Tamoxifen enhances the cytotoxic effects of nelfinavir in breast cancer cells

Introduction: The HIV protease inhibitor nelfinavir is currently under investigation as a new anti-cancer drug. Several studies have shown that nelfinavir induces cell cycle arrest, endoplasmic reticulum stress, autophagy, and apoptosis in cancer cells. In the present article, the effect of nelfinavir on human breast cancer cells is examined and potential combination treatments are investigated. Methods: The effects of nelfinavir and tamoxifen on the human breast cancer cell lines MCF7, T47 D, MDA-MB-453, and MDA-MB-435 were tested by analysing their influence on cell viability (via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay), apoptosis (annexin binding, poly(ADP-ribose) polymerase cleavage), autophagy (autophagy marker light chain 3B expression), endoplasmic reticulum stress (binding protein and activating transcription factor 3 expression), and the occurrence of oxidative stress (intracellular glutathione level). Results: Nelfinavir induced apoptosis in all four breast cancer cell lines tested, although the extent of autophagy and endoplasmic reticulum stress varied among the cell lines. The concentration of nelfinavir needed for an efficient induction of apoptosis in breast cancer cells could be reduced from 15 mu g/ml to 6 mu g/ml when combined with tamoxifen. At a concentration of 6 mu g/ml, tamoxifen substantially enhanced the endoplasmic reticulum stress reaction in those cell lines that responded to nelfinavir with binding protein (BiP) upregulation (MCF7, T47D), and enhanced autophagy in cell lines that responded to nelfinavir treatment with autophagy marker light chain 3B upregulation (MDA-MB-453). Although tamoxifen has been described to be able to induce oxidative stress at concentrations similar to those applied in this study (6 mu g/ml), we observed that nelfinavir but not tamoxifen reduced the intracellular glutathione level of breast cancer cells within hours of application by up to 32%, suggesting the induction of oxidative stress was an early event and an additional cause of the apoptosis induced by nelfinavir. Conclusions: The results demonstrate that nelfinavir may be an effective drug against breast cancer and could be combined with tamoxifen to enhance its efficacy against breast cancer cells. Moreover, the cytotoxic effect of a tamoxifen and nelfinavir combination was independent of the oestrogen receptor status of the analysed breast cancer cells, suggesting a potential benefit of a combination of these two drugs even in patients with no hormone-responsive tumours. We therefore recommend that clinical studies on nelfinavir with breast cancer patients should include this drug combination to analyse the therapeutic efficacy as well as the safety and tolerability of this potential treatment option

MicroRNAs in pulmonary arterial remodeling

Pulmonary arterial remodeling is a presently irreversible pathologic hallmark of pulmonary arterial hypertension (PAH). This complex disease involves pathogenic dysregulation of all cell types within the small pulmonary arteries contributing to vascular remodeling leading to intimal lesions, resulting in elevated pulmonary vascular resistance and right heart dysfunction. Mutations within the bone morphogenetic protein receptor 2 gene, leading to dysregulated proliferation of pulmonary artery smooth muscle cells, have been identified as being responsible for heritable PAH. Indeed, the disease is characterized by excessive cellular proliferation and resistance to apoptosis of smooth muscle and endothelial cells. Significant gene dysregulation at the transcriptional and signaling level has been identified. MicroRNAs are small non-coding RNA molecules that negatively regulate gene expression and have the ability to target numerous genes, therefore potentially controlling a host of gene regulatory and signaling pathways. The major role of miRNAs in pulmonary arterial remodeling is still relatively unknown although research data is emerging apace. Modulation of miRNAs represents a possible therapeutic target for altering the remodeling phenotype in the pulmonary vasculature. This review will focus on the role of miRNAs in regulating smooth muscle and endothelial cell phenotypes and their influence on pulmonary remodeling in the setting of PAH

Insulin signalling and the regulation of glucose and lipid metabolism

The epidemic of type 2 diabetes and impaired glucose tolerance is one of the main causes of morbidity and mortality worldwide. In both disorders, tissues such as muscle, fat and liver become less responsive or resistant to insulin. This state is also linked to other common health problems, such as obesity, polycystic ovarian disease, hyperlipidaemia, hypertension and atherosclerosis. The pathophysiology of insulin resistance involves a complex network of signalling pathways, activated by the insulin receptor, which regulates intermediary metabolism and its organization in cells. But recent studies have shown that numerous other hormones and signalling events attenuate insulin action, and are important in type 2 diabetes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62568/1/414799a.pd

Medium Chain Fatty Acids Are Selective Peroxisome Proliferator Activated Receptor (PPAR) γ Activators and Pan-PPAR Partial Agonists

Thiazolidinediones (TZDs) act through peroxisome proliferator activated receptor (PPAR) γ to increase insulin sensitivity in type 2 diabetes (T2DM), but deleterious effects of these ligands mean that selective modulators with improved clinical profiles are needed. We obtained a crystal structure of PPARγ ligand binding domain (LBD) and found that the ligand binding pocket (LBP) is occupied by bacterial medium chain fatty acids (MCFAs). We verified that MCFAs (C8–C10) bind the PPARγ LBD in vitro and showed that they are low-potency partial agonists that display assay-specific actions relative to TZDs; they act as very weak partial agonists in transfections with PPARγ LBD, stronger partial agonists with full length PPARγ and exhibit full blockade of PPARγ phosphorylation by cyclin-dependent kinase 5 (cdk5), linked to reversal of adipose tissue insulin resistance. MCFAs that bind PPARγ also antagonize TZD-dependent adipogenesis in vitro. X-ray structure B-factor analysis and molecular dynamics (MD) simulations suggest that MCFAs weakly stabilize C-terminal activation helix (H) 12 relative to TZDs and this effect is highly dependent on chain length. By contrast, MCFAs preferentially stabilize the H2-H3/β-sheet region and the helix (H) 11-H12 loop relative to TZDs and we propose that MCFA assay-specific actions are linked to their unique binding mode and suggest that it may be possible to identify selective PPARγ modulators with useful clinical profiles among natural products

Co-Regulation of the DAF-16 Target Gene, cyp-35B1/dod-13, by HSF-1 in C. elegans Dauer Larvae and daf-2 Insulin Pathway Mutants

Insulin/IGF-I-like signaling (IIS) has both cell autonomous and non-autonomous functions. In some cases, targets through which IIS regulates cell-autonomous functions, such as cell growth and metabolism, have been identified. In contrast, targets for many non-autonomous IIS functions, such as C. elegans dauer morphogenesis, remain elusive. Here, we report the use of genomic and genetic approaches to identify potential non-autonomous targets of C. elegans IIS. First, we used transcriptional microarrays to identify target genes regulated non-autonomously by IIS in the intestine or in neurons. C. elegans IIS controls expression of a number of stress response genes, which were differentially regulated by tissue-restricted IIS. In particular, expression of sod-3, a MnSOD enzyme, was not regulated by tissue-restricted IIS on the microarrays, while expression of hsp-16 genes was rescued back to wildtype by tissue restricted IIS. One IIS target regulated non-autonomously by age-1 was cyp-35B1/dod-13, encoding a cytochrome P450. Genetic analysis of the cyp-35B1 promoter showed both DAF-16 and HSF-1 are direct regulators. Based on these findings, we propose that hsf-1 may participate in the pathways mediating non-autonomous activities of age-1 in C. elegans

Lack of Wdr13 Gene in Mice Leads to Enhanced Pancreatic Beta Cell Proliferation, Hyperinsulinemia and Mild Obesity

WD-repeat proteins are very diverse, yet these are structurally related proteins that participate in a wide range of cellular functions. WDR13, a member of this family, is conserved from fishes to humans and localizes into the nucleus. To understand the in vivo function(s) of Wdr13 gene, we have created and characterized a mutant mouse strain lacking this gene. The mutant mice had higher serum insulin levels and increased pancreatic islet mass as a result of enhanced beta cell proliferation. While a known cell cycle inhibitor, p21, was downregulated in the mutant islets, over expression of WDR13 in the pancreatic beta cell line (MIN6) resulted in upregulation of p21, accompanied by retardation of cell proliferation. We suggest that WDR13 is a novel negative regulator of the pancreatic beta cell proliferation. Given the higher insulin levels and better glucose clearance in Wdr13 gene deficient mice, we propose that this protein may be a potential candidate drug target for ameliorating impaired glucose metabolism in diabetes

Prenatal Hypoxic-Ischemic Insult Changes the Distribution and Number of NADPH-Diaphorase Cells in the Cerebellum

Astrogliosis, oligodendroglial death and motor deficits have been observed in the offspring of female rats that had their uterine arteries clamped at the 18th gestational day. Since nitric oxide has important roles in several inflammatory and developmental events, here we evaluated NADPH-diaphorase (NADPH-d) distribution in the cerebellum of rats submitted to this hypoxia-ischemia (HI) model. At postnatal (P) day 9, Purkinje cells of SHAM and non-manipulated (NM) animals showed NADPH-d+ labeling both in the cell body and dendritic arborization in folia 1 to 8, while HI animals presented a weaker labeling in both cellular structures. NADPH-d+ labeling in the molecular (ML), and in both the external and internal granular layer, was unaffected by HI at this age. At P23, labeling in Purkinje cells was absent in all three groups. Ectopic NADPH-d+ cells in the ML of folia 1 to 4 and folium 10 were present exclusively in HI animals. This labeling pattern was maintained up to P90 in folium 10. In the cerebellar white matter (WM), at P9 and P23, microglial (ED1+) NADPH-d+ cells, were observed in all groups. At P23, only HI animals presented NADPH-d labeling in the cell body and processes of reactive astrocytes (GFAP+). At P9 and P23, the number of NADPH-d+ cells in the WM was higher in HI animals than in SHAM and NM ones. At P45 and at P90 no NADPH-d+ cells were observed in the WM of the three groups. Our results indicate that HI insults lead to long-lasting alterations in nitric oxide synthase expression in the cerebellum. Such alterations in cerebellar differentiation might explain, at least in part, the motor deficits that are commonly observed in this model

Blood Glucose Levels Regulate Pancreatic β-Cell Proliferation during Experimentally-Induced and Spontaneous Autoimmune Diabetes in Mice

Type 1 diabetes mellitus is caused by immune-mediated destruction of pancreatic beta-cells leading to insulin deficiency, impaired intermediary metabolism, and elevated blood glucose concentrations. While at autoimmune diabetes onset a limited number of beta-cells persist, the cells' regenerative potential and its regulation have remained largely unexplored. Using two mouse autoimmune diabetes models, this study examined the proliferation of pancreatic islet ss-cells and other endocrine and non-endocrine subsets, and the factors regulating that proliferation.We adapted multi-parameter flow cytometry techniques (including DNA-content measurements and 5'-bromo-2'-deoxyuridine [BrdU] incorporation) to study pancreatic islet single cell suspensions. These studies demonstrate that beta-cell proliferation rapidly increases at diabetes onset, and that this proliferation is closely correlated with the diabetic animals' elevated blood glucose levels. For instance, we show that when normoglycemia is restored by exogenous insulin or islet transplantation, the beta-cell proliferation rate returns towards low levels found in control animals, yet surges when hyperglycemia recurs. In contrast, other-than-ss endocrine islet cells did not exhibit the same glucose-dependent proliferative responses. Rather, disease-associated alterations of BrdU-incorporation rates of delta-cells (minor decrease), and non-endocrine islet cells (slight increase) were not affected by blood glucose levels, or were inversely related to glycemia control after diabetes onset (alpha-cells).We conclude that murine beta-cells' ability to proliferate in response to metabolic need (i.e. rising blood glucose concentrations) is remarkably well preserved during severe, chronic beta-cell autoimmunity. These data suggest that timely control of the destructive immune response after disease manifestation could allow spontaneous regeneration of sufficient beta-cell mass to restore normal glucose homeostasis