Urine micro-RNA signature as a potential non-invasive diagnostic biomarker in bladder cancer.

Background: Bladder cancer (BC) is one of the most prevalent malignancies worldwide, 70% of patients initially diagnosed with superficial BC. In addition, 20% of BC patients with recurrence experience disease progression. Thus, identification of novel biomarkers for diagnosis, prognosis and therapeutic targets of BC will help to advance clinical diagnosis and treatment of this disease. MicroRNAs (miRNAs) are single stranded, non coding RNAs that are hypothesized to regulate gene expression at the post transcriptional level. This study aimed to assess the urine and tissue expression levels of miR-200, miR-145 and miR-21 in BC patients o evaluate their potential as noninvasive biomarkers. Subjects and methods: Urine and their corresponding tissue samples were collected from 111 BC patients and from 25 healthy controls. A quantitative real-time polymerase chain reaction method based on a TaqMan probe was used to evaluate the expression levels of miR­200, miR­145 and miR-21, the correlations between these miRNA expression levels in urine and tissues and certain clinicopathological parameters were investigated. Results: The expression of the 3 studied miRNAs was significantly higher in urine of low and high tumor grade BC patients compared to the controls and the expression were increased in BC tissues compared with those in normal bladder tissues, the results proved that the 3 miRNAs function as oncogenes. A marked positive correlation was observed between the mRNA expression of miR-200 and miR 21, with a coefficient of 0.511 and P value of 0.02. Conclusion: The results of the present study indicated that miR-200, miR-145 and miR-21 may function as oncogenes and have a potential to serve as an early noninvasive diagnostic biomarkers and therapeutic targets for treatment of BC.

Therapeutic efficacy of Nano-formulation of lactoperoxidase and lactoferrin via promoting immunomodulatory and apoptotic effects.

This study identifies promising potential of a novel and safer nanocombination of bovine milk lactoperoxidase (LPO) and lactoferrin (LF) to target breast cancer in vitro and in adult female albino rat model. Favorable selective anticancer effects of the prepared nanocombination were observed, in a dose-dependent manner, against both MCF-7 and MDA cell lines, sparing normal HFB-4 cells. The administration of LPO + LFNPs markedly improved the induced-breast cancer disorders, prolonged survival and reduced the values of serum TNF-α, IL1ß, CD4+, ALAT, ASAT, urea, creatinine, cholesterol and triglycerides with remarkable elevation in mammary SOD and GPx activity and GSH level. Moreover, the histopathological findings showed that LPO + LFNPs succeeded in prevention of mammary gland tumorigenesis. Superior efficacy of LPO + LFNPs was observed against pro-inflammatory cytokines through their anti-inflammatory and immunomodulatory properties. The treatment of LPO + LFNPs more significantly modulated the apoptosis and enhanced the expression of cell cycle regulator genes, which demonstrates a successful tumor therapy in vitro and in vivo. Therefore, this study provided evidence that the chemo-preventive feature of LPO + LFNPs may offer a novel alternative therapy for the treatment of breast cancer through enhances apoptosis pathway, improvement of immune response, reduction of inflammation and restoration of the impaired oxidative stress.

Upregulation of Twist2 in Non-Muscle Invasive Urothelial Carcinoma of the Bladder Correlate with Response to Treatment and Progression.

BACKGROUND: Twist2 is a transcription factor and an epithelial-to-mesenchymal transition that plays an important role in cell polarity, cell adhesion, and has a role in tumour invasion and metastases. AIM: In this study, we examined the expression of Twist2 in non-muscle invasive bladder carcinoma (NMIBC) and correlated the expression with response to treatment and tumour progression. METHODS: Data of 305 patients with NMIBC of Ta, T1 were retrieved from hospitals archives. Twist2 expression was examined in tissue samples by immunohistochemistry at initial diagnosis and final follow-up, normal control was 10 normal urothelium, 10 patients with muscle-invasive bladder cancer (MIBC) were a positive control. Treatment of NMIBC was implemented according to the European Association of Urology guidelines on NMIBC. The descriptive statistical analysis included means, standard deviation, p-value; Univariate and multivariate Cox regression analyses. RESULTS: Twist2 expression score was identified as negative, low (1-15%); medium (15-40%); and high (40-100%). Patients who had low or low medium scores at the initial diagnosis had a good response and a favourable prognosis. Expression of a high score of Twist2 in patients having high-grade T1 tumours showed non-responsiveness to repeated courses of intravesical bacillus Calmette Guerin (BCG) therapy and was upstaged to MIBC. CONCLUSION: Twist2 expression in tissue samples of NMIBC would indicate the tumour response to therapy, upgrading and upstaging in the follow up after intravesical BCG therapy.

Transforming growth factor beta2 is negatively regulated by endogenous retinoic acid during early heart morphogenesis.

Vitamin A-deficient (VAD) quail embryos lack the vitamin A-active form, retinoic acid (RA) and are characterized by a phenotype that includes a grossly abnormal cardiovascular system that can be rescued by RA. Here we report that the transforming growth factor, TGFbeta2 is involved in RA-regulated cardiovascular development. In VAD embryos TGFbeta2 mRNA and protein expression are greatly elevated. The expression of TGFbeta receptor II is also elevated in VAD embryos but is normalized by treatment with TGFbeta2-specific antisense oligonucleotides (AS). Administration of this AS or an antibody specific for TGFbeta2 to VAD embryos normalizes posterior heart development and vascularization, while the administration of exogenous active TGFbeta2 protein to normal quail embryos mimics the excessive TGFbeta2 status of VAD embryos and induces VAD cardiovascular phenotype. In VAD embryos pSmad2/3 and pErk1 are not activated, while pErk2 and pcRaf are elevated and pSmad1/5/8 is diminished. We conclude that in the early avian embryo TGFbeta2 has a major role in the retinoic acid-regulated posterior heart morphogenesis for which it does not use Smad2/3 pathways, but may use other signaling pathways. Importantly, we conclude that retinoic acid is a critical negative physiological regulator of the magnitude of TGFbeta2 signals during vertebrate heart formation.

Retinoic acid is a negative physiological regulator of N-cadherin during early avian heart morphogenesis.

The vitamin A-deficient (VAD) early avian embryo has a grossly abnormal cardiovascular system that is rescued by treating the embryo with the vitamin A-active form, retinoic acid (RA). Here we examine the role of N-cadherin (N-cad) in RA-regulated early cardiovascular morphogenesis. N-cad mRNA and protein are expressed globally in the presomite through HH14 normal and VAD quail embryos. The expression in VAD embryos prior to HH10 is significantly higher than that in normal embryos. Functional analyses of the N-cad overproducing VAD embryos reveal N-cad involvement in the RA-regulated cardiovascular development and suggest that N-cad expression may be mediated by Msx1. We provide evidence that in the early avian embryo, endogenous RA is a negative physiological regulator of N-cad. We hypothesize that a critical endogenous level of N-cad is needed for normal early cardiovascular morphogenesis to occur and that this level is ensured by stage-specific, developmentally regulated RA signaling.

Fetal responses to epidural analgesia as evidenced by Doppler indices.

BACKGROUND: This study was designed to evaluate the maternal effects ofepidural analgesia by different local anesthetics and their impact on placental and fetal blood flow. METHODS: Depending on the type of local anesthetics used, sixty full-term parturients were randomly allocated into 3 equal groups in a randomized blind study; Group (1) received Bupivacaine (0.125%), Group (2) received Ropivacaine (0.2%) and, Group (3) received Levobupivacaine (0.125%). Epidural fentanyl (100 microg) was added to all groups. Safety was assessed by recording the mothers' characters and vital signs as well as the fetal Doppler indices while efficacy was assessed by measuring severity of pain, onset and duration of analgesia, and the motor blockade. Doppler velocimetry studies for fetus included monitoring of Umbilical Artery Pulsitility Indices (UAPI) and Middle Cerebral Artery Pulsitility Indices (MCAPI). RESULTS: Parturient in all groups were comparable. Pulse rate and arterial blood pressure were significantly decreased in all groups after analgesia, but remained within normal ranges. The pain score, had significant reduction in all groups with best results observed in Group 3. The onset of analgesia was relatively rapid in Group 2 followed by Group 3 then Group 1. The duration of analgesia was prolonged in Group 1 followed by Group 3 and then Group 2. There was no incidence of motor block except in 5 parturient (20%) in Group 1. UAPI was significantly decreased in the three studied groups after epidural analgesia. But, during uterine contraction, there was slight elevation in the UAPI in all groups. The best improvement in placental perfusion was observed in Group 3, then Group 1, and the least was Group 2. On the other hand, MCAPI was significantly increased in al groups after epidural analgesia. But, during uterine contraction, there was slight decrease in the MCAPI in the three groups. The best improvement in MCAPI was observed in Group 3, then Group 1, and the least was Group 2. CONCLUSION: All local anesthetics produced excellent analgesia during labor. The Doppler indices were improved in the three groups with the best results in levobupivacaine group.

Immunization against Egyptian Schistosoma mansoni infection by multivalent DNA vaccine.

The development of multivalent vaccines consisting of several antigens is a novel approach to creating broad-range protection against different parasite strains and parasite life cycle stages. We have previously confirmed that the schistosome Sm21.7 and SmFimbrin (SmFim) proteins could induce protection in mice. Therefore, this study aimed to construct the multivalent DNA vaccine Sm21.7-SmFim/pBudCE4.1 and evaluate its immune efficacy. The open reading frames of two Schistosoma mansoni genes, Sm21.7 and SmFim, were inserted into the eukaryotic expression plasmid pBudCE4.1 designed for the independent expression of two genes in mammalian cells. To evaluate the in vitro expression of the multivalent Sm21.7-SmFim/pBudCE4.1 DNA vaccine and its immunological effect in mice, the recombinant plasmid Sm21.7-SmFim/pBudCE4.1 was used to transfect 293T cells, and the expression of mRNA and proteins was examined using reverse transcription-polymerase chain reaction and Western blot analysis. Then the ability of Sm21.7-SmFim/pBudCE4.1 to protect against S. mansoni challenge infections was analyzed according to worm burden and egg reduction rates after vaccination of mice. Vaccinated mice showed a significant level of protection (56%), and a decrease in the number and size, and change in the cellular profile, of granulomas. Egg reduction in liver and intestine was 41.53% and 55.63%, respectively, as determined relative to mice that received the empty vector only. In addition to reductions in worm viability, worm fecundity and egg hatching ability were observed following challenge infection in the immunized group. Results showed that Sm21.7-SmFim/pBudCE4.1 could express Sm21.7 and SmFim mRNA and proteins. Enzyme-linked immunosorbent assay and Western blot analysis indicated that immunized mice generated specific immunoglobulin G against Sm21.7-SmFim/pBudCE4.1. These results suggest that vaccination with multivalent S. mansoni DNA vaccine (SmFim-Sm21.7/pBudCE4.1) not only induces a significant reduction in worm and egg burdens, but also significantly reduces the size of egg granulomas. In summary, the multivalent vaccine stimulated specific immunity with a significant level of protection and has anti-pathological effect.

Alterations of beta-catenin and Tcf-4 instead of GSK-3beta contribute to activation of Wnt pathway in hepatocellular carcinoma.

OBJECTIVE: The goal of this study is to investigate the inappropriate activation of Wnt pathway in the hepatocarcinogenesis. METHODS: We analyzed the alterations of three key components of Wnt pathway, beta-catenin, glycogen synthase kinase 3beta (GSK-3beta) and T cell factor 4 (Tcf-4), in 34 samples of hepatocellular carcinoma (HCC) and paracancerous normal liver by immunohistochemistry, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), direct sequencing, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. RESULTS: We found 61.8% (21/34) of all the HCCs examined showed an abnormal beta-catenin protein accumulation in the cytoplasm or nuclei. RT-PCR-SSCP and direct sequencing showed that beta-catenin exon 3 mutations existed in 44.1% (15/34) of the HCCs. No mutations of GSK-3beta or Tcf-4 were detected in HCCs. Moreover, mRNA of beta-catenin and Tcf-4 but not GSK-3beta was found to be over expressed in HCCs. On analyzing the relationship between alterations of beta-catenin or Tcf-4 and C-myc or Cyclin D1 expression, we found that the mutations of beta-catenin as well as over expression of beta-catenin or Tcf-4 gene were independently correlated with C-myc gene over expression in HCCs. CONCLUSIONS: Our present findings strongly suggest mutations of beta-catenin as well as over expression of beta-catenin and Tcf-4 gene activate the Wnt pathway in HCC independently with the target gene most likely to be C-myc.

Function of RARgamma and RARalpha2 at the initiation of retinoid signaling is essential for avian embryo survival and for distinct events in cardiac morphogenesis.

Avian embryogenesis requires retinoid receptor activation by the vitamin A active form, retinoic acid (RA), during neurulation. We conducted loss-of-function analysis in quail embryos by nutritional deprivation of RA and by blocking generation of retinoid receptors. Here we identify a distinct role for RARalpha2 in cardiac inflow tract morphogenesis and for RARgamma in cardiac left/right orientation and looping morphogenesis. Blocking normal embryos with antisense oligonucleotides to RARalpha2 or RXRalpha diminishes GATA-4 transcripts, while blocking RARgamma or RXRalpha diminishes nodal and Pitx2 transcripts; the expression of these genes in the heart forming region resembles that of the vitamin A-deficient embryo. Blocking the function of RARgamma, RARalpha2, and RXRalpha recapitulates the complete vitamin A-deficient phenotype. RARgamma is the most potent mediator of the retinoid signal at this time of development. Our studies provide strong evidence that critical RA-requiring developmental events in the early avian embryo are regulated by means of distinct retinoid receptor signaling pathways.

Wnt signaling in hepatocellular carcinoma: analysis of mutation and expression of beta-catenin, T-cell factor-4 and glycogen synthase kinase 3-beta genes.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is a common killer cancer in the world. Recently, abnormal activation of the Wnt pathway has been found to be involved in the carcinogenesis of several human cancers including HCC. The goal of the present study was to investigate the mechanism of inappropriate activation of the Wnt pathway in hepatocarcinogenesis. METHODS: We analyzed the alterations of three key components of the Wnt pathway: beta-catenin, glycogen synthase kinase (GSK)-3beta and T-cell factor (Tcf)-4 in 34 HCC and paracancerous normal liver by immunohistochemistry, polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP), direct sequencing, and quantitative real-time reverse transcription (RT)-PCR. RESULTS: We found that 61.8% (21/34) of all HCC examined showed an abnormal beta-catenin protein accumulation in the cytoplasm or nuclei. The RT-PCR-SSCP and direct sequencing showed that beta-catenin exon 3 mutations existed in 44.1% (15/34) of the HCC. No mutations of GSK-3beta or Tcf-4 were detected in HCC. Moreover, messenger RNA of beta-catenin and Tcf-4, but not GSK-3beta, was found to be overexpressed in HCC. On analyzing the relationship between alterations of beta-catenin or Tcf-4 and C-myc or Cyclin D1 expression, we found that mutations of beta-catenin, as well as overexpression of beta-catenin or the Tcf-4 gene were independently correlated with C-myc gene overexpression in HCC. CONCLUSION: Our present findings strongly suggest that mutations of beta-catenin, as well as overexpression of beta-catenin and the Tcf-4 gene, independently activate the Wnt pathway in HCC, with the target gene most likely to be C-myc.