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1.  MicroRNA ‘signature’ during estrogen-mediated mammary carcinogenesis and its reversal by ellagic acid intervention 
Cancer letters  2013;339(2):175-184.
Dysregulated miRNA expression has been associated with the development and progression of cancers, including breast cancer. The role of estrogen (E2) in regulation of cell proliferation and breast carcinogenesis is well-known. Recent reports have associated several miRNAs with estrogen receptors in breast cancers. Investigation of the regulatory role of miRNAs is critical for understanding the effect of E2 in human breast cancer, as well as for developing strategies for cancer chemoprevention. In the present study we used the well-established ACI rat model that develops mammary tumors upon E2 exposure and identified a ‘signature’ of 33 significantly modulated miRNAs during the process of mammary tumorigenesis. Several of these miRNAs were altered as early as 3 weeks after initial E2 treatment and their modulation persisted throughout the mammary carcinogenesis process, suggesting that these molecular changes are early events. Furthermore, ellagic acid, which inhibited E2-induced mammary tumorigenesis in our previous study, reversed the dysregulation of miR-375, miR-206, miR-182, miR-122, miR- 127 and miR-183 detected with E2 treatment and modulated their target proteins (ERα, cyclin D1, RASD1, FoxO3a, FoxO1, cyclin G1, Bcl-w and Bcl-2). This is the first systematic study examining the changes in miRNA expression associated with E2 treatment in ACI rats as early as 3 week until tumor time point. The effect of a chemopreventive agent, ellagic acid in reversing miRNAs modulated during E2-mediated mammary tumorigenesis was also established. These observations provide mechanistic insights into the new molecular events behind the chemoprevention action of ellagic acid in and treatment of breast cancer.
doi:10.1016/j.canlet.2013.06.012
PMCID: PMC3775863  PMID: 23791885
Breast cancer; microRNAs; Ellagic acid; Estrogen; Carcinogenesis
2.  Modified silicone sling assisted temporalis muscle transfer in the management of lagophthalmos 
Indian Journal of Ophthalmology  2014;62(2):176-179.
Aim:
To evaluate the efficacy of modified temporalis muscle transfer (TMT) by silicone sling for the management of paralytic lagophthalmos.
Settings and Design:
Prospective interventional study.
Materials and Methods:
Ten patients of lagophthalmos due to facial palsy underwent modified TMT using silicone sling. The patients were followed-up for a period of 3 months. Palpebral aperture in primary gaze and during eye closure were assessed both pre- and postoperatively along with problems associated with lagophthalmos like exposure keratopathy and lacrimation.
Statistical Analysis:
Paired t-test was applied to measure the statistical outcome.
Results:
Eight patients achieved full correction of lagophthalmos with no lid gap on closing the eye. The mean (standard deviation (SD)) lid gap on eye closure was 7.7 (0.86) mm preoperatively, 0.5 (0.47) mm at 1st postoperative day, and 0.7 (0.75) mm at 3rd month. There was a reduction in mean lid gap on eye closure of 7 mm at 3 months (P < 0.0001) which is highly significant. The mean (SD) vertical interpalpebral distance during primary gaze was 12.05 (1.12) mm preoperatively, 10 (0.94) mm at 1st postoperative day, and 10.35 (1.08) mm at 3rd month. There was a reduction in mean vertical inter palpebral distance of 1.7 mm at 3 months (P = 0.001) which is significant. Exposure keratitis decreased in five out of six patients at 3 months.
Conclusion:
Modified TMT by silicone sling is a useful procedure with lesser morbidity and good outcomes for the treatment of paralytic lagophthalmos due to long standing facial palsy.
doi:10.4103/0301-4738.128629
PMCID: PMC4005234  PMID: 24618488
Lagophthalmos; silicone sling; temporalis muscle
3.  Controlled-release systemic delivery - a new concept in cancer chemoprevention 
Carcinogenesis  2012;33(8):1608-1615.
Many chemopreventive agents have encountered bioavailability issues in pre-clinical/clinical studies despite high oral doses. We report here a new concept utilizing polycaprolactone implants embedded with test compounds to obtain controlled systemic delivery, circumventing oral bioavailability issues and reducing the total administered dose. Compounds were released from the implants in vitro dose dependently and for long durations (months), which correlated with in vivo release. Polymeric implants of curcumin significantly inhibited tissue DNA adducts following the treatment of rats with benzo[a]pyrene, with the total administered dose being substantially lower than typical oral doses. A comparison of bioavailability of curcumin given by implants showed significantly higher levels of curcumin in the plasma, liver and brain 30 days after treatment compared with the dietary route. Withaferin A implants resulted in a nearly 60% inhibition of lung cancer A549 cell xenografts, but no inhibition occurred when the same total dose was administered intraperitoneally. More than 15 phytochemicals have been tested successfully by this formulation. Together, our data indicate that this novel implant-delivery system circumvents oral bioavailability issues, provides continuous delivery for long durations and lowers the total administered dose, eliciting both chemopreventive/chemotherapeutic activities. This would also allow the assessment of activity of minor constituents and synthetic metabolites, which otherwise remain uninvestigated in vivo.
doi:10.1093/carcin/bgs209
PMCID: PMC3499062  PMID: 22696595
4.  Controlled Systemic Delivery by Polymeric Implants Enhances Tissue and Plasma Curcumin Levels Compared with Oral Administration 
Curcumin possess potent anti-inflammatory and anti-proliferative activities but with poor biopharmaceutical attributes. To overcome these limitations, curcumin implants were developed and tissue (plasma, brain and liver) curcumin concentrations were measured in female ACI rats for 3 months. Biological efficacy of tissue levels achieved was analyzed by modulation of hepatic cytochromes. Curcumin implants exhibited diffusion-mediated biphasic release pattern with ~2-fold higher in vivo release as compared to in vitro. Plasma curcumin concentration from implants was ~3.3 ng/ml on day 1 which dropped to ~0.2 ng/ml after 3 months whereas only 0.2–0.3 ng/ml concentration was observed from 4–12 days with diet and was undetected subsequently. Almost 10 fold higher curcumin levels were observed in brain on day 1 from implants compared with diet (30.1±7.3 vs 2.7±0.8 ng/g) and were higher even after 90 days (7.7±3.8 vs 2.2±0.8 ng/g). Although, curcumin levels were similar in liver from both the routes (~25–30 ng/g from day 1–4 and ~10–15 ng/g at 90 days), implants were more efficacious in altering hepatic CYP1A1 levels and CYP3A4 activity at ~28 fold lower doses. Curcumin implants provided much higher plasma and tissue concentrations and are a viable alternative for delivery of curcumin to various organs like brain.
doi:10.1016/j.ejpb.2011.12.009
PMCID: PMC3345811  PMID: 22227368
Curcumin; Implants; Bioavailability; Chemoprevention; Controlled Release; Tissue curcumin levels
5.  Sustained expression of CYPs and DNA adduct accumulation with continuous exposure to PCB126 and PCB153 through a new delivery method: Polymeric implants 
Toxicology reports  2014;1:820-833.
A new delivery method via polymeric implants was used for continuous exposure to PCBs. Female Sprague-Dawley rats received subcutaneous polymeric implants containing PCB126 (0.15% load), PCB153 (5% load), or both, for up to 45 days and release kinetics and tissue distribution were measured. PCB153 tissue levels on day 15 were readily detected in lung, liver, mammary and serum, with highest levels in the mammary tissue. PCB126 was detected only in liver and mammary tissues. However, a completely different pharmacokinetics was observed on co-exposure of PCB153 and PCB126, with a 1.8-fold higher levels of PCB153 in the liver whereas a 1.7-fold lower levels in the mammary tissue. PCB126 and PCB153 caused an increase in expression of key PCB-inducible enzymes, CYP 1A1/2 and 2B1/2, respectively. Serum and liver activities of the antioxidant enzymes, PON1 and PON3, and AhR transcription were also significantly increased by PCB126. 32P-Postlabeling for polar and lipophilic DNA-adducts showed significant quantitative differences: PCB126 increased 8-oxodG, an oxidative DNA lesion, in liver and lung tissues. Adduct levels in the liver remained upregulated up to 45 days, while some lung DNA adducts declined. This is the first demonstration that continuous low-dose exposure to PCBs via implants can produce sustained tissue levels leading to the accumulation of DNA-adducts in target tissue and induction of indicator enzymes. Collectively, these data demonstrate that this exposure model is a promising tool for long-term exposure studies.
doi:10.1016/j.toxrep.2014.09.010
PMCID: PMC4266188  PMID: 25530946
polychlorinated biphenyls (PCBs); PCB126 (3,3’,4,4’,5-pentachlorobiphenyl); PCB153 (2,2’4,4’,5,5’-hexachlorobiphenyl); Polymeric implants; DNA adducts; 32P-Postlabeling; CYPs; paraoxonase 1 (PON1)
6.  Vagus nerve stimulation improves left ventricular function in a canine model of chronic heart failure 
European Journal of Heart Failure  2013;15(12):1319-1326.
Aims
Autonomic dysfunction is a feature of chronic heart failure (HF). This study tested the hypothesis that chronic open-loop electrical vagus nerve stimulation (VNS) improves LV structure and function in canines with chronic HF.
Methods and results
Twenty-six canines with HF (EF ∼35%) produced by intracoronary microembolizations were implanted with a bipolar cuff electrode around the right cervical vagus nerve and connected to an implantable pulse generator. The canines were enrolled in Control (n = 7) vs. VNS therapy (n = 7) or a crossover study, with crossovers occurring at 3 months (C × VNS, n = 6; VNS × C, n = 6). After 6 months of VNS, LVEF and LV end-systolic volume (ESV) were significantly improved compared with Control (ΔEF Control –4.6 ± 0.9% vs. VNS 6.0 ± 1.6%, P < 0.001) and (ΔESV Control 8.3 ± 1.8 mL vs. VNS –3.0 ± 2.3 mL, P = 0.002. Plasma and tissue biomarkers were also improved. In the crossover study, VNS also resulted in a significant improvement in EF and ESV compared with Control (ΔEF Control –2.3 ± 0.65% vs. VNS 6.7 ± 1.1 mL, P < 0.001 and ΔESV Control 3.2 ± 1.2 mL vs. VNS –4.0 ± 0.9 mL, P < 0.001). Initiation of therapy in the Control group at 3 months resulted in a significant improvement in EF (Control –4.7 ± 1.4% vs. VNS 3.7 ± 0.74%, P < 0.001) and ESV (Control 1.5 ± 1.2 mL vs. NS –5.5 ± 1.6 mL, P = 0.003) by 6 months.
Conclusions
In canines with HF, long-term, open-looped low levels of VNS therapy improves LV systolic function, prevents progressive LV enlargement, and improves biomarkers of HF when compared with control animals that did not receive therapy.
doi:10.1093/eurjhf/hft118
PMCID: PMC3895958  PMID: 23883651
Vagal; Autonomic nervous system; Parasympathetic; Neurostimulation; Heart failure
7.  Chemopreventive and Therapeutic Activity of Dietary Blueberry against Estrogen-Mediated Breast Cancer 
Berries are gaining increasing importance lately for their chemopreventive and therapeutic potential against several cancers. In earlier studies, a blueberry-supplemented diet has shown protection against 17β-estradiol (E2)-mediated mammary tumorigenesis. This study tested both preventive and therapeutic activities of diet supplemented with whole blueberry powder (50:50 blend of Tifblue and Rubel). Animals received 5% blueberry diet, either 2 weeks prior to or 12 weeks after E2 treatment in preventive and therapeutic groups, respectively. Both interventions delayed the tumor latency for palpable mammary tumors by 28 and 37 days, respectively. Tumor volume and multiplicity were also reduced significantly in both modes. The effect on mammary tumorigenesis was largely due to down-regulation of CYP 1A1 and ER-α gene expression and also favorable modulation of microRNA (miR-18a and miR-34c) levels. These data suggest that the blueberry blend tested is effective in inhibiting E2-mediated mammary tumorigenesis in both preventive and therapeutic modes.
doi:10.1021/jf403734j
PMCID: PMC4334276  PMID: 24245576
blueberry; estrogen; breast cancer; chemoprevention; chemotherapy; ACI rats; miRNA modulation; blood chemistry; hematopoietic parameters
8.  Nitroxyl (HNO) a Novel Approach for the Acute Treatment of Heart Failure 
Circulation. Heart failure  2013;6(6):1250-1258.
Background
The nitroxyl (HNO) donor, Angeli’s salt (AS), exerts positive inotropic, lusitropic, and vasodilator effects in vivo that are cyclic AMP-independent. Its clinical utility is limited by chemical instability and co-generation of nitrite that itself has vascular effects. Here we report on effects of a novel, stable, pure HNO donor (CXl-1020) in isolated myoctyes, and intact hearts in experimental models and in patients with heart failure (HF).
Methods and Results
CXL-1020 converts solely to HNO and inactive CXL-1051 with a t1/2 of 2 minutes. In adult mouse ventricular-myocytes, it dose-dependently increased sarcomere shortening by 75–210% (50–500 µM), with a ~30% rise in the peak Ca2+ transient only at higher doses. Neither protein-kinase-A or soluble guanylate-cyclase inhibition altered this contractile response. Unlike isoproterenol, CXL-1020 was equally effective in myocytes from normal or failing hearts. In anesthetized dogs with coronary microembolization-induced HF, CXL-1020 reduced LV end-diastolic pressure and myocardial oxygen-consumption while increasing ejection fraction from 27 to 40% and maximal ventricular power index by 42% (both p<0.05). In conscious dogs with tachypacing-induced HF, CXL-1020 increased contractility assessed by end-systolic elastance, and provided veno-arterial dilation. Heart rate was minimally altered. In patients with systolic HF, CXL-1020 reduced both left and right heart filling pressures and systemic vascular resistance, while increasing cardiac and stroke volume index. Heart rate was unchanged, and arterial pressure declined modestly.
Conclusions
These data show the functional efficacy of a novel pure HNO donor to enhance myocardial function, and show first-in-man evidence for potential utility in heart failure.
Clinical Trial Registration
URL: http://www.clinicaltrials.gov. Unique identifiers: NCT01096043, NCT01092325.
doi:10.1161/CIRCHEARTFAILURE.113.000632
PMCID: PMC4110196  PMID: 24107588
nitroxyl; cardiomyopathy; contractility; myocyte; pharmacology; human; canine
9.  Association of Genetic Variation with Gene Expression and Protein Abundance within the Natriuretic Peptide Pathway 
The natriuretic peptide (NP) system is a critical physiologic pathway in heart failure with wide individual variability in functioning. We investigated the genetic component by testing the association of single nucleotide polymorphisms (SNP) with RNA and protein expression. Samples of DNA, RNA, and tissue from human kidney (n=103) underwent genotyping, RT-PCR, and protein quantitation (in lysates), for four candidate genes (NP-receptor 1 [NPR1], NPR2, NPR3 and membrane metallo-endopeptidase [MME]). The association of genetic variation with expression was tested using linear regression for individual SNPs, and a principal components (PC) method for overall gene variation. Eleven SNPs in NPR2 were significantly associated with protein expression (false discovery rate ≤0.05), but not RNA quantity. RNA and protein quantity correlated poorly with each other. The PC analysis showed only NPR2 as significant. Assessment of the clinical impact of NPR2 genetic variation is needed.
doi:10.1007/s12265-013-9491-y
PMCID: PMC3795918  PMID: 23835779
Natriuretic peptide; heart failure; gene expression; pharmacogenomics; nesiritide; genetic polymorphisms
10.  Promising molecular targeted therapies in breast cancer 
Indian Journal of Pharmacology  2011;43(3):236-245.
In recent years, there has been a significant improvement in the understanding of molecular events and critical pathways involved in breast cancer. This has led to the identification of novel targets and development of anticancer therapies referred to as targeted therapy. Targeted therapy has high specificity for the molecules involved in key molecular events that are responsible for cancer phenotype such as cell growth, survival, migration, invasion, metastasis, apoptosis, cell-cycle progression, and angiogenesis. Targeted agents that have been approved for breast cancer include trastuzumab and lapatinib, directed against human epidermal growth factor receptor 2 (HER2) and bevacizumab, directed against vascular endothelial growth factor (VEGF). Several other targeted agents currently under evaluation in preclinical and clinical trials include inhibitors of epidermal growth factor receptor (EGFR), dual EGFR and HER2 inhibitors, VEGF/VEGFR inhibitors, and agents that interfere with crucial signaling pathways such as PI3K/AKT/mTOR and RAS/MEK/ERK; agents against other tyrosine kinases such as Src, insulin-like growth factor (IGF)/IGF-receptor (IGFR); agents that promote apoptosis such as Poly ADP ribose polymerase inhibitors; agents that target invasion and metastasis such as matrix metalloproteinases inhibitors and others. In this review, we highlight the most promising targeted agents and their combination with mainstream chemotherapeutic drugs in clinical trials.
doi:10.4103/0253-7613.81497
PMCID: PMC3113372  PMID: 21713084
Breast cancer; chemotherapy; targeted therapy
11.  Effects of Acute Intravenous Infusion of Apelin on Left Ventricular Function in Dogs with Advanced Heart Failure 
Journal of cardiac failure  2013;19(7):509-516.
Background
Apelin-13 (APLN) through apelin receptor (APJ) exerts peripheral vasodilatory and potent positive inotropic effects. We examined the effects of exogenous intravenous infusion of APLN on left ventricular (LV) systolic function in dogs with heart failure (HF, LV ejection fraction, EF~30%).
Methods and Results
Studies were performed in 7 dogs with microembolization-induced HF. Each dog received an intravenous infusion of low dose and high dose APLN followed by washout period. LV end-diastolic volume (EDV), end-systolic volume (ESV) and LV EF were measured at specified time points. APLN protein level was determined in plasma at all time points. mRNA and protein levels of APLN and APJ in LV tissue were also measured in 7 normal (NL) and 7 heart failure (HF) dogs. APLN reduced EDV only at the high dose, significantly reduced ESV and increased EF with both doses. In plasma of HF dogs, APLN levels were reduced significantly compared to NL dogs. APLN treatment in HF dogs significantly increased the plasma APLN levels at both low and high doses. Expression of APLN, but not of APJ, was reduced in LV tissue of HF dogs compared to NL.
Conclusion
Exogenous administration of APLN improved LV systolic function in dogs with advanced HF.
doi:10.1016/j.cardfail.2013.05.004
PMCID: PMC3706995  PMID: 23834927
12.  Augmentation of Left Ventricular Wall Thickness With Alginate Hydrogel Implants Improves Left Ventricular Function and Prevents Progressive Remodeling in Dogs With Chronic Heart Failure 
JACC. Heart failure  2013;1(3):252-258.
Objectives
The study tested the hypothesis that augmentation of the left ventricular (LV) wall thickness with direct intramyocardial injections of alginate hydrogel implants (AHI) reduces LV cavity size, restores LV shape, and improves LV function in dogs with heart failure (HF).
Background
Progressive LV dysfunction, enlargement, and chamber sphericity are features of HF associated with increased mortality and morbidity.
Methods
Studies were performed in 14 dogs with HF produced by intracoronary microembolizations (LV ejection fraction [EF] <30%). Dogs were randomized to AHI treatment (n = 8) or to sham-operated control (n = 6). During an open-chest procedure, dogs received either intramyocardial injections of 0.25 to 0.35 ml of alginate hydrogel (Algisyl-LVR, LoneStar Heart, Inc., Laguna Hills, California) or saline. Seven injections were made ∼1.0 to 1.5 cm apart (total volume 1.8 to 2.1 ml) along the circumference of the LV free wall halfway between the apex and base starting from the anteroseptal groove and ending at the posteroseptal groove. Hemodynamic and ventriculographic measurements were made before treatment (PRE) and repeated post-surgery for up to 17 weeks (POST).
Results
Compared to control, AHI significantly reduced LV end-diastolic and end-systolic volumes and improved LV sphericity. AHI treatment significantly increased EF (26 ± 0.4% at PRE to 31 ± 0.4% at POST; p < 0.05) compared to the decreased EF seen in control dogs (27 ± 0.3% at PRE to 24 ± 1.3% at POST; p < 0.05). AHI treatment was well tolerated and was not associated with increased LV diastolic stiffness.
Conclusions
In HF dogs, circumferential augmentation of LV wall thickness with AHI improves LV structure and function. The results support continued development of AHI for the treatment of patients with advanced HF.
doi:10.1016/j.jchf.2013.02.006
PMCID: PMC3756288  PMID: 23998003
animal models; congestive heart failure; functional mitral regurgitation; left ventricular function; pressure-volume relationship
13.  Punicalagin and Ellagic Acid Demonstrate Antimutagenic Activity and Inhibition of Benzo[a]pyrene Induced DNA Adducts 
BioMed Research International  2014;2014:467465.
Punicalagin (PC) is an ellagitannin found in the fruit peel of Punica granatum. We have demonstrated antioxidant and antigenotoxic properties of Punica granatum and showed that PC and ellagic acid (EA) are its major constituents. In this study, we demonstrate the antimutagenic potential, inhibition of BP-induced DNA damage, and antiproliferative activity of PC and EA. Incubation of BP with rat liver microsomes, appropriate cofactors, and DNA in the presence of vehicle or PC and EA showed significant inhibition of the resultant DNA adducts, with essentially complete inhibition (97%) at 40 μM by PC and 77% inhibition by EA. Antimutagenicity was tested by Ames test. PC and EA dose-dependently and markedly antagonized the effect of tested mutagens, sodium azide, methyl methanesulfonate, benzo[a]pyrene, and 2-aminoflourine, with maximum inhibition of mutagenicity up to 90 percent. Almost all the doses tested (50–500 μM) exhibited significant antimutagenicity. A profound antiproliferative effect on human lung cancer cells was also shown with PC and EA. Together, our data show that PC and EA are pomegranate bioactives responsible for inhibition of BP-induced DNA adducts and strong antimutagenic, antiproliferative activities. However, these compounds are to be evaluated in suitable animal model to assess their therapeutic efficacy against cancer.
doi:10.1155/2014/467465
PMCID: PMC4052943  PMID: 24949451
14.  Multi-layer polymeric implants for sustained release of chemopreventives 
Cancer letters  2012;326(1):33-40.
Poor oral bioavailability limits the use of many chemopreventives in the prevention and treatment of cancer. To overcome this limitation, we report an improvised implant formulation (“coated” implants) using curcumin, individual curcuminoids, withaferin A and oltipraz. This method involves the coating of blank polycaprolactone implants with 20–30 layers of 10–20% polycaprolactone solution in dichloromethane containing 0.5–2% of the test agent. The in vitro release showed that while oltipraz was released with almost zero-order kinetics over eight weeks, curcumin, individual curcuminoids and withaferin A were released with some initial burst. The in vivo release was determined by grafting implants subcutaneously in A/J mice. When delivered by coated implants, oltipraz significantly diminished lung DNA adducts in mice treated with dibenzo[a, l]pyrene compared with sham treatment (28±7 versus 54±17 adducts/109 nucleotides). Withaferin A also diminished DNA adducts, but it was insignificant. Curcumin and individual curcuminoids were ineffective. Analysis of lung, liver and brain by UPLC-fluorescence showed the presence of the three test curcuminoids indicating effectiveness of the implant delivery system. Further, based on its known antitumor activity in vivo, withaferin A given via the implants significantly inhibited human lung cancer A549 xenograft in athymic nude mice, while it was ineffective when the same total dose was administered i.p. and required over 2-fold higher dose to elicit effectiveness. Together, our data suggest that coated polymeric implants can accommodate heat-labile compounds, can furnish sustained release for long duration, and elicit DNA damage-inhibiting and anti-tumor activities.
doi:10.1016/j.canlet.2012.07.017
PMCID: PMC3462657  PMID: 22820161
Polycaprolactone; Curcumin; Withaferin A; Oltipraz; DNA adducts; Lung tumor xenograft; Bioavailability
15.  TRC210258, a novel TGR5 agonist, reduces glycemic and dyslipidemic cardiovascular risk in animal models of diabesity 
Background
Patients with diabesity have a significantly increased risk of developing cardiovascular disease. Therefore, therapy addressing the multiple metabolic abnormalities linked with diabesity and leading to further reduction of cardiovascular risk is highly desirable. Activation of the TGR5 receptor holds therapeutic potential for diabesity. In the present study, we evaluated the efficacy of TRC210258, a novel TGR5 agonist, in clinically relevant animal models of diabesity.
Methods
A novel small molecule, TRC210258 (N-(4-chlorophenyl)-2-(4-fluorophenoxy)-N-methylimidazo (1, 2-a) pyrimidine-3-carboxamide), was synthesized. The in vitro TGR5 receptor activation potential of TRC210258 was assessed by cyclic adenosine monophosphate (cAMP) assay and cAMP-responsive element reporter assay using cells overexpressing the human TGR5 receptor. The effect of TRC210258 on glucagon-like peptide-1 release was evaluated in vitro using a human enteroendocrine cell line. The effect of TRC210258 on energy expenditure and glycemic control was evaluated in high-fat diet-induced obese mice. Additionally, the effect of TRC210258 on dyslipidemic parameters was determined in high fat-fed hamsters.
Results
TRC210258 demonstrated potent TGR5 agonist activity, with enhanced glucagon-like peptide-1 release and energy expenditure. Treatment with TRC210258 resulted in better glycemic control and improved parameters of dyslipidemia such as plasma triglyceride, low-density lipoprotein cholesterol, and non-high-density lipoprotein cholesterol levels. Treatment with TRC210258 also improved emerging dyslipidemic cardiovascular risk parameters, including remnant cholesterol and triglyceride clearance.
Conclusion
This study highlights the potential of TRC210258, a novel TGR5 agonist, to improve dyslipidemic cardiovascular risk beyond glycemic control in patients with type 2 diabetes.
doi:10.2147/DMSO.S50209
PMCID: PMC3873233  PMID: 24379686
TGR5; diabesity; atherogenic dyslipidemia; triglyceride clearance; remnant cholesterol; cardiovascular risks
16.  Vaginal cells of smokers are more resistant to human papillomavirus infection than that of non-smokers 
To evaluate effect of HPV and smoking on DNA double-strand breaks in vaginal samples, vaginal specimens collected from participants (n=76) were classified based on HPV and smoking status and DNA double-strand breaks measured using comet assay. Mean tail length (31.2 +/− 18.7 μm) and tail moment (2.4 +/− 2.8 arbitrary units) for HPV-positive patients were lower (p<0.001) compared with HPV-negative patients (61.7 +/− 22.6 μm; 8.7 +/− 4.9). Never-smokers were found to have higher level (p<0.001) of double-strand breaks (57.7 +/− 24.5 μm, 7.5 +/− 5.5 AU) compared with ever smokers (35.3 +/− 21.9 μm; 3.4 +/− 3.7 AU). Among HPV infected patients, never-smokers have more double-strand breaks compared to smokers (p<0.001) which correlated with age (p<0.001). Highly differentiated vaginal epithelium may be resistant to DNA damage associated with HPV infection and smoking, which may be attributed to adoptive survival mechanisms of vaginal epithelium.
doi:10.1016/j.yexmp.2012.10.016
PMCID: PMC3534756  PMID: 23137616
cigarette smoking; comet assay; DNA double-strand breaks; HPV infection; vaginal abnormalities
17.  Anti-proliferative activity and protection against oxidative DNA damage by punicalagin isolated from pomegranate husk 
Ellagitannins are the most abundant polyphenols in pomegranate (Punica granatum) husk and contribute greatly towards its biological properties. A pre-enriched pomegranate husk powder was extracted with water and then further purified by an Amberlite XAD-16 column. Punicalagin (PC) anomers were eluted using a gradient of methanol and water. Fractions eluted with 20% and 25% methanol yielded 1.08 g of light brown powder (purity > 97%) from a total of 40 g of extract. This fraction was identified as PC by HPLC-UV using reference compounds and confirmed by FTICR-MS analysis. PC (10–40 µM) was found to significantly inhibit oxidative DNA products, about 70% inhibition at 40 µM (p=0.0017), resulting from Cu2+-catalyzed redox cycling of 4-hydroxy-17β-estradiol as analyzed by 32P-postlabeling. Evidence of high antioxidant activity of PC was also obtained based on ORAC assay (1556±79 µmol of TE/g), as well as by 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS)-, 2,2-diphenyl-1-picrylhydrazyl (DPPH)-, hydrogen peroxide (H2O2) scavenging and ferrous ion-chelating activities (IC50=1.1, 17.1, 24 and 45.4 µg/ml, respectively). Further, PC exhibited strong anti-proliferative activity against the human lung, breast and cervical cancer cell lines. Together, these data suggest that PC can be isolated in its purified form by simple column chromatography, inhibits oxidative DNA damage and possesses high anti-proliferative activity.
doi:10.1016/j.foodres.2012.07.059
PMCID: PMC3593254  PMID: 23493479
Punica granatum; Punicalagin; Antioxidant activity; Antiproliferative activity; Oxidative DNA damage; 32P-postlabeling
18.  Detection of Proteinuria in Pregnancy: Comparison of Qualitative Tests for Proteins and Dipsticks with Urinary Protein Creatinine Index 
Background and Objectives: Excretion of urinary protein increases to 300 mg/d (from up to 150 mg/d) in normal pregnancy. Values above this may be due to disorders that can endanger the patient or her pregnancy. Quantitative analysis of 24-hour urine is considered the gold standard for ascertaining daily protein excretion. Routine laboratory tests performed on spot urine samples indicate protein concentration in the particular sample, and can lead to diagnostic error if urine output is less or more than 1L/d. The Protein Creatinine Index (PCI) shows good correlation with 24-hour protein estimation. However, PCI varies with sex and race. We have correlated the results of qualitative estimation procedures and the dipstick values with protein creatinine index.
Material and Methods: We measured protein and creatinine in spot urine samples obtained from 57 pregnant and 80 non–pregnant healthy women of 18–36 years, and calculated PCI. We also tested the samples qualitatively for proteins by routine tests and dipsticks.
Results: Normal range of PCI in non–pregnant women, determined by a non–parametric method was 30–150. PCI was increased significantly in pregnancy (maximum increase in the third trimester). Amongst the qualitative tests, heat coagulation test gave the lowest percentage of false positives and a slightly higher percentage of false negatives compared to Heller’s nitric acid and sulphosalicylic acid tests, and dipsticks.
Interpretations and Conclusions: We conclude that heat coagulation test be used for initial screening, with PCI being performed on all samples testing positive to rule out false positives.
doi:10.7860/JCDR/2013/6656.3330
PMCID: PMC3809617  PMID: 24179878
PCI; Pregnancy; Non-Pregnant Women; Dipsticks
19.  Pleiotropic Effects of Long-term Monotherapy with Rosuvastatin in Dogs with Moderate Heart Failure 
Cardiology  2012;123(3):160-167.
Objective
The objective of this study was to investigate potential pleiotropic effects of rosuvastatin (RSV) in left ventricular (LV) myocardium of dogs with moderate heart failure (HF).
Methods
LV tissue was obtained from HF dogs randomized to 3 months therapy with low dose (LD) RSV (n=7), high dose (HD) RSV (n=7) or to no therapy (Control, n=7), and from 7 normal (NL) dogs. mRNA and protein expression of pro-hypertrophic mediators NGFI-A binding protein 1 (Nab1), phosphatase and tensin homolog (PTEN), phosphoinositide-3 kinase (PI3K) and mammalian target of rapamycin (mTOR); pro-inflammatory cytokine interleukin-6 (IL-6); bone marrow-derived stem cells (BMSCs) markers cKit and Sca1; vascular endothelial (VEGF) and fibroblast (FGF) growth factors and nitric oxide synthase (NOS) isoforms were measured.
Results
Nab1, PTEN, PI3K, mTOR, and IL-6 increased in Controls. HD RSV reduced expression of Nab1, PTEN, PI3K, mTOR, and IL-6 to near normal levels. cKit and Sca1 significantly increased while VEGF and FGF decreased in Controls compared to NL. RSV therapy further increased expression of cKit, Sca1, VEGF and FGF. HD RSV normalized expression of NOS isoforms.
Conclusion
These pleiotropic effects of RSV may account, in part, for the observed beneficial effect of RSV on LV function and structural remodeling.
doi:10.1159/000342082
PMCID: PMC3544002  PMID: 23128666
Inflammation; Cytokines; Growth factors; Nitric oxide synthase; Hypertrophy; Stem cells
20.  Chronic Therapy with a Partial Adenosine A1 Receptor Agonist, Improves Left Ventricular Function and Remodeling in Dogs with Advanced Heart Failure 
Circulation. Heart failure  2013;6(3):563-571.
Background
Adenosine (AD) elicits cardioprotection through A1-receptor (A1R) activation. Therapy with AD A1R agonists, however, is limited by undesirable actions of full agonism such as bradycardia. This study examined the effects of capadenoson (CAP), a partial AD A1R agonist, on left ventricular (LV) function and remodeling in dogs with heart failure (HF).
Methods and Results
12 dogs with microembolization-induced HF were randomized to 12 weeks oral therapy with CAP (7.5 mg Bid, n=6) or to no therapy (Control, n=6). LV end-diastolic (EDV) and end-systolic (ESV) volumes, ejection fraction (EF), plasma norepinephrine (NE) and n-terminal pro-brain natriuretic peptide (nt-pro BNP) were measured before (PRE) and 1 and 12 weeks after therapy (POST). LV tissue obtained at POST was used to assess volume fraction of interstitial fibrosis (VFIF), SERCA-2a activity, expression of mitochondria uncoupling proteins (UCP) and glucose transporters (GLUT). In controls, EDV and ESV increased and EF decreased significantly from PRE to POST (EF: 30±2 vs. 27±1 %, p<0.05). In CAP-treated dogs, EDV was unchanged; EF increased significantly after one week (36±2 vs. 27±2 %, p<0.05) with a further increase at POST (39±2 %, p<0.05) while ESV decreased. CAP significantly decreased VFIF, normalized SERCA-2a activity and expression of UCP-2 and -3, and GLUT-1 and -2 and significantly decreased NE and nt-pro BNP.
Conclusion
In HF dogs, CAP improves LV function and prevents progressive remodeling. Improvement of LV systolic function occurs early after initiating therapy. The results support development of partial AD A1R agonists for the treatment of chronic HF.
doi:10.1161/CIRCHEARTFAILURE.112.000208
PMCID: PMC3790141  PMID: 23564604
Heart failure; Ventricular remodeling; Protein expression; Adenosine receptors
21.  Vagus Nerve Stimulation in Experimental Heart Failure 
Heart failure reviews  2011;16(2):171-178.
Chronic heart failure (HF) is associated with autonomic dysregulation characterized by a sustained increase of sympathetic drive and by withdrawal of parasympathetic activity. Sympathetic overdrive and increased heart rate are predictors of poor long-term outcome in patients with HF. Considerable evidence exists that supports the use of pharmacologic agents that partially inhibit sympathetic activity as effective long-term therapy for patients with HF; the classic example is the wide use of selective and non-selective beta-adrenergic receptor blockers. In contrast, modulation of parasympathetic activation as potential therapy for HF has received only limited attention over the years given its complex cardiovascular effects. In this article, we review results of recent experimental animal studies that provide support for the possible use of electrical Vagus nerve stimulation (VNS) as a long-term therapy for the treatment of chronic HF. In addition to exploring the effects of chronic VNS on left ventricular (LV) function, the review will also address the effects of VNS on potential modifiers of the HF state that include cytokine production and nitric oxide elaboration. Finally, we will briefly review other nerve stimulation approaches also currently under investigation as potential therapeutic modalities for treating chronic HF.
doi:10.1007/s10741-010-9209-z
PMCID: PMC3784341  PMID: 21128115
Ventricular function; Ventricular remodeling; Electrical nerve stimulation; Sympathetic activity; Parasympathetic activity; Animal models of disease; Cytokines; Nitric oxide
22.  Cigarette smoke condensate-induced oxidative DNA damage and its removal in human cervical cancer cells 
International journal of oncology  2011;39(4):941-947.
Exposure to cigarette smoke is well documented to increase oxidative stress and could account for higher risk of cervical cancer in smokers. Cervical pre-cancerous lesions that are initiated by human papillomavirus (HPV) infection generally regress in the absence of known risk factors such as smoking. 8-oxodeoxyguanosine (8-oxodG) is a highly mutagenic oxidative DNA lesion that is formed by the oxidation of deoxyguanosine. In the present study, we examined: a) the effect of cigarette smoke condensate (CSC) on 8-oxodG formation in and its removal from HPV-transfected (ECT1/E6 E7), HPV-positive (CaSki) and HPV-negative (C33A) human cervical cancer cells, and b) the cell cycle progression and apoptosis in CSC-treated ECT1/E6 E7 cells. CSC induced 8-oxodG in a dose-(p=0.03) and time (p=0.002)-dependent fashion in ECT1/E6 E7 cells as determined by flow cytometry. A 2.4-fold higher level of 8-oxodG was observed in HPV-positive compared with HPV-negative cells. However, 8-oxodG lesions were almost completely removed 72 h post-exposure in all cell lines as determined by ImageStream analysis. This observation correlates with the 2- and 5-fold increase in the p53 levels in ECT1/E6 E7 and CaSki cells with no significant change in C33A cells. We conclude that: a) cigarette smoke constituents induce oxidative stress with higher burden in HPV-positive cervical cancer cells and b) the significant increase observed in p53 levels in wild-type cervical cells (ECT1/E6 E7 and CaSki) may be attributed to the p53-dependent DNA repair pathway while a p53-independent pathway in C33A cells cannot be ruled out.
doi:10.3892/ijo.2011.1106
PMCID: PMC3760590  PMID: 21720711
cigarette smoke condensate; human cervical cancer cells; HPV; oxidative DNA damage; DNA repair
23.  Antioxidant and antiproliferative activities of anthocyanin/ellagitannin-enriched extracts from Syzygium cumini L. (‘jamun’, the Indian Blackberry) 
Nutrition and Cancer  2012;64(3):428-438.
Colored fruits, particularly berries, are highly chemoprotective because of their antioxidant, anti-proliferative and anti-inflammatory activities. We report cancer chemoprotective potential of Syzygium cumini L., commonly known as ‘jamun’ or Indian blackberry. Anthocyanins and other polyphenolics were extracted with acidic ethanol, and enriched by amberlite XAD7/HP20 (1:1). The pulp powder was found to contain 0.54% anthocyanins, 0.17% ellagic acid/ellagitannins and 1.15% polyphenolics. Jamun seed contained no detectable anthocyanins, but had higher amounts of ellagic acid/ellagitannins (0.5%) and total polyphenolics (2.7%) than the pulp powder. Upon acid hydrolysis, the pulp extract yielded five anthocyanidins by HPLC: malvidin (44.4%), petunidin (24.2%), delphinidin (20.3%), cyanidin (6.6%), and peonidin (2.2%). Extracts of both jamun pulp (1,445±64 μmol of trolox equivalent (TE)/g) and seeds (3,379±151 μM of TE/g) showed high oxygen radical absorbance capacity (ORAC). Their high antioxidant potential was also reflected by 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS)- and 2,2-diphenyl-1-picrylhydrazyl (DPPH)-scavenging, and ferrous ion-chelating activities. We also analyzed anti-proliferative activity of jamun extracts against human lung cancer A549 cells. The hydrolyzed pulp and seed extracts showed significant antiproliferative activity. However, unhydrolyzed extracts showed much less activity. These data showed that in addition to five anthocyanidins, jamun contains appreciable amounts of ellagic acid/ellagitannins, with high antioxidant and antiproliferative activities.
doi:10.1080/01635581.2012.657766
PMCID: PMC3351850  PMID: 22420901
Anthocyanins; Syzygium cumini; ellagic acid and ellagitannins; antioxidant activity; anti-proliferative activity
24.  Structure-activity relationship study of novel anticancer aspirin-based compounds 
Molecular medicine reports  2011;4(5):891-899.
We performed a structure-activity relationship (SAR) study of a novel aspirin (ASA) derivative, which shows strong anticancer activity in vitro and in vivo. A series of ASA-based benzyl esters (ABEs) were synthesized and their inhibitory activity against human colon (HT-29 and SW480) and pancreatic (BxPC-3 and MIA PaCa-2) cancer cell lines was evaluated. The ABEs that we studied largely comprise organic benzyl esters bearing an ASA or acyloxy group (X) at the meta or para position of the benzyl ring and one of four different leaving groups. The nature of the salicyloyl/acyloxy function, the leaving group, and the additional substituents affecting the electron density of the benzyl ring, all were influential determinants of the inhibitory activity on cancer cell growth for each ABE. Positional isomerism also played a significant role in this effect. The mechanism of action of these compounds appears consistent with the notion that they generate either a quinone methide or an m-oxybenzyl zwitterion (or an m-hydroxybenzyl cation), which then reacts with a nucleophile, mediating their biological effect. Our SAR study provides an insight into the biological properties of this novel class of compounds and underscores their potential as anticancer agents.
doi:10.3892/mmr.2011.534
PMCID: PMC3565583  PMID: 21805049
anticancer compounds; phosphoaspirin; nonsteroidal anti-inflammatory drugs; cancer prevention
25.  Protective effects of antioxidants and anti-inflammatory agents against manganese-induced oxidative damage and neuronal injury 
Toxicology and applied pharmacology  2011;256(3):219-226.
Exposure to excessive manganese (Mn) levels leads to neurotoxicity, referred to as manganism, which resembles Parkinson’s disease (PD). Manganism is caused by neuronal injury in both cortical and subcortical regions, particularly in the basal ganglia. The basis for the selective neurotoxicity of Mn is not yet fully understood. However, several studies suggest that oxidative damage and inflammatory processes play prominent roles in the degeneration of dopamine-containing neurons. In the present study, we assessed the effects of Mn on reactive oxygen species (ROS) formation, changes in high-energy phosphates and associated neuronal dysfunctions both in vitro and in vivo. Results from our in vitro study showed a significant (P<0.01) increase in biomarkers of oxidative damage, F2-isoprostanes (F2-IsoPs), as well as the depletion of ATP in primary rat cortical neurons following exposure to Mn (500 µM) for 2 hours. These effects were protected when neurons were pretreated for 30 min with 100 µM of an antioxidant, the hydrophilic vitamin E analog, trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), or an anti-inflammatory agent, indomethacin. Results from our in vivo study confirmed a significant increase in F2-IsoPs levels in conjunction with the progressive spine degeneration and dendritic damage of the striatal medium spiny neurons (MSNs) of mice exposed to Mn (100 mg/kg, s.c.) 24 hours. Additionally, pretreatment with vitamin E (100 mg/kg, i.p.) or ibuprofen (140 µg/ml in the drinking water for two weeks) attenuated the Mn-induced increase in cerebral F2-IsoPs and protected the MSNs from dendritic atrophy and dendritic spine loss. Our findings suggest that the mediation of oxidative stress/mitochondrial dysfunction and the control of alterations in biomarkers of oxidative injury, neuroinflammation and synaptodendritic degeneration may provide an effective, multi-pronged therapeutic strategy for protecting dysfunctional dopaminergic transmission and slowing of the progression of Mn-induced neurodegenerative processes.
doi:10.1016/j.taap.2011.06.001
PMCID: PMC3205299  PMID: 21684300
Manganese; oxidative stress; medium spiny neurons; neurodegeneration; vitamin E; trolox; ibuprofen

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