GraphPad Prism5 Research Articles

Evaluation of antiangiogenic potential of lubiprostone, a clc-2 channel agonist by chorioallantoic Membrane assay. (cam)

Angiogenesis is the formation of new blood vessels. Abundant ion channels are located on the endothelial cell surface. Ion channels have significant role in cell proliferation and thus in vascular angiogenesis. The antiangiogenic activity of a specific voltage gated Chloride channel opener ClC2 (Lubiprostone) was evaluated by Chorioallantoic Membrane (CAM) Assay (in ovo).The standard antiangiogenic drug used for comparison was Bevacizumab. All the data obtained from the experimental groups have been compared to the control group. The data was analyzed by one-way ANOVA followed by post hoc test, the Dunnett's test to compare mean of every group with the control mean, using graph pad prism5.0 software. In the CAM assay, the parameters evaluated were number of branches per unit area and angiogenic score. Medium (Lubiprostone 10 -5 M /egg) and high (Lubiprostone 10 -4 M /egg) doses of both the channel blockers gave significant results. From the results obtained, Lubiprostone has effective anti-angiogenesis which may be therapeutically beneficial .The molecular pathways may be due to negative regulation of cell volume, cell migration and thus proliferation.

Abstract 4632: Effects of Hsp90 inhibitors on triple-negative breast cancer: BRCA1 as a therapeutic target for TNBC

Abstract Background: Breast cancer is the second leading cause of death for woman. Within breast cancer, those classified as Triple Negative Breast Cancer (TNBC) exhibit dismal survival rates due to their propensity to develop distant metastases. Heat shock protein 90 (Hsp90) is a molecular chaperone that aids in the folding and maturation of various proteins involved in breast cancer progression and resistance to therapy. The aim of this study was to elucidate whether the two natural inhibitors of Hsp90, celastrol and triptolide inhibit triple negative breast cancer growth. Both these compounds are terpenoids and were obtained from the Chinese herb “Thunder God of Vine” (Tripterygium wilfordii). Methods: BT20, BT549, MDA-MB-157 and MDA-MB-231 cells (all TNBC cells), and immortalized human mammary epithelial cells (HMLE) were grown in DMEM containing 10% FBS as per ATCC recommendations. Cell proliferation was assessed by hexoseaminidase activity, and IC50 values calculated using GraphPad Prism5. For clonogenicity, 500 cells were incubated with IC50 concentrations of each compound for 24 h, after which they were allowed to grow and form colonies. For in vivo, BT20 cells were injected into flanks of athymic nude mice and treated with celastrol and triptolide at 3 mg/Kg bw and 0.25 mg/Kg bw, respectively. Results: Celastrol and triptolide treatment suppressed proliferation and colony forming ability of all four TNBC cell lines, but not that of the immortalized HMLE cells. The compounds increased apoptotic cell death, based on increased Annexin V staining. Moreover, there was increased expression of the pro-apoptotic protein Bax but decreased expression of the anti-apoptotic protein, Bcl2 and BclXL. Immunoprecipitation-coupled western blots also showed that the compounds inhibit HSP90/CDC37 complex formation. Interestingly, the coupled immunoprecipitation-western blot analyses showed increased HSP90-BRCA1 interaction after treatment with the compounds. Coupled to this, western blot and immunostaining assays showed increased cytosolic levels and reduced nuclear levels of BRCA1 protein. Similar results were obtained in vivo with BT20 xenografts. In addition to decreased tumor size in response to treatment with celastrol or triptolide, the xenograft tissues showed an increase in cytoplasmic BRCA1 levels following treatment. In addition, there was increased in HSP90-BRCA1 complex formation in the treated xenograft tissues. Finally, knockdown of BRCA1 using specific silencer RNA resulted in partial inhibition in cell growth reduction after celastrol and triptolide treatment in both BT20 and MDA-MB-231 cells. Conclusion: Taken together, these data suggest that both celastrol and triptolide suppress TNBC cell growth, in part through increasing cytosolic HSP90/BRCA1 complex formation. Citation Format: Prabhu Ramamoorthy, Parthasarathy Rangarajan, Ossama Tawfik, Shrikant Anant, Roy A. Jensen. Effects of Hsp90 inhibitors on triple-negative breast cancer: BRCA1 as a therapeutic target for TNBC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4632.

Prevalence and Relationship Between Maternal Periodontal Disease and Preterm Low Birth Weight Baby

AbstractThe objective of this study was to examine the prevalence and relationship between maternal periodontal disease and preterm low birth weight (LBW) babies among women delivering at Hindu Rao Hospital, Delhi, India. A case control study was performed on 150 women fulfilling the selection criteria over a period of one year. The study consisted of 50 cases, (women delivering preterm babies weighing ≤2.5 kg, Group I) and 100 controls (women delivering babies at ≥37 weeks and weighing >2.5 kg, Group II). Associated risk factors for preterm low birth weight (PT-LBW) were ascertained by means of a structured questionnaire and maternal notes. Women having any of the possible risk factors for preterm LBW were either excluded or confounded in the study. The prevalence of periodontal disease was assessed by the community periodontal index of treatment needs (CPITN) scoring method, with scores ranging from 0 to 4. Data were analyzed using Graphpad Prism-5 software. P value and odds ratio (OR) with 95 % confidence interval were calculated as and when required for statistical analysis. Prevalence of periodontal disease was 84.66 % (n = 150) in the study population. Prevalence of periodontal disease was high, at least in some form or other, in cases (100 %) as compared to controls (77 %). The prevalence of severe periodontal disease (CPITN score-4) was 8 % in cases and 3 % in controls. Periodontal disease is an avoidable risk factor for LBW as almost all of the known risk factors for LBW were either excluded or confounded during the study. Hence, routine periodontal examination and advice on good oral hygiene should be included as part of preconceptional care and antenatal checkups during pregnancy. Any dysfunction should be thoroughly investigated and treated for the sake of health of both mother and baby.

MAPPING IN VITRO AND IN VIVO DERIVED CYP2C9 AND CYP2C19 ONTOGENY FUNCTIONS: A CRITICAL COMPARISON BETWEEN VARIOUS ONTOGENY MODELS

In vivo derived ontogeny profiles for CYP1A2 and CYP3A4, show improved clearance (CL) predictions within a paediatric physiologically based pharmacokinetic (p-PBPK) model1. The aim of this study is to derive ontogeny functions (OF) for CYP2C9 and CYP2C19 based on age related CL data on ibuprofen and pantoprazole & lansoprazole, respectively.A literature review was undertaken to collect age related CL data for these probes, the values were deconvoluted back to intrinsic CL values (per mg of liver microsomal protein) as described previously. The 'best-fit' algorithm for ratio of paediatric to mean adult intrinsic CL with age was determined in Graphpad Prism5 to obtain in vivo OF for CYP2C9 and CYP2C19. These were compared for performance with previously established ‘in vitro' OF in Simcyp Paediatric simulator (v14) using validation datasets.CYP2C9 and CYP2C19 enzyme activities showed an increase with age to values higher than adults by ages 2 and 1 month respectively, maximum values were reached at 1.5 years and 6 months, respectively before declining to typical adult levels by around 25 years.The CYP2C9 in vivo derived OF led to improved predictions of diclofenac and S-Warfarin CL compared to in vitro derived OF across the age range. For CYP2C19 there is a dearth of suitable validation compounds due to lack of clinical data with a possibility of using omeprazole or voriconazole. The reasons for discrepancy between in vitro and in vivo derived OF require further investigation.

Abstract PR05: Effects of Hsp90 inhibitors on triple-negative breast cancer: Notch as a therapeutic target for stem cells

Abstract Background: Breast cancer is the second leading cause of death for woman. Within breast cancer subtypes, those classified as Triple Negative Breast Cancer (TNBC) exhibit dismal survival rates due to their propensity to develop distant metastases. Tumors contain heterogeneous cell populations and it has numerically rare cancer stem cells with indefinite proliferative potential that is responsible for tumor invasiveness, heterogeneity, and therapy resistance. Heat shock protein 90 (Hsp90) is a molecular chaperone that aids in the folding and maturation of various proteins involved in breast cancer progression and resistance to therapy. The aim of this study was to elucidate whether the two natural inhibitors of Hsp90, celastrol and triptolide inhibit triple negative breast cancer growth. Both these compounds are terpenoids and were obtained from the Chinese herb “Thunder God of Vine” (Tripterygium wilfordii). Methods: BT20, BT549, MDA-MB-231 and MDA-MB-157 cells (all TNBC cells) were obtained and grown in DMEM containing 10% FBS as per ATCC recommendations. Cell proliferation was assessed by hexoseaminidase activity, and IC50 values calculated using GraphPad Prism5. For clonogenicity, 500 cells were treated with IC50 concentration of each compound for 24h, and then allowed to grow and form colonies. Mammosphere assay was performed using 5000 cells/ml in ultra low attachment plates. Images were captured after 5 days. For in vivo, BT20 cells were injected into flanks of athymic nude mice and treated with celastrol and triptolide at 3 mg/Kg bw and 0.25 mg/Kg bw, respectively. Results: Celastrol and triptolide treatment suppressed the proliferation and colony formation ability of all four TNBC cell lines BT20, BT549, MDA-MB-231 and MDA-MB-157. Interestingly, the mammosphere assay (an assay used to evaluate the self-renewal capacity of the cancer stem cells) revealed that celastrol or triptolide significantly reduces the size and number of spheroids. Furthermore, expression of breast cancer stem cell markers ALDH1 and CD133 were significantly reduced in BT20 cells upon the treatment. Recently, Notch signaling has been shown to be critical for self-renewal of cancer stem cells. Activation of the Notch receptor, a membrane spanning receptor involves the interaction with a ligand resulting in a series of proteolytic cleavage events culminating in the release of the Notch intracellular domain (NICD). This NICD translocates to the nucleus, and together with its interacting partner CSL/RBPJ binds to cognate element and activates the expression of downstream target genes such as Hes-1. In cells treated with either celastrol or triptolide, there was a significant reduction in NICD, and its downstream target Hes-1. Furthermore, there was a reduction in ALDH+ cells. However, in cells where we ectopically overexpressed NICD, neither compound was as potent as control vector transfected cells in reducing proliferation, colony formation or mammosphere formation, suggesting the direct role for inhibiting Notch activation as a mechanism of action for the two compounds. We confirmed these finding in vivo using BT20 tumor xenografts grown in athymic nude mice. There was a reduction in the size of tumors in mice treated with celastrol or triptolide. In addition, western blot and immunohistochemistry analyses demonstrated a reduction in the number of ALDH+ and CD133+ cells. Conclusion: Taken together these data suggest that both celastrol and triptolide affect cancer stem cells in TNBC, in part through inhibition of Notch signaling. This abstract is also presented as Poster B31. Citation Format: Prabhu Ramamoorthy, Sydney Byrne, Satish Ramalingam, Parthasarathy Rangarajan, Dharmalingam Subramaniam, Scott Weir, Shrikant Anant, Roy Jensen. Effects of Hsp90 inhibitors on triple-negative breast cancer: Notch as a therapeutic target for stem cells. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr PR05.

Peroxisome Proliferator-activated Receptor γ (PPARγ) Mediates a Ski Oncogene-induced Shift from Glycolysis to Oxidative Energy Metabolism

Overexpression of the Ski oncogene induces oncogenic transformation of chicken embryo fibroblasts (CEFs). However, unlike most other oncogene-transformed cells, Ski-transformed CEFs (Ski-CEFs) do not display the classical Warburg effect. On the contrary, Ski transformation reduced lactate production and glucose utilization in CEFs. Compared with CEFs, Ski-CEFs exhibited enhanced TCA cycle activity, fatty acid catabolism through β-oxidation, glutamate oxidation, oxygen consumption, as well as increased numbers and mass of mitochondria. Interestingly, expression of PPARγ, a key transcription factor that regulates adipogenesis and lipid metabolism, was dramatically elevated at both the mRNA and protein levels in Ski-CEFs. Accordingly, PPARγ target genes that are involved in lipid uptake, transport, and oxidation were also markedly up-regulated by Ski. Knocking down PPARγ in Ski-CEFs by RNA interference reversed the elevated expression of these PPARγ target genes, as well as the shift to oxidative metabolism and the increased mitochondrial biogenesis. Moreover, we found that Ski co-immunoprecipitates with PPARγ and co-activates PPARγ-driven transcription.

Cardiac adaptation to endurance training in young adult

Context: Regular physical exercise is known to cause improvement of the cardiovascular function. This adaptation is studied here with the help of non-invasive methods. Aims: To evaluate morphological changes in heart by echocardiography, to see the effect of exercise on autonomic function, on aerobic power and to assess the sequence of changes. Settings and Design: Study comprises of 12-week duration and was done on the students of physical education. Materials and Methods: This study was a longitudinal study in which 100 subjects (51 male, 20.18 yrs±1.147, 49 female, 19.91 yrs±1.89) were assessed using electrocardiography, echocardiography and Queen's College Step test (for VO 2max ) within 7 days of admission to their college and re-examined after 12 weeks. Statistical Analysis: Paired t-test using Graph pad prism5 software. Results: Electrocardiographic evaluation was suggestive of significant decrease in heart rate, significant increase in RR interval and t-wave amplitude in cardiac leads in males and similar but not significant result in females. No significant change was found in left ventricular morphology and ejection fraction after exercise program. Conclusions: The results of this study suggest that the exercise training over a period of 3 months does not influence cardiovascular morphology, but causes changes in parasympathetic and sympathetic tone and improves aerobic power.

Conditional Disruption of Pkd1 in Osteoblasts Results in Osteopenia Due to Direct Impairment of Bone Formation

PKD1 (polycystin-1), the disease-causing gene for ADPKD, is widely expressed in various cell types, including osteoblasts, where its function is unknown. Although global inactivation of Pkd1 in mice results in abnormal skeletal development, the presence of polycystic kidneys and perinatal lethality confound ascertaining the direct osteoblastic functions of PKD1 in adult bone. To determine the role of PKD1 in osteoblasts, we conditionally inactivated Pkd1 in postnatal mature osteoblasts by crossing Oc (osteocalcin)-Cre mice with floxed Pkd1 (Pkd1(flox/m1Bei)) mice to generate conditional heterozygous (Oc-Cre;Pkd1(flox/+)) and homozygous (Oc-Cre;Pkd1(flox/m1Bei)) Pkd1-deficient mice. Cre-mediated recombination (Pkd1(Delta flox)) occurred exclusively in bone. Compared with control mice, the conditional deletion of Pkd1 from osteoblasts resulted in a gene dose-dependent reduction in bone mineral density, trabecular bone volume, and cortical thickness. In addition, mineral apposition rates and osteoblast-related gene expression, including Runx2-II (Runt-related transcription factor 2), osteocalcin, osteopontin, and bone sialoprotein, were reduced proportionate to the reduction of Pkd1 gene dose in bone of Oc-Cre;Pkd1(flox/+) and Oc-Cre;Pkd1(flox/m1Bei) mice. Primary osteoblasts derived from Oc-Cre;Pkd1(flox/m1Bei) displayed impaired differentiation and suppressed activity of the phosphatidylinositol 3-kinase-Akt-GSK3beta-beta-catenin signaling pathways. The conditional deletion of Pkd1 also resulted in increased adipogenesis in bone marrow and in osteoblast cultures. Thus, PKD1 directly functions in osteoblasts to regulate bone formation.