Raloxifene and n-Acetylcysteine Ameliorate TGF-Signalling in Fibroblasts from Patients with Recessive Dominant Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin disease caused by mutation of the COL7A1 gene. RDEB is associated with high levels of TGF-ß1, which is likely to be involved in the fibrosis that develops in this disease. Endoglin (CD105) is a type III coreceptor for TGF-ß1 and its overexpression in fibroblasts deregulates physiological Smad/Alk1/Alk5 signalling, repressing the synthesis of TGF-ß1 and extracellular matrix (ECM) proteins. Raloxifene is a specific estrogen receptor modulator designated as an orphan drug for hereditary hemorrhagic telangiectasia, a rare vascular disease. Raloxifene stimulates endoglin synthesis, which could attenuate fibrosis. By contrast, the antioxidant N-acetylcysteine may have therapeutic value to rectify inflammation, fibrosis and endothelial dysfunction. Thus, we present here a repurposing strategy based on the molecular and functional screening of fibroblasts from RDEB patients with these drugs, leading us to propose the repositioning of these two well-known drugs currently in clinical use, raloxifene and N-acetylcysteine, to counteract fibrosis and inflammation in RDEB. Both compounds modulate the profibrotic events that may ultimately be responsible for the clinical manifestations in RDEB, suggesting that these findings may also be relevant for other diseases in which fibrosis is an important pathophysiological event.

11PS04 is a new chemical entity identified by microRNA-based biosensing with promising therapeutic potential against cancer stem cells.

Phenotypic drug discovery must take advantage of the large amount of clinical data currently available. In this sense, the impact of microRNAs (miRs) on human disease and clinical therapeutic responses is becoming increasingly well documented. Accordingly, it might be possible to use miR-based signatures as phenotypic read-outs of pathological status, for example in cancer. Here, we propose to use the information accumulating regarding the biology of miRs from clinical research in the preclinical arena, adapting it to the use of miR biosensors in the earliest steps of drug screening. Thus, we have used an amperometric dual magnetosensor capable of monitoring a miR-21/miR-205 signature to screen for new drugs that restore these miRs to non-tumorigenic levels in cell models of breast cancer and glioblastoma. In this way we have been able to identify a new chemical entity, 11PS04 ((3aR,7aS)-2-(3-propoxyphenyl)-7,7a-dihydro-3aH-pyrano[3,4-d]oxazol-6(4H)-one), the therapeutic potential of which was suggested in mechanistic assays of disease models, including 3D cell culture (oncospheres) and xenografts. These assays highlighted the potential of this compound to attack cancer stem cells, reducing the growth of breast and glioblastoma tumors in vivo. These data demonstrate the enhanced chain of translatability of this strategy, opening up new perspectives for drug-discovery pipelines and highlighting the potential of miR-based electro-analytical sensors as efficient tools in modern drug discovery.

Multifunctional Albumin-Stabilized Gold Nanoclusters for the Reduction of Cancer Stem Cells.

Controlled delivery of multiple chemotherapeutics can improve the effectiveness of treatments and reduce side effects and relapses. Here in, we used albumin-stabilized gold nanoclusters modified with doxorubicin and SN38 (AuNCs-DS) as combined therapy for cancer. The chemotherapeutics are conjugated to the nanostructures using linkers that release them when exposed to different internal stimuli (Glutathione and pH). This system has shown potent antitumor activity against breast and pancreatic cancer cells. Our studies indicate that the antineoplastic activity observed may be related to the reinforced DNA damage generated by the combination of the drugs. Moreover, this system presented antineoplastic activity against mammospheres, a culturing model for cancer stem cells, leading to an efficient reduction of the number of oncospheres and their size. In summary, the nanostructures reported here are promising carriers for combination therapy against cancer and particularly to cancer stem cells.

Better Nutritional Status Is Positively Associated with mRNA Expression of SIRT1 in Community-Dwelling Older Adults in the Toledo Study for Healthy Aging.

Background: The expression of certain genes involved in response to oxidative stress is likely related to aging-related outcomes, such as frailty in old age. Given nutrition's substantial impact in aging and age-related diseases, one of its mechanisms might be through influencing gene expression. Objective: This study aimed to investigate the association between nutrition and the expression of 15 genes related to cellular response to stress in older community-dwelling individuals. Methods: A nested case-control study of 350 participants (mean ± SEM age: 76.5 ± 6.9 y, 42.9% men, 22% frail according to Fried's criteria) was selected from the Toledo Study for Healthy Aging. Blood-derived RNA was retro-transcribed into complementary DNA. TaqMan Arrays were used for determining gene expression. The Mini Nutritional Assessment (MNA) and the PREDIMED (PREvención con DIeta MEDiterranea) questionnaire measured nutritional status and adherence to the Mediterranean diet (MD), respectively. Data were reweighed so that inference from linear and logistic regression models applied to the original sampling population. Results: Higher MNA scores were associated with higher expressions of the gene coding for sirtuin-1 (SIRT1), regardless of age, sex, and Charlson comorbidity score (P = 0.04) and even after adjusting for frailty status (P = 0.016) and level of adherence to the MD (P = 0.04). Malnutrition risk and SIRT1 gene expression were inversely associated (P = 0.0045) independently of frailty status (P = 0.0045) and level of adherence to the MD (P = 0.0075). Conclusions: In older individuals, improvement in nutritional status is positively associated with SIRT1 gene expression independently of frailty status or adherence to the MD. These findings may provide potential biomarkers and targets for health interventions among the elderly.

Frailty Is Associated With Lower Expression of Genes Involved in Cellular Response to Stress: Results From the Toledo Study for Healthy Aging.

BACKGROUND: Specific mechanisms underlying frailty syndrome are not well known. Frailty can be viewed as a loss of functional reserve resulting in increased vulnerability to stressors. We hypothesize that pathways regulating cellular response to stress are potential players in the development of frailty. The aim of this study was to evaluate the association of the expression of certain genes related to cellular response to stress with the presence of frailty in older patients. METHODS: A sample of 350 individuals aged 65 years or older (22% frail) was selected from the Toledo Study of Healthy Aging. RNA was extracted from blood and retro-transcribed into complementary DNA. TaqMan Low density Arrays were used for the measurement of expression of genes implicated in cellular response to oxidative stress, genes implicated in inflammation, genes implicated in vascular physiology, and genes related to hypoxia. For data analysis, a logistic regression model was used to assess the relationship of gene expression and frailty. RESULTS: Among the analyzed genes, lower expression of genes related to cellular response to hypoxia (hypoxia inducible factor-1α) or to cellular response to oxidative stress (nuclear factor erythroid 2-related factor 2 and its target genes heme oxygenase-2, thioredoxin reductase-1, and superoxide dismutase-2), but not to those related to inflammation or vascular physiology, were significantly associated with the presence of frailty after adjustment for age and sex. These associations remained significant after adjustment by type 2 diabetes and Charlson index of comorbidities. Lower expressions of genes involved in cellular response to stress were also associated with increased risk of functional impairment. CONCLUSIONS: Reduced expression of several genes implicated in cellular response to oxidative stress or hypoxia is significantly associated with the presence of frailty. These results help to fill the gap of knowledge of this evolving field and provide targets for intervention to promote health and independence in the elderly.

Structure and Function of Prokaryotic UDP-Glucose Pyrophosphorylase, A Drug Target Candidate.

UDP-glucose is an essential metabolite for a variety of processes in the cell physiology in all organisms. In prokaryotes, it is involved in the synthesis of trehalose, an osmoprotectant, in galactose utilization via the Leloir pathway and it plays a key role in the synthesis of the components of the bacterial envelope, particularly the lipopolysaccharide and the capsule, which represent necessary virulence factors of many bacterial pathogens. UDP-glucose is synthesized in bacteria by the prokaryotic UDP-glucose pyrophosphorylase (UGP, EC 2.7.7.9), an enzyme belonging to the family of sugar:nucleotidyl transferases. Despite the ubiquitous distribution of UGP activity in all domains of life, prokaryotic UGPs are evolutionarily unrelated to their eukaryotic counterparts. Taken together, these features make of bacterial UGP an attractive target candidate for the discovery and development of new generation antibiotics. This review summarizes the current knowledge on structure and function of bacterial UGPs, underlying their potential as drug target candidates.

Hypoxia-inducible factor 1 alpha contributes to cardiac healing in mesenchymal stem cells-mediated cardiac repair.

Mesenchymal stem cells (MSC) are effective in treating myocardial infarction (MI) and previous reports demonstrated that hypoxia improves MSC self-renewal and therapeutics. Considering that hypoxia-inducible factor-1 alpha (HIF-1α) is a master regulator of the adaptative response to hypoxia, we hypothesized that HIF-1α overexpression in MSC could mimic some of the mechanisms triggered by hypoxia and increase their therapeutic potential without hypoxia stimulation. Transduction of MSC with HIF-1α lentivirus vectors (MSC-HIF) resulted in increased cell adhesion and migration, and activation of target genes coding for paracrine factors. When MSC-HIF were intramyocardially injected in infarcted nude rats, significant improvement was found (after treatment of infarcted rats with MSC-HIF) in terms of cardiac function, angiogenesis, cardiomyocyte proliferation, and reduction of fibrotic tissue with no induction of cardiac hypertrophy. This finding provides evidences for a crucial role of HIF-1α on MSC biology and suggests the stabilization of HIF-1α as a novel strategy for cellular therapies.

The biology of HIFα proteins in cell differentiation and disease.

The biology of the α subunits of the hypoxia-inducible factors (HIFα) has expanded in the past years from their original role in angiogenesis to their nowadays position in the self-renewal and differentiation of stem cells. Hypoxia is a physiological condition in different tissues-including tumors-and, may cause stem cells in the onset of genomic instability, this last associated in the scientific literature with the acquisition of a malignant phenotypes. HIFα proteins have been the subjects of excellent scientific contributions in the past years, providing new paradigms in the biology of these key proteins and their pivotal role in cell homeostasis. Over other therapeutic implications, the relevance of studies focused on the etiology of tumor-initiating cells and the characterization of the mechanisms that could lead to their malignancy, is gaining significance in the health areas of cancer and regenerative medicine.

FM19G11: A new modulator of HIF that links mTOR activation with the DNA damage checkpoint pathways.

The network consisting of mTOR and p53 pathways is crucial to understanding a wide variety of physiological and pathological events, including cancer and aging. In addition, the HIF1alpha protein, a downstream target of mTOR, is a hallmark of different tumor types and was the desired strategy of many drug discovery efforts. Here we present the novel chemical entity FM19G11, a new modulator of HIF1alpha expression, which was used as a molecular tool to dissect and further characterize the cross-talk between these signaling cascades in human colon carcinoma cell lines. To our knowledge, FM19G11 is the first drug that triggers a DNA damage response (DDR) associated with G(1)/S-phase arrest in a p53-dependent manner, due to rapid hyper-activation of the growth signaling pathway through mTOR. Assessment of colonies demonstrated that FM19G11 decreases the clonogenicity of HT29, HCT116/p53(+/+) and HCT116/p53(-/-) cells. Moreover, FM19G11 causes significant lower colony growth in soft agar of p53-proficient human colon cancer cells. Consequently, p53 sensitizes human colon cancer cells to FM19G11 by significant reduction of their viability, lessening their colony formation capability and shrinking their anchorage-independent growth. Cell signaling studies served to assign a new mode of action to FM19G11, whose tumor-suppressant activity compromises the survival of functional p53 malignant cells.

Activated spinal cord ependymal stem cells rescue neurological function.

Spinal cord injury (SCI) is a major cause of paralysis. Currently, there are no effective therapies to reverse this disabling condition. The presence of ependymal stem/progenitor cells (epSPCs) in the adult spinal cord suggests that endogenous stem cell-associated mechanisms might be exploited to repair spinal cord lesions. epSPC cells that proliferate after SCI are recruited by the injured zone, and can be modulated by innate and adaptive immune responses. Here we demonstrate that when epSPCs are cultured from rats with a SCI (ependymal stem/progenitor cells injury [epSPCi]), these cells proliferate 10 times faster in vitro than epSPC derived from control animals and display enhanced self renewal. Genetic profile analysis revealed an important influence of inflammation on signaling pathways in epSPCi after injury, including the upregulation of Jak/Stat and mitogen activated protein kinase pathways. Although neurospheres derived from either epSPCs or epSPCi differentiated efficiently to oligodendrocites and functional spinal motoneurons, a better yield of differentiated cells was consistently obtained from epSPCi cultures. Acute transplantation of undifferentiated epSPCi or the resulting oligodendrocyte precursor cells into a rat model of severe spinal cord contusion produced a significant recovery of motor activity 1 week after injury. These transplanted cells migrated long distances from the rostral and caudal regions of the transplant to the neurofilament-labeled axons in and around the lesion zone. Our findings demonstrate that modulation of endogenous epSPCs represents a viable cell-based strategy for restoring neuronal dysfunction in patients with spinal cord damage.

Hypoxia causes downregulation of mismatch repair system and genomic instability in stem cells.

The DNA mismatch repair (MMR) system maintains genomic integrity by correcting replication errors: its malfunction causes genomic instability in several tumor types. Hypoxia-inducible factor-1alpha (HIF1alpha), the major regulator of the processes that occur in hypoxia and certain epigenetic events downregulate the expression of MMR genes in cancer cells. However, there is a lack of information regarding MMR regulation and the genetic stability of stem cells under hypoxic conditions. The expression of the MMR system is downregulated in murine and human stem cells cultured in hypoxia, which correlates with lower DNA repair activity in neural stem cells. We observed, through the use of short hairpin loop RNAi expression constructs, that HIF1alpha positively regulated MLH1 and MSH6 when the C17.2 neural stem cells were exposed to short-term hypoxia. However, in prolonged exposure to oxygen depletion, the reduced transcriptional activation of MMR genes was directed by specific epigenetic events. Chromatin immunoprecipitation experiments showed a hypoacetylated/hypermethylated histone H3 and lower SP1 binding within MLH1 and MSH6 adjacent promoter regions. Treatment with the histone deacetylase inhibitor trichostatin A increased histone H3 acetylation and SP1 occupancy and enhanced MMR expression. Sequencing of microsatellite markers revealed genomic instability in the murine and human stem cells grown under hypoxia. Thus, the present article reports, for the first time in the stem cell field, experimental data that indicate that hypoxic niches are an environment in which stem cells might undergo genomic instability, which could lie at the origin of subpopulations with cancer stem cell properties. Disclosure of potential conflicts of interest is found at the end of this article.

New cytotoxic cembranes from the sea pen Gyrophyllum sibogae.

Two new cembrane-type diterpenoids have been isolated from the 2-propanol extract of the sea pen Gyrophyllum sibogae collected in South Africa: 7,8-dihydroflabellatene A (1) and 7,8-dihydroflabellatene B (2). Their structures were determined on the basis of detailed spectroscopic analysis and by single-crystal X-ray analysis of the major metabolite 1, which showed strong in vitro cytotoxicity against a panel of 13 tumor cell lines.

Aplidin induces apoptosis in human cancer cells via glutathione depletion and sustained activation of the epidermal growth factor receptor, Src, JNK, and p38 MAPK.

We report that Aplidin, a novel antitumor agent of marine origin presently undergoing Phase II clinical trials, induced growth arrest and apoptosis in human MDA-MB-231 breast cancer cells at nanomolar concentrations. Aplidin induced a specific cellular stress response program, including sustained activation of the epidermal growth factor receptor (EGFR), the non-receptor protein-tyrosine kinase Src, and the serine/threonine kinases JNK and p38 MAPK. Aplidin-induced apoptosis was only partially blocked by the general caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone and was also sensitive to AG1478 (an EGFR inhibitor), PP2 (an Src inhibitor), and SB203580 (an inhibitor of JNK and p38 MAPK) in MDA-MB-231 cells. Supporting a role for EGFR in Aplidin action, EGFR-deficient mouse embryo fibroblasts underwent apoptosis upon treatment more slowly than wild-type EGFR fibroblasts and also showed delayed JNK and reduced p38 MAPK activation. N-Acetylcysteine and ebselen (but not other antioxidants such as diphenyleneiodonium, Tiron, catalase, ascorbic acid, and vitamin E) reduced EGFR activation by Aplidin. N-Acetylcysteine and PP2 also partially inhibited JNK and p38 MAPK activation. The intracellular level of GSH affected Aplidin action; pretreatment of cells with GSH or N-acetylcysteine inhibited, whereas GSH depletion caused, hyperinduction of EGFR, Src, JNK, and p38 MAPK. Remarkably, Aplidin also induced apoptosis and activated EGFR, JNK, and p38 MAPK in two cell lines (A-498 and ACHN) derived from human renal cancer, a neoplasia that is highly refractory to chemotherapy. These data provide a molecular basis for the anticancer activity of Aplidin.

Aplidin induces the mitochondrial apoptotic pathway via oxidative stress-mediated JNK and p38 activation and protein kinase C delta.

Aplidin, a new antitumoural drug presently in phase II clinical trials, has shown both in vitro and in vivo activity against human cancer cells. Aplidin effectively inhibits cell viability by triggering a canonical apoptotic program resulting in alterations in cell morphology, caspase activation, and chromatin fragmentation. Pro-apoptotic concentrations of Aplidin induce early oxidative stress, which results in a rapid and persistent activation of both JNK and p38 MAPK and a biphasic activation of ERK. Inhibition of JNK and p38 MAPK blocks the apoptotic program induced by Aplidin demonstrating its central role in the integration of the cellular stress induced by the drug. JNK and p38 MAPK activation results in downstream cytochrome c release and activation of caspases -9 and -3 and PARP cleavage, demonstrating the mediation of the mitochondrial apoptotic pathway in this process. We also demonstrate that protein kinase C delta (PKC-delta) mediates the cytotoxic effect of Aplidin and that it is concomitantly processed and activated late in the apoptotic process by a caspase mediated mechanism. Remarkably, cells deficient in PKC-delta show enhanced survival upon drug treatment as compared to its wild type counterpart. PKC-delta thus appears as an important component necessary for full caspase cascade activation and execution of apoptosis, which most probably initiates a positive feedback loop further amplifying the apoptotic process.