Notch-1 induces epithelial-mesenchymal transition consistent with cancer stem cell phenotype in pancreatic cancer cells

Activation of Notch-1 is known to be associated with the development and progression of human malignancies including pancreatic cancer. Emerging evidence suggest that the acquisition of epithelial-mesenchymal transition (EMT) phenotype and induction of cancer stem cell (CSC) or cancer stem-like cell phenotype are interrelated and contributes to tumor recurrence and drug resistance. The molecular mechanism(s) by which Notch-1 contributes to the acquisition of EMT phenotype and CSC self-renewal capacity has not been fully elucidated. Here we show that forced over-expression of Notch-1 leads to increased cell growth, clonogenicity, migration and invasion of AsPC-1 cells. Moreover, over-expression of Notch-1 led to the induction of EMT phenotype by activation of mesenchymal cell markers such as ZEB1, CD44, EpCAM, and Hes-1. Here we also report, for the first time, that over-expression of Notch-1 leads to increased expression of miR-21, and decreased expression of miR-200b, miR-200c, let-7a, let-7b, and let-7c. Re-expression of miR-200b led to decreased expression of ZEB1, and vimentin, and increased expression of E-cadherin. Over-expression of Notch-1 also increased the formation of pancreatospheres consistent with expression of CSC surface markers CD44 and EpCAM. Finally, we found that genistein, a known natural anti-tumor agent inhibited cell growth, clonogenicity, migration, invasion, EMT phenotype, formation of pancreatospheres and expression of CD44 and EpCAM. These results suggest that the activation of Notch-1 signaling contributes to the acquisition of EMT phenotype, which is in part mediated through the regulation of miR-200b and CSC self-renewal capacity, and these processes could be attenuated by genistein treatment.     

microRNA response elements-regulated TRAIL expression shows specific survival-suppressing activity on bladder cancer

Background

Bladder transitional cell carcinoma greatly threatens human health all over the world. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) shows a strong apoptosis-inducing effect on a variety of cancer cells including bladder cancer. However, adenovirus-mediated TRAIL expression still showed cytotoxicity to normal cells mainly due to lack of tumor specificity.

Methods

To solve the problem, we applied miRNA response elements (MREs) of miR-1, miR-133 and miR-218 to confer TRAIL expression with specificity to bladder cancer cells.

Results

Expression of miR-1, miR-133 and miR-218 was greatly decreased in bladder cancer than normal bladder tissue. Luciferase assay showed that application of the 3 MREs was able to restrain exogenous gene expression to within bladder cancer cells. Subsequently, we constructed a recombinant adenovirus with TRAIL expression regulated by MREs of miR-1, miR-133 and miR-218, namely Ad-TRAIL-MRE-1-133-218. qPCR, immunoblotting and ELISA assays demonstrated that Ad-TRAIL-MRE-1-133-218 expressed in bladder cancer cells, rather than normal bladder cells. The differential TRAIL expression also led to selective apoptosis-inducing and growth-inhibiting effect of Ad-TRAIL-MRE-1-133-218 on bladder cancers. Finally, bladder cancer xenograft in mouse models further confirmed that Ad-TRAIL-MRE-1-133-218 effectively suppressed the growth of bladder cancers.

Conclusions

Collectively, we demonstrated that MREs-based TRAIL delivery into bladder cancer cells was feasible and efficient for cancer gene therapy.     

Sensitization of human breast cancer cells to gemcitabine by the protein kinase C modulator bryostatin 1

PURPOSE: Protein kinase C (PKC) plays an important role in cell proliferation, differentiation, and apoptosis. The interaction between the PKC modulator bryostatin 1 (BRYO), and gemcitabine in human breast cancer MCF-7 and MDA-MB-231 cells and in the non-transformed MCF-10A human breast epithelial cells was investigated. METHODS: Immunoblotting was used to determine the expression of PKC isoenzymes and proteins involved in the cell cycle and apoptosis. MTT, ELISA and flow cytometry assays were used to determine cell survival. RESULTS: Treatment of cells with BRYO 200 n M resulted in a significant downregulation of cytoplasmic PKC in all three cell lines. However, the expression of membranous PKC was differentially affected in these cells. BRYO (1-200 n M) had no significant effects on cell viability in any of the cell lines. Nevertheless, BRYO significantly enhanced the antiproliferative and apoptotic effects of gemcitabine in the MCF-7 and MDA-MB-231 cells, but not in the MCF-10A cells. This was associated with significant reduction in the bcl-2/bax ratio. There was a significant upregulation of p53, p21(waf1), and p27 in MCF7 and MCF-10A cells treated with the combination of gemcitabine and BRYO compared to gemcitabine-treated cells. CONCLUSIONS: The potentiation of the effect of gemcitabine by BRYO was demonstrated in MCF-7 and MDA-MB-231 cells and was associated with a specific pattern of PKC modulation. Further investigation of the role of specific isoforms of PKC in the downstream molecular events of gemcitabine-induced cytotoxicity is warranted.     

Direct comparison of microarray gene expression profiles between non-amplification and a modified cDNA amplification procedure applicable for needle biopsy tissues

Global gene expression profiling by cDNA microarray analysis has been used to discover the biomarkers for early diagnosis of various cancers, subclassing cancer type, and prediction of patient's treatment outcome. The information provided by gene expression profiling may contribute to the design of molecular mechanism-based strategies for cancer prevention and/or treatment. However, the standard procedure for cDNA microarray analysis requires 5mug of good quality total RNA as starting material for each target preparation reaction. Thus, there is a limit for needle biopsy samples, laser capture microdissected tissues, or flow-sorted cells to successfully utilize the microarray technology. In order to profile the gene expression of needle biopsy tissue, we have modified the standard protocol by carrying out cDNA amplification after cDNA synthesis. We compared percentage present calls, absent calls, reproducibility, and concordance in needle biopsy samples processed by standard microarray protocol (cDNA non-amplification method) and our modified protocol (cDNA amplification method). The results showed that cDNA amplification method provided high reproducibility, representation, and concordance with the standard cDNA non-amplification method. We have successfully analyzed the gene expression profiles of needle biopsy tissues using the modified method without significantly changing the expression profiles. These results suggest that the global gene expression profiles of small biopsy samples can be achieved by our modified method to facilitate the analysis of gene expression profiles for clinical application.     

Zolpidem for postanoxic spasticity

A 28-year-old male sustained anoxic brain damage following aborted cardiac arrest, and subsequently developed severe muscular rigidity and spasticity involving all extremities. The spasticity was refractory to the standard regimens used for spastic hypertonia. Zolpidem dramatically inhibited muscular rigidity, spasticity, and dystonic posturing in a dose-dependent manner, resulting in a sustained improvement of his global performance over four years. The authors postulate a central mechanism of action by selective inhibition of GABAergic inhibitory neurons, and suggest a controlled clinical study to investigate the potential efficacy of zolpidem in relieving spasticity related to postanoxic brain injury.     

Melatonin effects on luteinizing hormone in postmenopausal women: a pilot clinical trial NCT00288262

Background  

The post marketing safety surveillance program for a drug containing a new chemical entity should assess both, the safety outcomes that relate specifically to the targeted population, as well as those that could potentially be related to special pharmacological characteristics of the drug. Active safety surveillance using valid epidemiological study designs has been proven to be a pertinent and reliable method to approach this endeavor.     

Cytochrome p450 and glutathione transferase expression in squamous cell cancer.

PURPOSE: The cytochrome P-450 (CYP) and glutathione S-transferase (GST) enzyme systems modulate the carcinogenic effects of tobacco. Therefore, the expression of these enzymes may be in part responsible for the observed interindividual and inter-racial differences in the risk of development of squamous cell carcinoma of the head and neck (SCCHN). The first aim of this study was to evaluate the feasibility of measuring the expression of the CYP and GST in target tissue from the head and neck. The second aim was to compare the expression of CYPs 1A1, 2E1, and 3A4 in squamous epithelium from African-American and Caucasian pediatric patients. The third aim was to compare the expression of CYPs 1A1, 2E1, 3A4, and GST-pi on the p16 expression in patients with SCCHN. EXPERIMENTAL DESIGN: The expression of CYP 1A1, 2E1, 3A4, GST-pi, and p16 was quantified by immunoblotting. Expression of CYPs 1A1, 2E1, and 3A4 was quantified in tissue from 160 pediatric patients undergoing tonsillectomy. Expression of CYPs 1A1, 2E1, 3A4, GST-pi, and p16 was determined in 46 resected SCCHN patients. RESULTS: Large interindividual variability in the expression of these enzymes was observed in the pediatric and adult populations. No significant difference was observed in CYP 1A1, 2E1, and 3A4 expression of Caucasian and African-American patients. There was no correlation between p16 and enzyme expression in patients with SCCHN. CONCLUSION: Evaluation of CYP expression in the target tissue of interest is feasible. The clinical significance of CYPs and GST-pi alterations in the risk of developing SCCHN will need to be investigated in larger trials.     

Assessment of Preventive Behavior for Cervical Cancer with the Health Belief Model

Introduction: Cervical cancer is a leading cause of death in developing countries. However, taking regular Pap smearsscreening, one of the most effective screening tests, can reduce chance of cervical cancer remarkably. The first step inhealth education is choosing the right model, one of the best known being the Health Belief Model (HBM). Here, weevaluated different HBM factors with regard to cervical cancer preventive behavior in Fasa, Iran. Materials and methods:This cross-sectional study included 200 married women between the ages of 17 and 64 in Fasa during 2013. Participantswere selected through stratified sampling from urban health centers. The questionnaire of the HBM included four sectionsand was filled out by interview. Data analysis was with SPSS 21, ANOVA and t-tests and Internal correlations betweencomponents of model were analyzed in terms of the Spearman Pearson correlation coefficient. Results: The mean age ofthe participants was 35.6±9.89 years. Some 52% had undergone a Pap test. The percentage values for participants’knowledge and perceived susceptibility were 49.5% and 46%, respectively, considered as moderate. Perceived severityand benefits were good at 56.5% and 73%, respectively, while perceived barriers was moderate (46.5%). Also, 57.1%demonstrated a good self-efficacy and 61% good behavior. Discussion: We found that the most predictable factors forknowledge and behavior were age, income level, perceived benefits, perceived severity and self-efficacy according tothe HBM. These factors should be taken into account for achieving acceptable preventive behaviors in health programs.     

Exploration of health professional stakeholders’ views and experiences regarding minor ailments services’ education,training and assessment

Background Minor ailments services are structured pharmacy-based primary health care services that manage minor conditions. Limited training, education and assessment exists to promote the delivery of minor ailments services by pharmacy staff and it is unclear if the existing training and education processes meet professional requirements. Objective To explore the views and experiences of health professional stakeholders such as community pharmacists, intern pharmacists, medicines counter assistants and general medical practitioners with regards to minor ailments services education, training and assessment practices and preferences. Setting This study explored the views and experiences of health professional stakeholders in Australia. Method Semi-structured interviews were conducted, audio recorded, transcribed verbatim and then coded thematically using QSR Nvivo12. Main outcome measure Stakeholders’ views and experiences regarding minor ailments services education, training and assessment practices and preferences. Results Twenty-eight interviews were conducted (community pharmacists n?=?12; medicines counter assistants n?=?4; intern pharmacists n?=?9; general medical practitioners n?=?3). Thematic analysis generated three themes: (1) pharmacy staff who require minor ailment service training; (2) acceptability and willingness to complete additional training; (3) learning preferences and approaches. Stakeholders reported considerations for the diverse roles in service delivery and fit for purpose tailored training. Conclusion Detailed practice guidelines may facilitate clarity of an individual staff member’s role. Education and training in both clinical and non-clinical aspects of the service may be beneficial and may improve minor ailments service uptake and outcomes.