Increasing evidence shows the beneficial effects of fish oil on breast cancer growth and invasion in vitro and in animal models. Expression of CSF-1 (colony stimulating factor-1) by breast cancer cells acts as potent activator of malignancy and metastasis. In this report, we used two human breast cancer cell lines, MDA-MB-231 and MCF-7, to show that the bioactive fish oil component DHA (docosahexaenoic acid) inhibits expression of CSF-1 and its secretion from these cancer cells. We found that the tumor suppressor protein PTEN regulates CSF-1 expression through PI 3 kinase/Akt signaling via a transcriptional mechanism. The enhanced abundance of microRNA-21 (miR-21) in breast cancer cells contributes to the growth and metastasis. Interestingly, DHA significantly inhibited expression of miR-21. miR-21 Sponge, which derepresses the miR-21 targets, markedly decreased expression of CSF-1 and its secretion. Furthermore, miR-21-induced upregulation of CSF-1 mRNA and its transcription were prevented by expression of PTEN mRNA lacking 3′-untranslated region (UTR) and miR-21 recognition sequence. Strikingly, miR-21 reversed DHA-forced reduction of CSF-1 expression and secretion. Finally, we found that expression of miR-21 as well as CSF-1 was significantly attenuated in breast tumors of mice receiving a diet supplemented with fish oil. Our results reveal a novel mechanism for the therapeutic function of fish oil diet that blocks miR-21, thereby increasing PTEN levels to prevent expression of CSF-1 in breast cancer.
The data derived from epidemiological and animal models confirm a beneficial effect of fish oil (rich in ω-3 polyunsaturated fatty acids) in the amelioration of tumor growth and progression, including breast cancer. The breast cancer patients often develop bone metastasis evidenced by osteolytic lesions, leading to severe pain and bone fracture. Using a mouse model of MDA-MB-231 human breast cancer cell metastasis to bone, here we show that fish oil diet enriched in DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid) prevents the formation of osteolytic lesions in bone, indicating suppression of cancer cell metastasis to bone. These results are supported by our data showing both DHA and EPA significantly attenuate the migration/invasion of MDA-MB-231 breast cancer cells in culture. The mechanism that limits breast cancer cells to selective metastasis to bone remains hitherto unexplored. Aberrant increased expression of CD44 is associated with generation of cancer stem cells, which contribute to metastasis of breast cancer cells. We demonstrate that DHA and EPA significantly inhibit the expression of CD44 protein and mRNA by a transcriptional mechanism. Furthermore, we show markedly reduced levels of CD44 mRNA and protein in the tumors of mice, which were fed fish oil diet than those in control diet. Our data provide the first evidence for a salutary effect of fish oil on breast cancer metastasis to bone. Our results identify a novel function of the fish oil active components, DHA and EPA, which target the cell-intrinsic pro-metastatic molecule CD44 to inhibit migration/invasion.
Sustained activation of Akt kinase acts as a focal regulator to increase cell growth and survival, which cause tumorigenesis including breast cancer. Statins, potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, display anticancer activity. The molecular mechanisms by which statins block cancer cell growth are poorly understood. We demonstrate that in the tumors derived from MDA-MB-231 human breast cancer cell xenografts, simvastatin significantly inhibited phosphorylation of Akt with concomitant attenuation of expression of the anti-apoptotic protein BclXL. In many cancer cells, BclXL is a target of NFκB. Simvastatin inhibited the DNA binding and transcriptional activities of NF κ B resulting in marked reduction in transcription of BclXL. Signals transmitted by anti-neoplastic mechanism implanted in the cancer cells serve to obstruct the initial outgrowth of tumors. One such mechanism represents the action of the tumor suppressor protein PTEN, which negatively regulates Akt kinase activity. We provide the first evidence for significantly increased levels of PTEN in the tumors of simvastatin-administered mice. Importantly, simvastatin markedly prevented binding of NFκB to the two canonical recognition elements, NFRE-1 and NFRE-2 present in the PTEN promoter. Contrary to the transcriptional suppression of BclXL, simvastatin significantly increased the transcription of PTEN. Furthermore, expression of NFκ B p65 subunit inhibited transcription of PTEN, resulting in reduced protein expression, which leads to enhanced phosphorylation of Akt. Taken together, our data present a novel bifaceted mechanism where simvastatin acts on a nodal transcription factor NFκ B, which attenuates the expression of anti-apoptotic BclXL and simultaneously derepresses the expression of anti-proliferative/proapoptotic tumor suppressor PTEN to prevent breast cancer cell growth.
Statin; Breast tumor; BclXL; Akt kinase
Up to 40% of acute ischaemic strokes in young adults are cryptogenic in nature, that is, no cause is determined. In more than half of these patients, patent foramen ovale (PFO) is seen along with an increased incidence of atrial septal aneurysm. The commonest method of investigation is echocardiography (preferably transoesophageal echocardiography). On the basis of available evidence, low risk patients are treated with antiplatelet agents and high risk patients with warfarin. There are inconclusive data on the efficacy of PFO closure to prevent stroke recurrence. However, if there is recurrent stroke or intolerance to medical therapy, percutaneous closure is carried out.
atrial septal defect; cryptogenic stroke; patent foramen ovale
Giardia lamblia is a microaerophilic human gastrointestinal parasite and considered as an early-diverged eukaryote. In vitro oxidative stress generation plays a significant role in cell cycle progression and cell death of this parasite. In the present study hydrogen peroxide, metronidazole, and a modified growth medium without cysteine and ascorbic acid have been chosen as oxidative stress-inducing agents. Cell cycle progression has been found to be regulated by different types of oxidative stresses. Apoptosis is not an established pathway in Giardia, which is devoid of ideal mitochondria, but in the present investigation, apoptosis-like programmed cell death has been found by the experiments like AnnexinV-FITC assay, DNA fragmentation pattern, etc. On the contrary, Caspase-9 assay, which confirms the caspase-mediated apoptotic pathway, has been found to be negative in all the stress conditions. Protease inhibitor assay confirmed that, even in absence of any proteases, programmed cell death does occur in this primitive eukaryote. All these results signify a novel pathway of programmed suicidal death in Giardia lamblia under oxidative stress. This is the first demonstration of protease-independent programmed cell death regulation in Giardia exclusive for its own specialties.
Giardia lamblia; oxidative stress; reactive oxygen species; programmed cell death; apoptosis; early branching eukaryotes
Using synthetic antisense RNA from the 5'-untranslated region of the beta-tubulin gene as probe in gel retardation assays, a heat stable RNA-binding factor was identified in promastigotes of the kinetoplastid protozoan Leishmania donovani. The same or similar factors interact with several small ribosomal RNA (srRNA) species and, more weakly, with tRNA, as shown by binding and competition experiments. Deletion analysis indicated involvement of repeated purine-rich motifs on the antisense RNA, in the reaction. Related, conserved motifs occur on at least two of the srRNAs. By a modified Western blot assay, the RNA-binding species was identified as a single, small polypeptide. The activity is apparently specific for the promastigote stage of the parasite, being undetectable in amastigotes. The properties of this RNA-binding factor suggest that it is a novel, previously uncharacterized protein.
Psoralea corylifolia (Somraji) and Trigonella foenum-graecum L. (Methi), important medicinal plants widely used in India as folk medicine. Local people of West Bengal traditionally used the seeds of these plants to cure diabetes.
Present study was designed to investigate the antidiabetic efficacy of aqueous extract of seeds of these plants in separate or in composite manner in streptozotocin (STZ)-induced diabetic rat.
Materials and Methods:
Diabetes was induced by intramuscular injection of STZ at the dose of 40 mg/ml of citrate buffer/kg body weight. Fasting blood glucose (FBG), glyclated hemoglobin (HbA1C) and activities of hexokinase, glucose-6-phosphate dehydrogenase and glucose-6-phosphatase of liver in experimental animals were assessed. Hyperlipidemic state developed in the experimental diabetic rat was assessed by measuring the levels of total cholesterol, triglyceride, and lipoproteins in serum.
There was significant increased in the levels of FBG, HbA1C and lipid profiles along with diminution (P < 0.001) in the activities of hepatic hexokinase, glucose-6-phosphate dehydrogenase and elevation in glucose-6-phosphatase in diabetic control animals in respect to the untreated control. Significant recovery (P < 0.05) in the activities of above mentioned enzymes along with the correction in the levels of FBG, HbA1C and serum lipid profiles were noted towards the control level after the treatment of composite extract (i.e. 100 mg of Somraji: 100 mg of Methi, total 200 mg/kg body weight) than the individual extract (i.e. 200 mg of Somraji or 200 mg of Methi, per kg body weight) treatment.
Results suggest that composite extract of above plant parts has more potent antidiabetic efficacy than the individual extract.
Carbohydrate metabolic enzymes; glycogen; lipid profiles; streptozotocin
The U3-LTR region of leukemia viruses transactivates cancer-related signaling pathways through the production of a non-coding RNA transcript although the role of this transcript in virus infection remains unknown. In this study we demonstrate for the first time that an LTR-specific small non-coding RNA is produced from a FeLV-infected feline cell line. RNA cloning identified this as a 104 base transcript that originates from the U3-LTR region. We also demonstrate that in in vitro assays this LTR RNA transcript activates NFκB signaling. Taken together, our findings suggest a possible role for this LTR transcript in FeLV pathogenesis.
FeLV; LTR; non-coding RNA
Despite the devastating effect of suicide on numerous lives, there is still a lack of knowledge concerning its neurochemical aspects. There is increasing evidence that brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are involved in the pathophysiology and treatment of depression through binding and activating their cognate receptors TrkB and TrkA respectively. The present study was performed to examine whether the expression profiles of BDNF and/or TrkB as well as NGF and/or TrkA were altered in the hippocampus of postmortem brain of the participants, who had committed suicide and whether these alterations were associated with specific psychopathologic conditions. These studies were performed on the hippocampus of 21 suicide victims and 19 non-psychiatric control individuals. The protein and mRNA levels of BDNF, TrkB, NGF, and TrkA were determined by sandwich enzyme-linked immunosorbent assay, Western blot and reverse transcription-PCR. Given the importance of BDNF and NGF and their cognate receptors in mediating physiological functions, including cell survival and synaptic plasticity, our findings of reduced expression of BDNF, TrkB, NGF, and TrkA on both the protein and mRNA levels of postmortem brains of suicide victims suggest that these molecules may play an important role in the pathophysiological aspects of suicidal behavior.
brain-derived neurotrophic factor; nerve growth factor; suicide; postmortem brain; hippocampus; TrkB; TrkA; RT-PCR; ELISA
Keratoconus (KC; Mendelian Inheritance in Man (OMIM) 14830) is a bilateral, progressive corneal defect affecting all ethnic groups around the world. It is the leading cause of corneal transplantation. The age of onset is at puberty, and the disorder is progressive until the 3rd–4th decade of life when it usually arrests. It is one of the major ocular problems with significant social and economic impacts as the disease affects young generation. Although genetic and environmental factors are associated with KC, but the precise etiology is still elusive. Results from complex segregation analysis suggests that genetic abnormalities may play an essential role in the susceptibility to KC. Due to genetic heterogeneity, a recent study revealed 17 different genomic loci identified in KC families by linkage mapping in various populations. The focus of this review is to provide a concise update on the current knowledge of the genetic basis of KC and genomic approaches to understand the disease pathogenesis.
Disease pathogenesis; genetic heterogeneity; genetics and genomics; genome-wide association study; genomic loci; keratoconus; linkage mapping; molecular mechanisms; whole exome-genome sequencing
Keratoconus is a progressive corneal thinning disease associated with significant tissue remodeling activities and activation of a variety of signaling networks. However, it is not understood how differential gene and protein expression direct function in keratoconus corneas to drive the underlying pathology, ectasia. Research in the field has focused on discovering differentially expressed genes and proteins and quantifying their levels and activities in keratoconus patient samples. In this study, both microarray analysis of total ribonucleic acid (RNA) and whole proteome analyses are carried out using corneal epithelium and tears from keratoconus patients and compared to healthy controls. A number of structural proteins, signaling molecules, cytokines, proteases, and enzymes have been found to be deregulated in keratoconus corneas. Together, the data provide clues to the complex process of corneal degradation which suggest novel ways to clinically diagnose and manage the disease. This review will focus on discussing these recent advances in the knowledge of keratoconus biology from a gene expression and function point-of-view.
Deregulation; ectasia; gene expression; keratoconus; mass spectroscopy; proteomics; signaling pathways
Yellow mosaic disease, caused by a whitefly transmitted New World Begomovirus, named Corchorus golden mosaic virus (CoGMV), is emerging as a serious biotic constraint for jute fibre production in Asia. For rapid and sensitive diagnosis of the Begomovirus associated with this disease, a non-radiolabelled diagnostic probe, developed against the DNA A component of the east Indian isolate of CoGMV, detected the presence of the virus in infected plants and viruliferous whiteflies following Southern hybridization and nucleic acid spot hybridization tests. Presence of the virus was also confirmed when polymerase chain reaction amplification was performed using virus-specific primers on DNA templates isolated from infected plants and viruliferous whiteflies.
New world; Begomovirus; PCR; Probe; Hybridization
Rheumatoid arthritis (RA) is an independent risk factor for adverse cardiovascular (CV) events that accounts for a significant proportion of mortality among these patients. Anti-CCP antibodies are associated with higher frequency of extra-articular manifestations and poorer outcomes in RA.
To determine the role of anti-cyclic citrullinated peptide (CCP) antibody as an independent risk factor for developing CV complications as documented by carotid intima medial thickness and abnormal echocardiography in established RA patients.
Materials and methods
Eighty patients of RA having disease duration of at least 3 years participated in this hospital-based, cross-sectional, and observational study. Forty patients were anti-CCP antibody positive. Patients of established RA having known CV risk factors, known heart disease, or family history of premature ischemic heart disease were excluded.
Anti-CCP positive group had early morning stiffness, tender and swollen joint count, and c-reactive protein (CRP) level significantly higher than those in anti-CCP negative group. Average intima-medial thicknesses of common carotid arteries were also significantly higher among anti-CCP positive group (P = 0.029) and were positively correlated with patients' age and disease duration. Lower left ventricular ejection fraction and left ventricular diastolic dysfunction were more commonly dispersed among the anti-CCP positive patients with P values of 0.01 and 0.034, respectively. Mild pericardial thickening was documented among 12.5% patients of anti-CCP positive group, while none of the anti-CCP negative patients had similar findings in echocardiography.
This study stressed on the important role of anti-CCP antibody in myocardial dysfunction due to inflammation in RA patients. Both atherosclerotic vascular involvement and cardiac abnormalities including pericardial, myocardial, and endocardial involvements were higher among anti-CCP positive RA patients. Hence, patients with high titer of anti-CCP antibody associated with prolonged disease duration and increased disease activity should be evaluated for CV morbidity more meticulously.
Anti-CCP antibody; Cardiovascular complications; Carotid intima-medial thickness; Rheumatoid arthritis
The aim of this study is to understand the mechanism of EGFR overexpression in head and neck squamous cell carcinoma (HNSCC). For this reason, expression/mutation of EGFR were analyzed in 30 dysplastic head and neck lesions and 148 HNSCC samples of Indian patients along with 3 HNSCC cell lines. In addition, deletion/methylation/mutation/expression of SH3GL2 (associated with EGFR degradation) and CDC25A (associated with dephosphorylation of EGFR) were analyzed in the same set of samples. Our study revealed high frequency of EGFR overexpression (66–84%), low frequency of gene amplification (10–32.5%) and absence of functional mutation in the dysplastic lesions and HNSCC samples. No correlation was found between protein overexpression and mRNA expression/gene amplification status of EGFR. On the other hand, frequent alterations (deletion/methylation) of SH3GL2 (63–77%) and CDC25A (37–64%) were seen in the dysplastic and HNSCC samples. Two novel single nucleotide polymorphism (SNPs) were found in the promoter region of SH3GL2. Reduced expression of these genes showed concordance with their alterations. Overexpression of EGFR and p-EGFR were significantly associated with reduced expression and alterations of SH3GL2 and CDC25A respectively. In-vitro demethylation experiment by 5-aza-2′-deoxycytidine (5-aza-dC) showed upregulation of SH3GL2 and CDC25A and downregulation of EGFR expression in Hep2 cell line. Poor patient outcome was predicted in the cases with alterations of SH3GL2 and CDC25A in presence of human papilloma virus (HPV) infection. Also, low SH3GL2 and high EGFR expression was a predictor of poor patient survival. Thus, our data suggests that overexpression of EGFR due to its reduced degradation and dephosphorylation is needed for development of HNSCC.
The International Consortium (FTDC) that revised the diagnostic criteria for behavioural variant frontotemporal dementia (bvFTD) did not have an Asian representation. Whether the revised criteria are equally useful in the early detection of Asian bvFTD patients therefore remains largely unexplored. Earlier studies have indicated differences in clinical manifestations in Indian and other Asian bvFTD patients when compared to western groups. There is an urgent need for clarification, given the projected exponential rise in dementia in these countries and the imminent clinical trials on bvFTD.
To assess how Indian bvFTD patients fulfil the FTDC criteria, hypothesizing that our patients might present differently early in the illness.
In a hospital-based retrospective observational study, we assessed 48 probable bvFTD patients, diagnosed according to the FTDC criteria, for the speed with which these criteria were fulfilled, the frequency of individual symptoms and their order of appearance during the illness.
Most of our patients presented with moderate to severe dementia, in spite of having relatively short onset to diagnosis times. Patients on average took 1.4 years from onset to meet the FTDC criteria, with 90% of them presenting with four or more symptoms at diagnosis. Disinhibition was the commonest symptom and the first symptom in most patients.
With most patients presenting with advanced and florid disease, the FTDC criteria have little additional impact in early identification of bvFTD in India. Modifying the criteria further could allow detection of Indian patients early enough for their inclusion in future clinical trials.
Obesity; Overweight; Children; Adolescent
Joubert syndrome (JS) is a very rare, autosomal-recessive condition. It is characterized by agenesis of cerebellar vermis, abnormal eye movements with nystagmus, episodes of hyperpnea and apnea, delayed generalized motor development, retinal coloboma and dystrophy and, sometimes, multicystic kidney disease. The importance of recognizing JS is related to the outcome and its potential complications. Prenatal diagnosis by ultarsonography and antenatal magnetic resonance imaging (MRI) is also possible. We have diagnosed a case of JS in a male infant with history of delayed mental and motor milestone development, history of abnormal breathing pattern, abnormal limb movement, generalized hypotonia and abnormal head movements with nystagmus. MRI showed hypoplastic cerebellar vermis with hypoplasia of the superior cerebellar peduncle resembling the “Molar Tooth Sign” in the mid-brain.
Joubert syndrome; Molar tooth sign; Vermian agenesis
Acquired perforating dermatosis (APD) is a rare disorder characterized by transepidermal elimination of contents from dermis with minimal disruption of surrounding structures, believed to be due to altered expression of dermal proteins. Its occurrence in patients with systemic mycosis has never been reported. We report a 60-y gentleman who presented with features of adrenal insufficiency (nausea vomiting, hypotension and increased pigmentation) for 4 mo, multiple hyperpigmented pruritic nodules with central keratinous plug over extensor surface of both lower limbs along with hepatosplenomegaly of one month duration. Investigations revealed low cortisol (2.3 μg/dl; normal: 5–34 μg/dl), elevated ACTH (68 pg/ml; normal: 5–15 pg/ml), enlarged bilateral adrenals with hepatosplenomegaly on CT. Methanamine silver staining of fine needle aspiration from the adrenals and bone marrow aspiration showed numerous oval yeast cells suggestive of histoplasma. Histopathology of biopsy of one of the skin nodules revealed transepidermal elimination process characterized by invagination of epidermis with extrusion of collagen bundles suggestive of APD. Patient improved with hydrocortisone replacement and there was clinical improvement with resolution of skin lesions following amphotericin-B and itraconazole therapy. This is probably the first reported case of APD in a patient with disseminated histoplasmosis who had presented with Addison’s disease.
Addison’s disease; acquired perforating dermatosis; amphotericin-B; histoplasmosis; itraconazole
During intracavitary brachytherapy (ICBT) for cervical cancer, the choice of applicator system remains rather arbitrary. However, as the applicator geometry may play an important role in dose distribution, thereby improving the therapeutic ratio, this study was conducted to compare the Manchester-style and Fletcher-style applicator systems.
Material and methods
After completion of EBRT, 22 patients with cervical cancer (stage IIA-IIIB) underwent intracavitary brachytherapy. Two different types of applicators: Manchester-style and Fletcher-style were used for each patient for alternate insertions. The purpose was to compare the dose distribution obtained when two different applicators were applied to the same patient. CT based computerized treatment planning was done and dose was prescribed to point A. After optimization, height, width and thickness of the 100% isodose curve, as well as the 100% isodose volume were noted. Dose received by the urinary bladder and rectum were noted.
The 100% isodose volume and its maximum width were significantly greater (P value < 0.0001 in both occasions) when Manchester-style applicator was used. However, the dose received by 0.1 cc, 1.0 cc and 2.0 cc of the urinary bladder were all significantly greater (P value < 0.0001) with the Manchester-style applicator. No significant difference was found in rectal doses.
The larger 100% isodose volume, as well as the greater width achieved with the use of Manchester-style applicator can be helpful in circumstances where the tumour is large in size. However, this must be balanced against the increased dose received by the urinary bladder.
brachytherapy; cervical cancer; Fletcher-suit applicator; Manchester-style applicator
The tumor suppressor gene, Von Hippel-Lindau (VHL), is frequently mutated in the most common form of kidney cancer, clear cell renal cell carcinoma (CCRCC). In hypoxic conditions, or when there is a VHL mutation, the hypoxia inducible factors, HIF1α and HIF2α, are stabilized and transcribe a panel of genes associated with cancer such as vascular endothelial growth factor receptor (VEGFR), platelet derived growth factor (PDGF), and glucose transporter 1 (GLUT1). Recent studies in clear cell kidney cancer have suggested that HIF2α, but not HIF1α, is the critical oncoprotein in the VHL pathway. Therefore, targeting HIF2α could provide a potential therapeutic approach for patients with advanced CCRCC. Since iron regulatory protein 1 (IRP1) is known to inhibit the translation of HIF2α, we investigated whether Tempol, a stable nitroxide that activates IRP1 towards IRE-binding, might have a therapeutic effect on a panel of human CCRCC cells expressing both HIF1α and HIF2α. We first evaluated the protein expression of HIF1α and HIF2α in 15 different clear cell renal carcinoma cell lines established from patient tumors in our laboratory. Tempol decreased the expression of HIF2α, and its downstream targets in all the cell lines of the panel. This effect was attributed to a dramatic increase of IRE-binding activity of IRP1. Several cell lines were found to have an increased IRP1 basal activity at 20% O2 compared to 5% O2, which may lower HIF2α expression in some of the cell lines in a VHL-independent manner. Taken together our data identify Tempol as an agent with potential therapeutic activity targeting expression of HIF2α in VHL-deficient clear cell kidney cancer and illustrate the importance of studying biochemical processes at relevant physiological O2 levels.
HIF; Tempol; RCC; VHL; IRP1; iron metabolism
Progressive loss of lung function and reversibility characterize chronic asthma. The conventional therapy is targeted to control the disease without targeting the loss of lung function or reversibility. In a prospective real-world observation of long-term use of add-on doxycycline as a matrix-metalloproteinase inhibitor, we documented significant improvement in lung function with possible reversal of remodeling.
Chronic asthma shows progressive decline in lung function with reduction or even loss of reversibility secondary to remodeling. A set of endopeptidase enzymes known as matrix metalloproteinases are intimately related to the pathogenesis of asthma and remodeling. The inhibition of matrix metalloproteinases is recognized as a prospective way of treating asthma and its corresponding structural remodeling.
In a randomized, prospective, real-world study, we have observed the change in lung function (spirometry) with an add-on of long-term doxycycline to standard asthma therapy as per the Global Initiative for Asthma guidelines in a small asthmatic population. The change in terms of forced expiratory volume (FEV1), forced vital capacity (FVC), percent of FEV1 (FEV1%), and forced expiratory flow (FEF25–75) were noted following variable duration of doxycycline therapy.
There has been a global improvement in all the parameters in all the six patients suggesting improvement in obstruction, and reduction in air trapping following a treatment of add-on doxycycline for a mean duration of 162.83 ± 83.07 days. Of the changes seen, the post bronchodilator FEV1, the FVC, and the FEF25–75 showed significant improvements with the P-value set at 0.004, 0.054, and 0.031, respectively. There was also evidence of the reversal of remodeling from the improvement in the FEV1/FVC ratio. Moreover there was a greater than expected improvement of pre-bronchodilator FEV1 after treatment that far surpassed the initial post-bronchodialator FEV1 value. Even after such a change, there were presences of some reversibility suggesting room for further improvement.
The results suggest significant improvements in the obstructive parameters used to evaluate asthma, with possible reversal of remodeling evident in chronic asthmatics when treated with doxycycline in addition to standard therapies. This observation needs further scientific validation.
chronic asthma; doxycycline; FEV1; FEV1/FVC; matrix metalliproteinase
Transforming growth factor-β (TGFβ) promotes glomerular hypertrophy and matrix expansion, leading to glomerulosclerosis. MicroRNAs are well suited to promote fibrosis because they can repress gene expression, which negatively regulate the fibrotic process. Recent cellular and animal studies have revealed enhanced expression of microRNA, miR-21, in renal cells in response to TGFβ. Specific miR-21 targets downstream of TGFβ receptor activation that control cell hypertrophy and matrix protein expression have not been studied. Using 3′UTR-driven luciferase reporter, we identified the tumor suppressor protein PTEN as a target of TGFβ-stimulated miR-21 in glomerular mesangial cells. Expression of miR-21 Sponge, which quenches endogenous miR-21 levels, reversed TGFβ-induced suppression of PTEN. Additionally, miR-21 Sponge inhibited TGFβ-stimulated phosphorylation of Akt kinase, resulting in attenuation of phosphorylation of its substrate GSK3β. Tuberin and PRAS40, two other Akt substrates, and endogenous inhibitors of mTORC1, regulate mesangial cell hypertrophy. Neutralization of endogenous miR-21 abrogated TGFβ-stimulated phosphorylation of tuberin and PRAS40, leading to inhibition of phosphorylation of S6 kinase, mTOR and 4EBP-1. Moreover, downregulation of miR-21 significantly suppressed TGFβ-induced protein synthesis and hypertrophy, which were reversed by siRNA-targeted inhibition of PTEN expression. Similarly, expression of constitutively active Akt kinase reversed the miR-21 Sponge-mediated inhibition of TGFβ-induced protein synthesis and hypertrophy. Furthermore, expression of constitutively active mTORC1 prevented the miR-21 Sponge-induced suppression of mesangial cell protein synthesis and hypertrophy by TGFβ. Finally, we show that miR-21 Sponge inhibited TGFβ-stimulated fibronectin and collagen expression. Suppression of PTEN expression and expression of both constitutively active Akt kinase and mTORC1 independently reversed this miR-21-mediated inhibition of TGFβ-induced fibronectin and collagen expression. Our results uncover an essential role of TGFβ-induced expression of miR-21, which targets PTEN to initiate a non-canonical signaling circuit involving Akt/mTORC1 axis for mesangial cell hypertrophy and matrix protein synthesis.
The Akt kinase signaling pathway is frequently deregulated in many human diseases including cancer, autoimmune disease and diabetes. In nephropathy, associated with diabetes, increased Akt signal transduction results in glomerular especially mesangial cell hypertrophy. The mechanism of Akt activation by elevated glucose is poorly understood. The oncogene DJ-1 prevents oxidative damage and apoptosis of dopaminergic neurons in animal models of Parkinson’s disease and in culture. We identified DJ-1 to increase in response to high glucose in renal glomerular mesangial cells concomitant with an increase in phosphorylation of Akt in a time-dependent manner. Plasmid-derived overexpression as well as downregulation of DJ-1 by siRNA showed the requirement of this protein in high glucose-stimulated Akt phosphorylation. The tumor suppressor protein PTEN acts as a negative regulator of Akt activation. Interestingly, DJ-1 was associated with PTEN and this interaction was significantly increased in response to high glucose. High glucose-induced increase in DJ-1 promoted phosphorylation of the PRAS40, a negative regulator of TORC1 kinase activity, resulting in activating and inactivating phosphorylation of S6 kinase and 4EBP-1, respectively. Furthermore, DJ-1 increased protein synthesis and hypertrophy of mesangial cells. Our results provide evidence for a unique mechanism whereby DJ-1 induces Akt/PRAS40/TORC1-mediated hypertrophy in response to high glucose.
Akt kinase; Kidney; Diabetes; mTOR
Metastatic renal cancer manifests multiple signatures of gene expression. Deviation in expression of mature miRNAs has been linked to human cancers. Importance of miR-21 in renal cell carcinomas is proposed from profiling studies using tumor tissue samples. However, the role of miR-21 function in causing renal cancer cell proliferation and invasion has not yet been shown. Using cultured renal carcinoma cells, we demonstrate enhanced expression of mature miR-21 along with pre-and pri-miR-21 by increased transcription compared to normal proximal tubular epithelial cells. Overexpression of miR-21 Sponge to quench endogenous miR-21 levels inhibited proliferation, migration and invasion of renal cancer cells. In the absence of mutation in the PTEN tumor suppressor gene, PTEN protein levels are frequently downregulated in renal cancer. We show that miR-21 targets PTEN mRNA 3′untranslated region to decrease PTEN protein expression and augments Akt phosphorylation in renal cancer cells. Downregulation of PTEN as well as overexpression of constitutively active Akt kinase prevented miR-21 Sponge-induced inhibition of renal cancer cell proliferation and migration. Moreover, we show that miR-21 Sponge inhibited the inactivating phosphorylation of the tumor suppressor protein tuberin and attenuated TORC1 activation. Finally, we demonstrate that expression of constitutively active TORC1 attenuated miR-21 Sponge-mediated suppression of proliferation and migration of renal cancer cells. Our results uncover a layer of post-transcriptional regulation of PTEN by transcriptional activation of miR-21 to force the canonical oncogenic Akt/TORC1 signaling conduit to drive renal cancer cell proliferation and invasion.
Vibrio cholerae is a bacterial pathogen that colonizes the chitinous exoskeleton of zooplankton as well as the human gastrointestinal tract. Colonization of these different niches involves an N-acetylglucosamine binding protein (GbpA) that has been reported to mediate bacterial attachment to both marine chitin and mammalian intestinal mucin through an unknown molecular mechanism. We report structural studies that reveal that GbpA possesses an unusual, elongated, four-domain structure, with domains 1 and 4 showing structural homology to chitin binding domains. A glycan screen revealed that GbpA binds to GlcNAc oligosaccharides. Structure-guided GbpA truncation mutants show that domains 1 and 4 of GbpA interact with chitin in vitro, whereas in vivo complementation studies reveal that domain 1 is also crucial for mucin binding and intestinal colonization. Bacterial binding studies show that domains 2 and 3 bind to the V. cholerae surface. Finally, mouse virulence assays show that only the first three domains of GbpA are required for colonization. These results explain how GbpA provides structural/functional modular interactions between V. cholerae, intestinal epithelium and chitinous exoskeletons.
Vibrio cholerae is the bacterium that causes cholera, a disease endemic in developing countries with poor sanitation. The bacterium colonizes aquatic organisms that serve as a reservoir of transmission to humans. Our work has focused on GbpA, a protein that is secreted by V. cholerae and appears to facilitate growth of the bacteria both in the human intestine and on the exoskeletons of marine organisms. We show that the protein possesses an unusual three-dimensional structure consisting of four separate domains. Two of the domains are similar to proteins that are known to bind chitin, an exoskeleton biopolymer, and our data show that these domains indeed harbour the chitin binding properties of GbpA. One of these domains is also capable of binding intestinal mucus. The two remaining domains are required for interacting with the bacterium itself, creating a stable interface between the bacterium and the human/marine host, facilitating colonization. Finally, work with a cholera mouse model shows that only the first three domains of GbpA are required for colonization. These results show how GbpA provides structural/functional modular interactions between V. cholerae, the intestinal epithelium and chitinous exoskeletons.