The Role of the α Cell in the Pathogenesis of Diabetes: A World beyond the Mirror.

Type 2 Diabetes Mellitus (T2DM) is one of the most prevalent chronic metabolic disorders, and insulin has been placed at the epicentre of its pathophysiological basis. However, the involvement of impaired alpha (α) cell function has been recognized as playing an essential role in several diseases, since hyperglucagonemia has been evidenced in both Type 1 and T2DM. This phenomenon has been attributed to intra-islet defects, like modifications in pancreatic α cell mass or dysfunction in glucagon's secretion. Emerging evidence has shown that chronic hyperglycaemia provokes changes in the Langerhans' islets cytoarchitecture, including α cell hyperplasia, pancreatic beta (ß) cell dedifferentiation into glucagon-positive producing cells, and loss of paracrine and endocrine regulation due to ß cell mass loss. Other abnormalities like α cell insulin resistance, sensor machinery dysfunction, or paradoxical ATP-sensitive potassium channels (KATP) opening have also been linked to glucagon hypersecretion. Recent clinical trials in phases 1 or 2 have shown new molecules with glucagon-antagonist properties with considerable effectiveness and acceptable safety profiles. Glucagon-like peptide-1 (GLP-1) agonists and Dipeptidyl Peptidase-4 inhibitors (DPP-4 inhibitors) have been shown to decrease glucagon secretion in T2DM, and their possible therapeutic role in T1DM means they are attractive as an insulin-adjuvant therapy.

Metabolic Syndrome: Is It Time to Add the Central Nervous System?

Metabolic syndrome (MS) is a set of cardio-metabolic risk factors that includes central obesity, hyperglycemia, hypertension, and dyslipidemias. The syndrome affects 25% of adults worldwide. The definition of MS has evolved over the last 80 years, with various classification systems and criteria, whose limitations and benefits are currently the subject of some controversy. Likewise, hypotheses regarding the etiology of MS add more confusion from clinical and epidemiological points of view. The leading suggestion for the pathophysiology of MS is insulin resistance (IR). IR can affect multiple tissues and organs, from the classic "triumvirate" (myocyte, adipocyte, and hepatocyte) to possible effects on organs considered more recently, such as the central nervous system (CNS). Mild cognitive impairment (MCI) and Alzheimer's disease (AD) may be clinical expressions of CNS involvement. However, the association between MCI and MS is not understood. The bidirectional relationship that seems to exist between these factors raises the questions of which phenomenon occurs first and whether MCI can be a precursor of MS. This review explores shared pathophysiological mechanisms between MCI and MS and establishes a hypothesis of a possible MCI role in the development of IR and the appearance of MS.

Advanced Glycation End Products: New Clinical and Molecular Perspectives.

Diabetes mellitus (DM) is considered one of the most massive epidemics of the twenty-first century due to its high mortality rates caused mainly due to its complications; therefore, the early identification of such complications becomes a race against time to establish a prompt diagnosis. The research of complications of DM over the years has allowed the development of numerous alternatives for diagnosis. Among these emerge the quantification of advanced glycation end products (AGEs) given their increased levels due to chronic hyperglycemia, while also being related to the induction of different stress-associated cellular responses and proinflammatory mechanisms involved in the progression of chronic complications of DM. Additionally, the investigation for more valuable and safe techniques has led to developing a newer, noninvasive, and effective tool, termed skin fluorescence (SAF). Hence, this study aimed to establish an update about the molecular mechanisms induced by AGEs during the evolution of chronic complications of DM and describe the newer measurement techniques available, highlighting SAF as a possible tool to measure the risk of developing DM chronic complications.

Caveolin-1 suppresses tumor formation through the inhibition of the unfolded protein response.

Caveolin-1 (CAV1), is a broadly expressed, membrane-associated scaffolding protein that acts both, as a tumor suppressor and a promoter of metastasis, depending on the type of cancer and stage. CAV1 is downregulated in human tumors, tumor cell lines and oncogene-transformed cells. The tumor suppressor activity of CAV1 is generally associated with its presence at the plasma membrane, where it participates, together with cavins, in the formation of caveolae and also has been suggested to interact with and inhibit a wide variety of proteins through interactions mediated by the scaffolding domain. However, a pool of CAV1 is also located at the endoplasmic reticulum (ER), modulating the secretory pathway in a manner dependent on serine-80 (S80) phosphorylation. In melanoma cells, CAV1 expression suppresses tumor formation, but the protein is largely absent from the plasma membrane and does not form caveolae. Perturbations to the function of the ER are emerging as a central driver of cancer, highlighting the activation of the unfolded protein response (UPR), a central pathway involved in stress mitigation. Here we provide evidence indicating that the expression of CAV1 represses the activation of the UPR in vitro and in solid tumors, reflected in the attenuation of PERK and IRE1α signaling. These effects correlated with increased susceptibility of cells to ER stress and hypoxia. Interestingly, the tumor suppressor activity of CAV1 was abrogated by site-directed mutagenesis of S80, correlating with a reduced ability to repress the UPR. We conclude that the tumor suppression by CAV1 involves the attenuation of the UPR, and identified S80 as essential in this context. This suggests that intracellular CAV1 regulates cancer through alternative signaling outputs.

Src-family kinase inhibitors block early steps of caveolin-1-enhanced lung metastasis by melanoma cells.

In advanced stages of cancer disease, caveolin-1 (CAV1) expression increases and correlates with increased migratory and invasive capacity of the respective tumor cells. Previous findings from our laboratory revealed that specific ECM-integrin interactions and tyrosine-14 phosphorylation of CAV1 are required for CAV1-enhanced melanoma cell migration, invasion and metastasis in vivo. In this context, CAV1 phosphorylation on tyrosine-14 mediated by non-receptor Src-family tyrosine kinases seems to be important; however, the effect of Src-family kinase inhibitors on CAV1-enhanced metastasis in vivo has not been studied. Here, we evaluated the effect of CAV1 and c-Abl overexpression, as well as the use of the Src-family kinase inhibitors, PP2 and dasatinib (more specific for Src/Abl) in lung metastasis of B16F10 melanoma cells. Overexpression of CAV1 and c-Abl enhanced CAV1 phosphorylation and the metastatic potential of the B16F10 murine melanoma cells. Alternatively, treatment with PP2 or dasatinib for 2 h reduced CAV1 tyrosine-14 phosphorylation and levels recovered fully within 12 h of removing the inhibitors. Nonetheless, pre-treatment of cells with these inhibitors for 2 h sufficed to prevent migration, invasion and trans-endothelial migration in vitro. Importantly, the transient decrease in CAV1 phosphorylation by these kinase inhibitors prevented early steps of CAV1-enhanced lung metastasis by B16F10 melanoma cells injected into the tail vein of mice. In conclusion, this study underscores the relevance of CAV1 tyrosine-14 phosphorylation by Src-family kinases during the first steps of the metastatic sequence promoted by CAV1. These findings open up potential options for treatment of metastatic tumors in patients in which Src-family kinase activation and CAV1 overexpression favor dissemination of cancer cells to secondary sites.

Design of a biodegradable UV-irradiated gelatin-chitosan/nanocomposed membrane with osteogenic ability for application in bone regeneration.

Guided bone regeneration membranes are used in oral surgery to protect the site of a lesion exposed to connective tissue invasion which, in turn, prevents new bone formation. Although non-degradable and degradable materials have been applied in clinical treatments, biodegradable membranes have the advantage that they do not require a secondary surgical procedure to be removed. However, they have a very low mechanical strength. As biodegradable membranes, biomaterials based on gelatin-chitosan have gained importance in clinical applications due to their unique properties. Gelatin contains RGD-like sequences, promoting cell adhesion/migration, and it can be blended with chitosan, which allows the immobilization of nanoparticles. In this work, we designed a new gelatin-chitosan polymeric membrane which contains hydroxyapatite and titania nanoparticles as two very well-documented osteoconductive materials. UV radiation was used as a non-toxic cross-linking agent to improve the thermophysical/mechanical characteristics and to control the biodegradability of the nanocomposed membrane. The microstructure, thermophysical and mechanical properties of the UV-irradiated material were studied by scanning electron microscopy, differential scanning calorimetry and Young's modulus, respectively. The in vitro biocompatibility of the new nanocomposite was evaluated by cell adhesion and proliferation assays. The osteoconductive ability was determined by an alkaline phosphatase production assay using mouse embryonic fibroblast (MEF) cells. The results show a homogeneous material with an appropriate distribution of nanoparticles. Cross-linking by UV radiation improved the mechanical and biological performance of the membrane. The presence of two osteoconductive nanoparticles, such as titania and hydroxyapatite, increased the osteogenic potential of the gelatin-based material in vitro, which confers a biological function, in addition to functioning as a physical barrier. The material obtained herein represents a good alternative to current guided bone regeneration membranes, with high potential for use in oral/orthopaedic applications in patients.

Current Evidence for the Use of Smoflipid® Emulsion in Critical Care Patients for Parenteral Nutrition.

There are strong data showing that malnutrition is highly prevalent in intensive care unit patients (20-50% in the worldwide), presenting a negative accumulated body energy balance. This results in an increased mortality, infections, and hospital length stay with high costs associated with the total treatment. Parenteral nutrition is the first option when the patient's physical condition is not suitable for oral nutrient intake. It is composed essentially by lipids as an energy source, metabolic, and structural function. However, these patients also require a mixture of essential and nonessential fatty acids (SMOF emulsions) to supply not only energy needs but also restore immunological, anti-inflammatory, and proregenerative functions. A revision of the safety and efficacy of Smoflipid® in patients requiring long-term parenteral nutrition was discussed here. Although controversial data are available indicating the contraindications or effectiveness of its use, most of studies presented indicate favorable benefits associated with improved clinical outcomes. The reported roles of this supplementation include positive immunomodulatory and anti-inflammatory effects, positive impact in liver function, reduction of hospital stay, and nosocomial infections as additional contributions to its energetic role, which in many cases results in reduced total costs per patient. Finally, many authors propose that the use of Smoflipid® should become a gold standard of parenteral nutrition in intensive unit care patients and that the costs associated with this supplement should not be limiting for its use, not only to improve the clinical outcome but also to reduce the treatment costs.

The streptozotocin-high fat diet induced diabetic mouse model exhibits severe skin damage and alterations in local lipid mediators.

BACKGROUND: Type 2 diabetes (T2D) can go undiagnosed for years, leading to a stage where produces complications such as delayed skin wound healing. Animal models have been developed in the last decades to study the pathological progression in this disease. Streptozotocin (STZ), that has a selective pharmacological toxicity toward pancreatic ß cells, in addition to high fat diet has been widely used to induce diabetes however no evidence has shown its effects on the skin integrity. METHODS: Eighteen C57BL/6J male mice, were divided in 3 groups; the first was fed with chow diet and the second was kept on a high fat diet and the third injected with STZ intraperitoneal for 5 days consecutively before starting the diet protocol with high fat. Mice were maintained 5 weeks in total. RESULTS: We show that animals treated with STZ-high fat diet exhibit skin injuries without significant alterations on basal insulin and triglycerides, compared to the control. The skin from these animals presents higher levels of oxidative stress, lower levels of adhesion proteins and alterations in lipid mediators, effects that are not produced by the high fat diet itself. CONCLUSION: Our results suggest that this in vivo model represents a relevant approach for studying skin damage induced by diabetes.

[Comparison Of Anthropometric Measures For Aggregation Of Multiple Risk Factors In Adults Of Cuenca, Ecuador]. / Comparación de Índices Antropométricos para Agregación de Múltiples Factores de Riesgo en Adultos de Cuenca, Ecuador.

OBJECTIVE: . The aim of this study is to compare the predictive capacity of different anthropometric indices in multiple risk factors aggregation (MRFA) determination in the adult population from Cuenca city, Ecuador. MATERIALS AND METHODS: . A cross- sectional descriptive study was performed with a random multi-stage sampling in 318 adult subjects who underwent a clinical, anthropometric and laboratory evaluation; being the abdominal circumference, body mass index (BMI) and waist height index (WHtR) evaluated. MRFA was defined as the presence of ≥2 components of the metabolic syndrome (excluding abdominal circumference). ROC curves were plotted to determine the area under the curve (AUC) for each index. RESULTS: . Of the 318 individuals, 54.1% (n=172) presented MRFA. According to ROC curves, the highest predictive capacity in women was observed with BMI and WHtR (AUC: 0.751 and 0.750, respectively), while in men abdominal circumference and WHtR showed a similar predictive power (AUC: 0.762). The multivariate analysis adjusted for sex and age showed that high WHtR (OR: 2.53, 95% CI: 1.12-5.71, p=0.026) was the best predictor of MRFA, followed by BMI (OR: 2.15, 95% CI: 1.19-3.88, p=0.010). CONCLUSIONS: . The predictive capacity of the anthropometric indexes is influenced by gender; nevertheless the WHtR is the best predictor of MRFA in our population.

OBJETIVOS.: El objetivo de este estudio es comparar la capacidad predictiva de diferentes índices antropométricos en la determinación de la agregación de múltiples factores de riesgo (AMFR) en la población adulta de la ciudad de Cuenca, Ecuador. MATERIALES Y MÉTODOS .: Se realizó un estudio descriptivo transversal con un muestreo aleatorio multietápico en 318 sujetos adultos a quienes se les realizó una evaluación clínica, antropométrica y de laboratorio; siendo la circunferencia abdominal, índice de masa corporal (IMC) e índice cintura altura (ICA) los índices evaluados. La AMFR se definió como la presencia de ≥ dos componentes del síndrome metabólico (excluyendo circunferencia abdominal). Se realizaron curvas COR para determinar el área bajo la curva (ABC) para cada índice. RESULTADOS.: De los 318 individuos, un 54,1% (n=172) presentaron AMFR. Según los resultados obtenidos por curvas COR, la mayor capacidad predictiva en mujeres se observó con el IMC y el ICA (ABC: 0,751 y 0,750, respectivamente) mientras que en hombres la circunferencia abdominal y el ICA mostraron una capacidad predictiva similar (ABC=0,762). El análisis multivariante ajustado por sexo y edad mostró que el ICA elevado (OR: 2,53; IC95%: 1,12-5,71; p=0,026) fue el mejor predictor de AMFR, seguido por el IMC (OR: 2,15; IC95%: 1,19-3,88; p=0,010). CONCLUSIONES.: La capacidad predictiva de los índices antropométricos está influenciada por el sexo, no obstante, el ICA es el mejor predictor de la AMFR en la población de Cuenca.

Comparación de Índices Antropométricos para Agregación de Múltiples Factores de Riesgo en Adultos de Cuenca, Ecuador / Comparison Of Anthropometric Measures For Aggregation Of Multiple Risk Factors In Adults Of Cuenca, Ecuador

RESUMEN Objetivos. El objetivo de este estudio es comparar la capacidad predictiva de diferentes índices antropométricos en la determinación de la agregación de múltiples factores de riesgo (AMFR) en la población adulta de la ciudad de Cuenca, Ecuador. Materiales y métodos . Se realizó un estudio descriptivo transversal con un muestreo aleatorio multietápico en 318 sujetos adultos a quienes se les realizó una evaluación clínica, antropométrica y de laboratorio; siendo la circunferencia abdominal, índice de masa corporal (IMC) e índice cintura altura (ICA) los índices evaluados. La AMFR se definió como la presencia de ≥ dos componentes del síndrome metabólico (excluyendo circunferencia abdominal). Se realizaron curvas COR para determinar el área bajo la curva (ABC) para cada índice. Resultados. De los 318 individuos, un 54,1% (n=172) presentaron AMFR. Según los resultados obtenidos por curvas COR, la mayor capacidad predictiva en mujeres se observó con el IMC y el ICA (ABC: 0,751 y 0,750, respectivamente) mientras que en hombres la circunferencia abdominal y el ICA mostraron una capacidad predictiva similar (ABC=0,762). El análisis multivariante ajustado por sexo y edad mostró que el ICA elevado (OR: 2,53; IC95%: 1,12-5,71; p=0,026) fue el mejor predictor de AMFR, seguido por el IMC (OR: 2,15; IC95%: 1,19-3,88; p=0,010). Conclusiones. La capacidad predictiva de los índices antropométricos está influenciada por el sexo, no obstante, el ICA es el mejor predictor de la AMFR en la población de Cuenca.

ABSTRACT Objective . The aim of this study is to compare the predictive capacity of different anthropometric indices in multiple risk factors aggregation (MRFA) determination in the adult population from Cuenca city, Ecuador. Materials and Methods . A cross- sectional descriptive study was performed with a random multi-stage sampling in 318 adult subjects who underwent a clinical, anthropometric and laboratory evaluation; being the abdominal circumference, body mass index (BMI) and waist height index (WHtR) evaluated. MRFA was defined as the presence of ≥2 components of the metabolic syndrome (excluding abdominal circumference). ROC curves were plotted to determine the area under the curve (AUC) for each index. Results . Of the 318 individuals, 54.1% (n=172) presented MRFA. According to ROC curves, the highest predictive capacity in women was observed with BMI and WHtR (AUC: 0.751 and 0.750, respectively), while in men abdominal circumference and WHtR showed a similar predictive power (AUC: 0.762). The multivariate analysis adjusted for sex and age showed that high WHtR (OR: 2.53, 95% CI: 1.12-5.71, p=0.026) was the best predictor of MRFA, followed by BMI (OR: 2.15, 95% CI: 1.19-3.88, p=0.010). Conclusions . The predictive capacity of the anthropometric indexes is influenced by gender; nevertheless the WHtR is the best predictor of MRFA in our population.

Influencia de la actividad física y el consumo calórico sobre la adiposidad visceral en adultos de la ciudad de Cuenca, Ecuador / Influence of physical activity and calorie intake on visceral adiposity in adults of the city of Cuenca, Ecuador

Introducción: El índice de adiposidad visceral (VAI) es un método sencillo y costo-efectivo en la determinación de adiposidad visceral. El objetivo de este estudio es evaluar la relación entre el VAI con diversos factores de riesgo cardiovascular, variables sociodemográficas y hábitos psicobiológicos en la población adulta de la ciudad de Cuenca, Ecuador. Materiales y métodos: Se realizó un estudio descriptivo transversal en 318 individuos adultos seleccionados mediante muestreo aleatorio y multietápico, a quienes se les realizó evaluación clínica, evaluación antropométrica y de laboratorio. El VAI se determinó utilizando las fórmulas propuestas que emplean circunferencia abdominal, el índice de masa corporal, los triacilglicéridos y HDL-C. Se realizó un modelo de regresión logística múltiple para determinar los principales factores asociados a adiposidad visceral en sus valores más elevados. Resultados: En los 318 individuos, el promedio del VAI fue 2,57 (1,66-3,94), con valores más elevados para el sexo femenino. En el modelo de regresión logística múltiple, los factores de riesgo significativos para VAI moderado-alto fueron: la edad (> 60 años: OR = 3,87; IC del 95%: 1,15-12,96; p = 0,03), el consumo calórico, la glucemia alterada en ayuno y la actividad física en ocio. Conclusión: El VAI es un método útil para definir a aquellos sujetos con adiposidad visceral en nuestra región. La edad, el consumo calórico diario y la glucemia alterada en ayuno son los principales factores asociados con los valores más elevados delíndice, mientras que la actividad física durante el ocio representó un factor protector para clasificar a los sujetos en los estadios más avanzados.

Introduction: The visceral adiposity index (VAI) is a simple and cost effective method for the determination of visceral adiposity. The objective of this study is to evaluate the relationship between VAI and different cardiovascular risk factors, sociodemographic variables, and psychobiological habits in the adult population of the city of Cuenca, Ecuador. Materials and methods: A descriptive cross-sectional study was performed on 318 adult individuals selected by multistage random sampling, who underwent a clinical, anthropometric and laboratory evaluation. VAI was determined using the proposed formula that used abdominal circumference, body mass index, triglycerides, and HDL-Cholesterol. A multiple logistic regression model was used to determine the main factors associated with the highest values of visceral adiposity. Results: The mean VAI was 2.57 (1.66-3.94) in the 318 individuals studied, with higher values for females. In the multiple logistic regression model, significant risk factors for moderatehigh VAI were: age (>60 years: OR = 3.87, 95% CI: 1.15-12.96, P=.03), calorie intake, impaired fasting glucose, and leisure time physical activity. Conclusion: VAI is a useful method to define those subjects with visceral adiposity in our region. Age, daily calorie intake, and impaired fasting glucose are the main factors associated with higher index values, while leisure time physical activity was a protective factor for classifying subjects in the more advanced stages.

Anti-neoplastic drugs increase caveolin-1-dependent migration, invasion and metastasis of cancer cells.

Expression of the scaffolding protein Caveolin-1 (CAV1) enhances migration and invasion of metastatic cancer cells. Yet, CAV1 also functions as a tumor suppressor in early stages of cancer, where expression is suppressed by epigenetic mechanisms. Thus, we sought to identify stimuli/mechanisms that revert epigenetic CAV1 silencing in cancer cells and evaluate how this affects their metastatic potential. We reasoned that restricted tissue availability of anti-neoplastic drugs during chemotherapy might expose cancer cells to sub-therapeutic concentrations, which activate signaling pathways and the expression of CAV1 to favor the acquisition of more aggressive traits. Here, we used in vitro [2D, invasion] and in vivo (metastasis) assays, as well as genetic and biochemical approaches to address this question. Colon and breast cancer cells were identified where CAV1 levels were low due to epigenetic suppression and could be reverted by treatment with the methyltransferase inhibitor 5'-azacytidine. Exposure of these cells to anti-neoplastic drugs for short periods of time (24-48 h) increased CAV1 expression through ROS production and MEK/ERK activation. In colon cancer cells, increased CAV1 expression enhanced migration and invasion in vitro via pathways requiring Src-family kinases, as well as Rac-1 activity. Finally, elevated CAV1 expression in colon cancer cells following exposure in vitro to sub-cytotoxic drug concentrations increased their metastatic potential in vivo. Therefore exposure of cancer cells to anti-neoplastic drugs at non-lethal drug concentrations induces signaling events and changes in transcription that favor CAV1-dependent migration, invasion and metastasis. Importantly, this may occur in the absence of selection for drug-resistance.

Extracellular matrix-specific Caveolin-1 phosphorylation on tyrosine 14 is linked to augmented melanoma metastasis but not tumorigenesis.

Caveolin-1 (CAV1) is a scaffolding protein that plays a dual role in cancer. In advanced stages of this disease, CAV1 expression in tumor cells is associated with enhanced metastatic potential, while, at earlier stages, CAV1 functions as a tumor suppressor. We recently implicated CAV1 phosphorylation on tyrosine 14 (Y14) in CAV1-enhanced cell migration. However, the contribution of this modification to the dual role of CAV1 in cancer remained unexplored. Here, we used in vitro [2D and transendothelial cell migration (TEM), invasion] and in vivo (metastasis) assays, as well as genetic and biochemical approaches to address this question in B16F10 murine melanoma cells. CAV1 promoted directional migration on fibronectin or laminin, two abundant lung extracellular matrix (ECM) components, which correlated with enhanced Y14 phosphorylation during spreading. Moreover, CAV1-driven migration, invasion, TEM and metastasis were ablated by expression of the phosphorylation null CAV1(Y14F), but not the phosphorylation mimicking CAV1(Y14E) mutation. Finally, CAV1-enhanced focal adhesion dynamics and surface expression of beta1 integrin were required for CAV1-driven TEM. Importantly, CAV1 function as a tumor suppressor in tumor formation assays was not altered by the Y14F mutation. In conclusion, our results provide critical insight to the mechanisms of CAV1 action during cancer development. Specific ECM-integrin interactions and Y14 phosphorylation are required for CAV1-enhanced melanoma cell migration, invasion and metastasis to the lung. Because Y14F mutation diminishes metastasis without inhibiting the tumor suppressor function of CAV1, Y14 phosphorylation emerges as an attractive therapeutic target to prevent metastasis without altering beneficial traits of CAV1.

Phosphorylation of caveolin-1 on tyrosine-14 induced by ROS enhances palmitate-induced death of beta-pancreatic cells.

A considerable body of evidence exists implicating high levels of free saturated fatty acids in beta pancreatic cell death, although the molecular mechanisms and the signaling pathways involved have not been clearly defined. The membrane protein caveolin-1 has long been implicated in cell death, either by sensitizing to or directly inducing apoptosis and it is normally expressed in beta cells. Here, we tested whether the presence of caveolin-1 modulates free fatty acid-induced beta cell death by reexpressing this protein in MIN6 murine beta cells lacking caveolin-1. Incubation of MIN6 with palmitate, but not oleate, induced apoptotic cell death that was enhanced by the presence of caveolin-1. Moreover, palmitate induced de novo ceramide synthesis, loss of mitochondrial transmembrane potential and reactive oxygen species (ROS) formation in MIN6 cells. ROS generation promoted caveolin-1 phosphorylation on tyrosine-14 that was abrogated by the anti-oxidant N-acetylcysteine or the incubation with the Src-family kinase inhibitor, PP2 (4-amino-5-(4-chlorophenyl)-7(dimethylethyl)pyrazolo[3,4-d]pyrimidine). The expression of a non-phosphorylatable caveolin-1 tyrosine-14 to phenylalanine mutant failed to enhance palmitate-induced apoptosis while for MIN6 cells expressing the phospho-mimetic tyrosine-14 to glutamic acid mutant caveolin-1 palmitate sensitivity was comparable to that observed for MIN6 cells expressing wild type caveolin-1. Thus, caveolin-1 expression promotes palmitate-induced ROS-dependent apoptosis in MIN6 cells in a manner requiring Src family kinase mediated tyrosine-14 phosphorylation.

Rab5 is required in metastatic cancer cells for Caveolin-1-enhanced Rac1 activation, migration and invasion.

Rab5 is a small GTPase that regulates early endosome trafficking and other cellular processes, including cell adhesion and migration. Specifically, Rab5 promotes Rac1 activation and cancer cell migration, but little is known about the upstream regulators of Rab5. We have previously shown that the scaffolding protein Caveolin-1 (CAV1) promotes Rac1 activation and migration of cancer cells. Here, we hypothesized that CAV1 stimulates Rab5 activation, leading to increased Rac1 activity and cell migration. Expression of CAV1 in B16-F10 mouse melanoma and HT-29(US) human colon adenocarcinoma cells increased the GTP loading of Rab5, whereas shRNA-mediated targeting of endogenous CAV1 in MDA-MB-231 breast cancer cells decreased Rab5-GTP levels. Accordingly, shRNA-mediated downregulation of Rab5 decreased CAV1-mediated Rac1 activation, cell migration and invasion in B16-F10 and HT-29(US) cells. Expression of CAV1 was accompanied by increased recruitment of Tiam1, a Rac1 guanine nucleotide exchange factor (GEF), to Rab5-positive early endosomes. Using the inhibitor NSC23766, Tiam1 was shown to be required for Rac1 activation and cell migration induced by CAV1 and Rab5. Mechanistically, we provide evidence implicating p85α (also known as PIK3R1), a Rab5 GTPase-activating protein (GAP), in CAV1-dependent effects, by showing that CAV1 recruits p85α, precluding p85α-mediated Rab5 inactivation and increasing cell migration. In summary, these studies identify a novel CAV1-Rab5-Rac1 signaling axis, whereby CAV1 prevents Rab5 inactivation, leading to increased Rac1 activity and enhanced tumor cell migration and invasion.

Rab5 activation promotes focal adhesion disassembly, migration and invasiveness in tumor cells.

Migration and invasion are essential steps associated with tumor cell metastasis and increasing evidence points towards endosome trafficking being essential in this process. Indeed, the small GTPase Rab5, a crucial regulator of early endosome dynamics, promotes cell migration in vitro and in vivo. Precisely how Rab5 participates in these events remains to be determined. Considering that focal adhesions represent structures crucial to cell migration, we specifically asked whether Rab5 activation promoted focal adhesion disassembly and thereby facilitated migration and invasion of metastatic cancer cells. Pulldown and biosensor assays revealed that Rab5-GTP loading increased at the leading edge of migrating tumor cells. Additionally, targeting of Rab5 by different shRNA sequences, but not control shRNA, decreased Rab5-GTP levels, leading to reduced cell spreading, migration and invasiveness. Re-expression in knockdown cells of wild-type Rab5, but not the S34N mutant (GDP-bound), restored these properties. Importantly, Rab5 association with the focal adhesion proteins vinculin and paxillin increased during migration, and expression of wild-type, but not GDP-bound Rab5, accelerated focal adhesion disassembly, as well as FAK dephosphorylation on tyrosine 397. Finally, Rab5-driven invasiveness required focal adhesion disassembly, as treatment with the FAK inhibitor number 14 prevented Matrigel invasion and matrix metalloproteinase release. Taken together, these observations show that Rab5 activation is required to enhance cancer cell migration and invasion by promoting focal adhesion disassembly.

Caveolin-1-enhanced motility and focal adhesion turnover require tyrosine-14 but not accumulation to the rear in metastatic cancer cells.

Caveolin-1 is known to promote cell migration, and increased caveolin-1 expression is associated with tumor progression and metastasis. In fibroblasts, caveolin-1 polarization and phosphorylation of tyrosine-14 are essential to promote migration. However, the role of caveolin-1 in migration of metastatic cells remains poorly defined. Here, caveolin-1 participation in metastatic cell migration was evaluated by shRNA targeting of endogenous caveolin-1 in MDA-MB-231 human breast cancer cells and ectopic expression in B16-F10 mouse melanoma cells. Depletion of caveolin-1 in MDA-MB-231 cells reduced, while expression in B16-F10 cells promoted migration, polarization and focal adhesion turnover in a sequence of events that involved phosphorylation of tyrosine-14 and Rac-1 activation. In B16-F10 cells, expression of a non-phosphorylatable tyrosine-14 to phenylalanine mutant failed to recapitulate the effects observed with wild-type caveolin-1. Alternatively, treatment of MDA-MB-231 cells with the Src family kinase inhibitor PP2 reduced caveolin-1 phosphorylation on tyrosine-14 and cell migration. Surprisingly, unlike for fibroblasts, caveolin-1 polarization and re-localization to the trailing edge were not observed in migrating metastatic cells. Thus, expression and phosphorylation, but not polarization of caveolin-1 favor the highly mobile phenotype of metastatic cells.

A molecular evaluation of germ cell death induced by etoposide in pubertal rat testes.

Etoposide is widely used in the treatment of patients with testicular cancer. The mechanism underlying apoptosis induction in cancer cells has been studied in different cell types, but it is not known whether the same factors participate in viable germ cells undergoing programmed cell death. Since testicular cancer primarily affects young males, we used pubertal rats (21 days old) as a model to determine different apoptotic parameters after etoposide treatment in healthy testes. We found that one intratesticular injection of etoposide (1.2 microg/testis) induced a significant increase in spermatocytes undergoing apoptosis, along with activation of caspase-9, -8 and -3 after 24 h of treatment. Spermatocyte apoptosis was inhibited when a general caspase inhibitor was added along with etoposide. Etoposide induces a significant stabilization/activation of p53, resulting in an increase level of this protein. The mRNA of Bcl-2 antagonist of cell death (BAD), a pro-apoptotic gene and a transcriptional target of p53, was significantly increased after etoposide treatment. Thus, our results suggest a single injection of etoposide induces apoptosis in healthy pachytene spermatocytes mediated by p53 and caspase activation. These findings will assist the search for new therapies to prevent the deleterious effect of cancer drugs upon normal cells.