Concerted loss of TGFß-mediated proliferation control and E-cadherin disrupts epithelial homeostasis and causes oral squamous cell carcinoma.

Although the etiology of squamous cell carcinomas of the oral mucosa is well understood, the cellular origin and the exact molecular mechanisms leading to their formation are not. Previously, we observed the coordinated loss of E-cadherin (CDH1) and transforming growth factor beta receptor II (TGFBR2) in esophageal squamous tumors. To investigate if the coordinated loss of Cdh1 and Tgfbr2 is sufficient to induce tumorigenesis in vivo, we developed two mouse models targeting ablation of both genes constitutively or inducibly in the oral-esophageal epithelium. We show that the loss of both Cdh1 and Tgfbr2 in both models is sufficient to induce squamous cell carcinomas with animals succumbing to the invasive disease by 18 months of age. Advanced tumors have the ability to invade regional lymph nodes and to establish distant pulmonary metastasis. The mouse tumors showed molecular characteristics of human tumors such as overexpression of Cyclin D1. We addressed the question whether TGFß signaling may target known stem cell markers and thereby influence tumorigenesis. From our mouse and human models, we conclude that TGFß signaling regulates key aspects of stemness and quiescence in vitro and in vivo. This provides a new explanation for the importance of TGFß in mucosal homeostasis.

Students' perceptions of a blended learning pharmacy seminar course in a Caribbean school of pharmacy.

BACKGROUND AND PURPOSE: Blended learning (BL) integrates face-to-face and online instructional methods, with applications in pharmacy education. This study aimed to assess pharmacy students' perceptions of BL in a pharmacy seminar course at The University of the West Indies, St. Augustine campus, Trinidad and Tobago. EDUCATIONAL ACTIVITY AND SETTING: Topics based on the use of medicines and public health were presented by student groups during live seminars, supplemented with online activities. An online survey of students' perceptions was administered at the end of the course. The usefulness of learning resources and course activities were assessed using 5-point Likert-like scales (1 = not helpful to 5 = very helpful). The effectiveness of the instructor, blended delivery, time value, and development of critical-thinking were rated on a 5-point Likert scale for agreement (1 = strongly disagree to 5 = strongly agree). Topics that were most instructive and additional topics of interest were also identified. FINDINGS: Approximately 51% of students (37/72) completed the questionnaire; 73% were female and mean age was 24 years. The learning resources and most course activities were generally helpful (median = 4) in facilitating learning. There was strong agreement (median = 5) on the ease of navigating the online platform, and instructor encouraging interest in pharmacy issues. Students agreed (median = 4) that the course facilitated critical thinking, the BL approach was effective, and the time spent was worthwhile. The most instructive topics included medication errors, antibiotic resistance, and medicines in children and the elderly. SUMMARY: BL in pharmacy seminars is a valuable approach to engage students learning about pharmacy and public health.

Aged garlic extract inhibits platelet activation by increasing intracellular cAMP and reducing the interaction of GPIIb/IIIa receptor with fibrinogen.

AIMS: Increased platelet aggregation plays an important role in the etiology of cardiovascular disease. Garlic inhibits platelet aggregation; however, the mechanisms involved have not clearly been defined. This study was undertaken to investigate the mechanisms by which an aged garlic extract (AGE) inhibits both the activation and aggregation of human platelets. MAIN METHODS: Isolated human platelets were stimulated with ADP and their adhesion to fibrinogen was assessed using Rose Bengal or (51)Cr uptake. Activation of platelets was assessed using fluorescence activated cell sorting (FACS) analysis along with measurement of intracellular cAMP. KEY FINDINGS: AGE at concentrations in the range of 3.12 to 12.5% (v/v) inhibited the binding of platelets to fibrinogen by approximately 40% when compared to control values in the Rose Bengal assay (P<0.05). In the (51)Cr experiments AGE significantly inhibited the binding of ADP-activated platelets to immobilized fibrinogen by 61.5% at 1.56% and 6.25% (v/v) of AGE respectively. At a concentration of 12.5% (v/v) the inhibition was 70.4% and at 25% (v/v) it was 64.5% respectively (P<0.05). In the fluorescence activated cell sorting (FACS) analysis, AGE significantly decreased the amount of PAC-1 binding to GPIIb/IIIa by approximately 72% compared with PBS control. In conjunction to these observations, AGE also increased platelet cAMP (P<0.01) levels. SIGNIFICANCE: These findings suggest that AGE inhibits platelet aggregation via inhibition of the GPIIb/IIIa receptor and an increase in cAMP.

CD44 upregulation in E-cadherin-negative esophageal cancers results in cell invasion.

E-cadherin is frequently lost during epithelial-mesenchymal transition and the progression of epithelial tumorigenesis. We found a marker of epithelial-mesenchymal transition, CD44, upregulated in response to functional loss of E-cadherin in esophageal cell lines and cancer. Loss of E-cadherin expression correlates with increased expression of CD44 standard isoform. Using an organotypic reconstruct model, we show increased CD44 expression in areas of cell invasion is associated with MMP-9 at the leading edge. Moreover, Activin A increases cell invasion through CD44 upregulation after E-cadherin loss. Taken together, our results provide functional evidence of CD44 upregulation in esophageal cancer invasion.

Cathepsin B is the driving force of esophageal cell invasion in a fibroblast-dependent manner.

Esophageal cancer, which frequently exhibits coordinated loss of E-cadherin (Ecad) and transforming growth factor beta (TGFbeta) receptor II (TbetaRII), has a high mortality rate. In a three-dimensional organotypic culture model system, esophageal keratinocytes expressing dominant-negative mutant versions of both Ecad and TbetaRII (ECdnT) invade into the underlying matrix embedded with fibroblasts. We also find that cathepsin B induction is necessary for fibroblast-mediated invasion. Furthermore, the ECdnT cells in this physiological context activate fibroblasts through the secretion of TGFbeta1, which, in turn, is activated by cathepsin B. These results suggest that the interplay between the epithelial compartment and the surrounding microenvironment is crucial to invasion into the extracellular matrix.

Aged garlic extract and its constituents inhibit platelet aggregation through multiple mechanisms.

Aged garlic extract (AGE) is a complex mixture. Its constituents include allin, cycloalliin, S-allyl-L-cysteine, S-methyl-L-cysteine, S-ethylcysteine, S-1-proponyl-L-cysteine, S-allylmercapto-L-cysteine, fructosyl-arginine, and beta-chlorogenin. It also contains L-arginine, L-cysteine, and L-methionine. AGE reduces the parameters associated with cardiovascular disease, including the inhibition of platelet aggregation. However, the underlying mechanisms have yet to be established and are the subject of this study. AGE inhibited platelet aggregation in a concentration-dependent manner, and this achieved significance between concentrations of 1.56-25% (v:v). The constituents of AGE, when tested as a mixture, displayed a biphasic pattern of inhibition, although this was not significant. L-Methionine displayed anti-aggregatory properties at concentrations of 0.00078, 0.00625, and 0.1 mmol/L, whereas L-arginine and L-cysteine were effective at 9 mmol/L. However, when the constituents and the amino acids were tested together, significant inhibition of platelet aggregation was observed only at a concentration of 1 mmol/L. AGE also displayed disaggregatory properties at concentrations of 12.5 and 25% (v:v). The constituents and the amino acids, when tested as a mixture, displayed disaggregatory properties at concentrations of 0.25 and 1 mmol/L. In contrast, a diethyl-ether extract of AGE had no effect on platelet aggregation. When platelets were stimulated with either A23187 or ADP, an increase in intraplatelet Ca2+ accompanied platelet aggregation. This increase in Ca2+ was abolished in the presence of AGE. It is likely that AGE works in a synergistic manner and exerts multiple effects on the biochemical pathways involved in platelet aggregation.

Aged garlic extract may inhibit aggregation in human platelets by suppressing calcium mobilization.

Cardiovascular disease is associated with multiple factors including the increased ability of platelets to aggregate. Aged garlic extract (AGE) was shown to inhibit platelet aggregation; however, the underlying mechanisms have yet to be established. Because calcium mobilization plays an important role in platelet aggregation, the effect of AGE was investigated in this preliminary study. ADP and the calcium ionophore A23187 both stimulated platelet aggregation with a concomitant increase in intracellular calcium ion concentration. When these experiments were repeated in the presence of AGE, both platelet aggregation and calcium mobilization were suppressed. In addition, when platelets were preincubated with AGE, the initial concentration of intracellular calcium was significantly reduced compared with platelets without AGE, confirming the metal-chelating properties of AGE. Platelets loaded with fura-2 acetoxymethyl ester (fura-2 AM) also displayed a reduction in platelet aggregation, and the addition of external calcium did not alter this observation. Although variable data were obtained in this study, these results taken together imply that AGE probably exerts its inhibitory effect on platelet aggregation either by suppressing the influx of calcium ions by chelating calcium within platelet cytosol or by altering other intracellular second messengers within the platelets.