PMI cells. mRNA levels of β1 and β2 adrenergic receptors were significantly higher in Nod1-/--PMI hearts vs WT-PMI hearts. Healthy cardiomyocytes pre-treated with the selective agonist of NOD1, iE-DAP, and perfused with isoproterenol showed diminished [Ca2+]i transients amplitude, cell contraction, and SR-Ca2+ load compared with vehicle-treated cells. iE-DAP-treated cells also presented increased diastolic Ca2+ leak under β-adrenergic stimulation. The selectivity of iE-DAP on Ca2+ handling was validated by pre-treatment with the inactive analog of NOD1, iE-Lys. Overall, our data establish that NOD1 deficiency improves the β-adrenergic modulation of Ca2+ handling in failing hearts.

TGFβ1 synergizes with FLT3 ligand to induce chemoresistant quiescence in acute lymphoblastic leukemia with MLL gene rearrangements.

Fms-like tyrosine kinase 3 (FLT3) is highly expressed in mixed-lineage leukemia (MLL) gene-rearranged acute lymphoblastic leukemia (MLL + ALL) with a dismal prognosis. We previously reported that FLT3 ligand (FL) stimulation induced cell cycle arrest in MLL + ALL cells leading to resistance against anti-leukemic agents. Given that FL stimulation enhanced transforming growth factor (TGF)β1 mRNA levels in MLL + ALL cells, we extensively examined the effect of TGFβ1 on the cell cycle progression and chemosensitivity in MLL + ALL cells, and found that TGFβ1 stimulation induced MLL + ALL cells into cell cycle arrest resistant to arabinosyl cytosine; its effect was markedly enhanced in synergy with FL. Thus, it is likely that TGFβ1 and FL, both abundantly produced by bone marrow stromal cells, function in a coordinated manner to render MLL + ALL cells chemoresistant, which should lead to the development of minimal residual disease (MRD) resulting in relapse. The use of inhibitors against FLT3 and TGFβ1 may become a useful strategy for eradicating MRD in MLL + ALL.

Protective Effect of Copper (I)-Nicotinate Complex on DAB-Induced Hepatocellular Carcinoma in Rats

The aim of the present work was to evaluate the effect of copper (I)-nicotinate complex (CNC) on experimentally 4-Dimethylaminoazobenzene (DAB)-induced hepatocellular carcinoma (HCC) in male rats. In addition to the histopathological examination we measured hepatic glutathione content, malondialdehyde, nitric oxide levels, lamin B1 m-RNA, caspase-3 activity and serum interleukin-12 level after 2, 4, 6, 8 months from commencement of the experiment. Results: histopathological examination showed HCC development after 4 months of DAB administration. The hepatic glutathione content, lamin B1 m-RNA and nitric oxide levels were significantly elevated, while malondialdehyde, interlukin-12 levels and caspase-3 activity were significantly decreased with the progression and development of the HCC. On the other hand, administration of CNC one month before DAB delayed the development of neoplastic growth to the 8th month. Interestingly, when CNC was administered one month after DAB, it successfully prevented HCC development throughout the whole experiment as confirmed by histopathological data and explained by biochemical markers, as glutathione, lamin B-1 and nitric oxide were significantly declined but malondialdehyde, interlukin-12 and caspase-3 activity were significantly elevated compared to that in corresponding control group. Conclusion: CNC was able to delay or prevent HCC development in rats fed with the potent liver carcinogen DAB. Our data shows that CNC exerts its anti-tumour effects through modulating oxidative stress status as well as the machinery of apoptosis and angiogenesis. Therefore, CNC may be used as a potential protective anticancer agent.

Effects of exercise training on kinin receptors expression in rats with myocardial infarction

Objectives: The objective of this study is to determine the role of kinin B1 and B2 receptors in exercise-induced cardiac muscle angiogenesis.Method: Thirty Wistar rats were randomly assigned to the control group, the myocardial infarction group and the exercise training group (myocardial infarction model was made and received 30 min exercise training on a treadmill). After 4 weeks of experiment, cardiac muscle was harvested.Results: B1 and B2 receptor mRNA and protein levels in the exercise-training group were significantly higher than those in the myocardial infarction group, which were higher than those in the control group. Capillary number in the cardiac muscle also showed the same tendency. There was a correlation between capillary number and B1 receptor protein (not B2 receptor protein) in the all groups.Conclusion: Kinin B1 and B2 receptors play roles in exercise-induced cardiac muscle angiogenesis. However, the B1 receptor appears to have a more prominent role.

Reg proteins promote acinar-to-ductal metaplasia and act as novel diagnostic and prognostic markers in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignant tumor. Acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) are both precursor lesions that lead to the development of PDAC. Reg family proteins (Reg1A, 1B, 3A/G, 4) are a group of calcium-dependent lectins that promote islet growth in response to inflammation and/or injuries. The aim of this study was to establish a role for Reg proteins in the development of PDAC and their clinical value as biomarkers. We found that Reg1A and Reg3A/G were highly expressed in the ADM tissues by immunohistochemistry. In the 3-dimensional culture of mouse acinar cells, Reg3A promoted ADM formation with concurrent activation of mitogen-acitvated protein kinase. Upregulation of Reg1A and Reg1B levels was observed as benign ductal epithelium progresses from PanIN to invasive PDAC. Patients with PDAC showed significantly higher serum levels of Reg1A and Reg1B than matching healthy subjects. These results were further validated by the quantification of Reg 1A and 1B mRNA levels in the microdissected tissues (22- and 6-fold increases vs. non-tumor tissues). Interestingly, patients with higher levels of Reg1A and 1B exhibited improved survival rate than those with lower levels. Furthermore, tissue expressions of Reg1A, Reg1B, and Reg4 could differentiate metastatic PDAC in the liver from intrahepatic cholangiocarcinoma with 92% sensitivity and 95% specificity. Overall, our results demonstrate the upregulation of Reg proteins during PDAC development. If validated in larger scale, Reg1A and Reg1B could become clinical markers for detecting early stages of PDAC, monitoring therapeutic response, and/or predicting patient's prognosis.

Silencing of TBX20 gene expression in rat myocardial and human embryonic kidney cells leads to cell cycle arrest in G2 phase.

Congenital heart diseases (CHDs) are the most common birth defects due to abnormal cardiac development. The T-box 20 (TBX20) gene is a member of the T-box family of transcription factors and encodes TBX20, which is essential for early heart development. In the present study, reduced TBX20 expression was observed in CHD tissue samples compared with normal tissues, and the function of TBX20 in Rattus norvegicus myocardial cells [H9c2(2-1)] and human embryonic kidney cells (HEK293) was investigated. TBX20 was silenced in H9c2 and HEK293 cells via transfection of small interfering RNA and short hairpin RNA duplexes, respectively, and TBX20 mRNA and protein levels were subsequently examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Cell proliferation was assessed using a cell counting kit and proliferating cell nuclear antigen expression was determined by western blotting. Analysis of cell apoptosis was achieved by annexin V-fluorescein isothiocyanate/propidium iodide staining and a fluorometric terminal deoxynucleotidyl transferase dUTP nick-end labeling system. Cell cycle analysis was achieved using fluorescence-activated cell sorting, and, an RT-qPCR array was used to profile the expression of TBX20-related genes. Silencing of TBX20 in H9c2 and HEK293 cells significantly inhibited cell proliferation, induced cell apoptosis and led to G2/M cell cycle arrest. A reduction in cyclin B1 mRNA levels and an increase in cyclin-dependent kinase inhibitor 1B mRNA levels was observed, which indicated that cells were arrested in G2 phase. Concurrently, the mRNA levels of GATA binding protein 4 were increased in both cell lines, which may provide an explanation for the abnormal cardiac hypertrophy observed in patients with congenital heart disease. These results suggest that TBX20 is required for heart morphogenesis, and inhibition of TBX20 expression may lead to the suppression of cell proliferation and cell cycle arrest.

Endometrial Receptivity Markers in Mice Stimulated With Raloxifene Versus Clomiphene Citrate and Natural Cycles.

Ovulation induction therapy with clomiphene citrate can suppress endometrial receptivity. Raloxifene may be an alternative therapeutic for women with ovulatory disorders. This study aimed to compare the expression of endometrial receptivity markers, including homeobox gene 10 (HOXA10), integrin β3, and leukemia inhibitory factor (LIF), as well as pinopode production during the implantation window in mice stimulated with raloxifene and clomiphene citrate and natural cycles. Thirty-six 8-week-old female Kunming mice were randomly divided into 3 groups (n = 12) and administered daily raloxifene (22 mg/kg), clomiphene citrate (18 mg/kg), and normal saline (1 mL), respectively, by gavage. Two days later, mice were injected with 5 IU human chorionic gonadotropin and mated. Successfully mated female animals were identified with vaginal plugs designated gestation day 1. At day 4.5, pregnant donor mice were euthanized, and uterus samples were collected for immunohistochemistry, quantitative polymerase chain reaction, Western blot, and scanning electron microscopy analyses. Homeobox gene 10, integrin β3, and LIF messenger RNA (mRNA) and protein levels were significantly higher in the raloxifene-treated animals compared with the clomiphene citrate group (all P < .05) but not significantly different from saline group values, except for LIF and integrin β3 mRNA levels (P < .05). Pinopodes were abundant and well developed in the raloxifene and saline groups; however, in the clomiphene citrate-treated mice, fewer and poorly developed pinopodes were obtained. In mice, raloxifene had no effect on HOXA10, integrin β3, and LIF expression as well as pinopode production, suggesting it has no adverse effects on endometrial receptivity. Raloxifene may provide a viable alternative oral ovulation induction agent to clomiphene citrate.

Hepatic insulin-like growth-factor binding protein (igfbp) responses to food restriction in Atlantic salmon smolts

The growth hormone (Gh)/insulin-like growth-factor (Igf) system plays a central role in the regulation of growth in fishes. However, the roles of Igf binding proteins (Igfbps) in coordinating responses to food availability are unresolved, especially in anadromous fishes preparing for seaward migration. We assayed plasma Gh, Igf1, thyroid hormones and cortisol along with igfbp mRNA levels in fasted and fed Atlantic salmon (Salmo salar). Fish were fasted for 3 or 10days near the peak of smoltification (late April to early May). Fasting reduced plasma glucose by 3days and condition factor by 10days. Plasma Gh, cortisol, and thyroxine (T4) were not altered in response to fasting, whereas Igf1 and 3,5,3′-triiodo-l-thyronine (T3) were slightly higher and lower than controls, respectively. Hepatic igfbp1b1, -1b2, -2a, -2b1 and -2b2 mRNA levels were not responsive to fasting, but there were marked increases in igfbp1a1 following 3 and 10days of fasting. Fasting did not alter hepatic igf1 or igf2; however, muscle igf1 was diminished by 10days of fasting. There were no signs that fasting compromised branchial ionoregulatory functions, as indicated by unchanged Na+/K+-ATPase activity and ion pump/transporter mRNA levels. We conclude that dynamic hepatic igfbp1a1 and muscle igf1 expression participate in the modulation of Gh/Igf signaling in smolts undergoing catabolism.