Gary L. Sanford

Inhibition of cyclooxygenase-2 decreases breast cancer cell motility, invasion and matrix metalloproteinase expression

BackgroundCyclooxygenase (COX) is the rate-limiting enzyme that catalyzes the formation of prostaglandins. The inducible isoform of COX (COX-2) is highly expressed in aggressive metastatic breast cancers and may play a critical role in cancer progression (i.e. growth and metastasis). However, the exact mechanism(s) for COX-2-enhanced metastasis has yet to be clearly defined. It is well established that one of the direct results of COX-2 action is increased prostaglandin production, especially prostaglandin E2 (PGE2). Here, we correlate the inhibition of COX-2 activity with decreased breast cancer cell proliferation, migration, invasion and matrix metalloproteinase (MMP) expression.MethodsBreast cancer cells (Hs578T, MDA-MB-231 and MCF-7) were treated with selective COX-2 inhibitors (NS-398 and Niflumic acid, NA). Cell proliferation was measured by staining with erythrosin B and counting the viable cells using a hemacytometer. Cell migration and invasion were measured using migration and invasion chamber systems. MMP expression was determined by enzyme immunoassay (secreted protein) and real-time quantitative polymerase chain reaction (mRNA).ResultsOur results show that there is a decline in proliferation, migration and invasion by the Hs578T and MDA-MB-231 breast cancer cell lines in the presence of either low concentrations (1 μM or lower) NA or NS-398. We also report that MMP mRNA and protein expression by Hs578T cells is inhibited by NS-398; there was a 50% decrease by 100 μM NS-398. PGE2 completely reversed the inhibitory effect of NS-398 on MMP mRNA expression.ConclusionOur data suggests that COX-2-dependent activity is a necessary component for cellular and molecular mechanisms of breast cancer cell motility and invasion. COX-2 activity also modulates the expression of MMPs, which may be a part of the molecular mechanism by which COX-2 promotes cell invasion and migration. The studies suggest that COX-2 assists in determining and defining the metastatic signaling pathways that promote the breast cancer progression to metastasis.

THREE-DIMENSIONAL GROWTH OF ENDOTHELIAL CELLS IN THE MICROGRAVITY-BASED ROTATING WALL VESSEL BIOREACTOR

SummaryWe characterized bovine aortic endothelial cells (BAEC) continuously cultured in the rotating wall vessel (RWV) bioreactor for up to 30 d. Cultures grew as large tissue-like aggregates (containing 20 or more beads) after 30 d. These cultures appeared to be growing in multilayers around the aggregates, where single beads were covered with confluent BAEC, which displayed the typical endothelial cell (EC) morphology. The 30-d multibead aggregate cultures have a different and smoother surface when viewed under a higher-magnification scanning electron microscope. Transmission electron microscopy of these large BAEC aggregates showed that the cells were viable and formed multilayered sheets that were separated by an extracellular space containing matrix-like material. These three-dimensional cultures also were found to have a basal production of nitric oxide (NO) that was 10-fold higher for the RWV than for the Spinner flask bioreactor (SFB). The BAEC in the RWV showed increased basal NO production, which was dependent on the RWV rotation rate: 73% increase at 8 rpm, 262% increase at 15 rpm, and 500% increase at 20 rpm as compared with control SFB cultures. The addition of l-arginine to the RWV cultures resulted in a fourfold increase in NO production over untreated RWV cultures, which was completely blocked by L-NAME [N(G)-nitro-L-arginine-methylester]. Cells in the SFB responded similarly. The RWV cultures showed an increase in barrier properties with an up-regulation of tight junction protein expression. We believe that this study is the first report of a unique growth pattern for ECs, resulting in enhanced NO production and barrier properties, and it suggests that RWV provides a unique model for investigating EC biology and differentiated function.

Plasma 25-hydroxyvitamin D concentrations are inversely associated with blood pressure of Dahl salt-sensitive rats.

Dietary salt is a contributing factor to the development of hypertension in individuals who are salt-sensitive. The vitamin D endocrine system has been reported to modulate vascular structure and function. Since elderly hypertensive females with low plasma renin activity, typical of salt-sensitivity, had significantly lower 25-hydroxyvitamin D concentrations compared with normotensive elderly and young females, we have used Dahl salt-sensitive and salt-resistant rats fed high (80 g/kg diet) and low (3 g/kg diet) salt diets as models to examine the relationship between salt-sensitivity and 25-hydroxyvitamin D, the precursor of the hormonal form of vitamin D, 1,25-dihydroxyvitamin D. Plasma 25-hydroxyvitamin D concentrations of salt-resistant rats were unaffected by a high salt diet, but plasma 25-hydroxyvitamin D concentrations of salt-sensitive rats were significantly reduced within three weeks to lower than 25%. There was a negative association between plasma 25-hydroxyvitamin D concentrations of salt-sensitive rats and the number of days that the rats were fed a high salt diet (r = -0.98, P < 0.02) and a positive association between blood pressure and the number of days that the rats were fed a high salt diet (r = 0.97, P < 0.05). An inverse relationship was found between plasma 25-hydroxyvitamin D concentrations and blood pressure (r = -0.99, P < 0.01). Spontaneously hypertensive rats did not have low plasma 25-hydroxyvitamin D concentrations, suggesting that reduction of plasma 25-hydroxyvitamin D concentration might be specific to salt-induced hypertension.

Lipids, lipoproteins and coronary heart disease in minority populations

Despite recent advances in both prevention and treatment, cardiovascular disease (CVD) remains the leading cause of mortality in the US. The Framingham Study was a landmark in defining CHD-related risk factors; unfortunately, very few minorities were included. A major preventable risk factor for CHD continues to be lipid abnormalities, but its association within minority populations is unclear. The few studies that have examined the association of hyperlipidemia with CHD in minorities have shown that total cholesterol was a predictor of CHD risk (e.g., black men aged 35-64). Several researchers have reported higher levels of HDL for black men and women compared to white men and women. Since HDL was shown to be inversely related to CHD, this discrepancy in HDL is hypothesized to account for the lower than expected mortality rate. Lipoprotein(a) has been identified as an independent risk factor for CHD; blacks have considerably higher levels than whites. Data also indicate the following: Hispanics have lower CVD mortality rates than the general population despite having known risk factors (e.g., obesity, diabetes, low socioeconomic status); Hispanic women have lower levels of HDL cholesterol; Native-American populations have lower prevalence of CHD associated with lower LDL-cholesterol and higher HDL-cholesterol. Understanding epidemiologic and pathophysiologic data regarding differences between various racial groups should help reduce CVD-related morbidity and mortality in minority populations.

Three-Dimensional Model of Angiogenesis: Coculture of Human Retinal Cells with Bovine Aortic Endothelial Cells in the NASA Bioreactor

Ocular angiogenesis is the leading cause of blindness and is associated with diabetic retinopathy and age-related macular degeneration. We describe, in this report, our preliminary studies using a horizontally rotating bioreactor (HRB), developed by the National Aeronautics and Space Administration (NASA), to explore growth and differentiation-associated events in the early phase of ocular angiogenesis. Human retinal (HRet) cells and bovine endothelial cells (ECs) were cocultured on laminin-coated Cytodex-3 microcarrier beads in an HRB for 1-36 days. Endothelial cells grown alone in the HRB remained cuboidal and were well differentiated. However, when HRet cells were cocultured with ECs, cordlike structures formed as early as 18-36 h and were positive for von Willebrand factor. In addition to the formation of cords and capillary-like structures, ECs showed the beginning of sprouts. The HRB seems not only to promote accelerated capillary formation, but also to enhance differentiation of retinal precursor cells. This leads to the formation of rosette-like structures (which may be aggregates of photoreceptors that were positive for rhodopsin). Upregulation of vascular endothelial growth factor and basic fibroblast growth factor was seen in retinal cells grown in the HRB as compared with monolayers and could be one of the factors responsible for accelerated capillary formation. Hence, the HRB promotes three-dimensional assembly and differentiation, possibly through promoting cell-to-cell interaction and/or secretion of growth and differentiation factors.

Co-culture of Retinal and Endothelial Cells Results in the Modulation of Genes Critical to Retinal Neovascularization

BackgroundNeovascularization (angiogenesis) is a multistep process, controlled by opposing regulatory factors, which plays a crucial role in several ocular diseases. It often results in vitreous hemorrhage, retinal detachment, neovascularization glaucoma and subsequent vision loss. Hypoxia is considered to be one of the key factors to trigger angiogenesis by inducing angiogenic factors (like VEGF) and their receptors mediated by hypoxia inducible factor-1 (HIF-1α) a critical transcriptional factor. Another factor, nuclear factor kappa B (NFκB) also regulates many of the genes required for neovascularization, and can also be activated by hypoxia. The aim of this study was to elucidate the mechanism of interaction between HRPC and HUVEC that modulates a neovascularization response.MethodsHuman retinal progenitor cells (HRPC) and human umbilical vein endothelial cells (HUVEC) were cultured/co-cultured under normoxia (control) (20% O2) or hypoxia (1% O2) condition for 24 hr. Controls were monolayer cultures of each cell type maintained alone. We examined the secretion of VEGF by ELISA and influence of conditioned media on blood vessel growth (capillary-like structures) via an angiogenesis assay. Total RNA and protein were extracted from the HRPC and HUVEC (cultured and co-cultured) and analyzed for the expression of VEGF, VEGFR-2, NFκB and HIF-1α by RT-PCR and Western blotting. The cellular localization of NFκB and HIF-1α were studied by immunofluorescence and Western blotting.ResultsWe found that hypoxia increased exogenous VEGF expression 4-fold in HRPC with a further 2-fold increase when cultured with HUVEC. Additionally, we found that hypoxia induced the expression of the VEGF receptor (VEGFR-2) for HRPC co-cultured with HUVEC. Hypoxia treatment significantly enhanced (8- to 10-fold higher than normoxia controls) VEGF secretion into media whether cells were cultured alone or in a co-culture. Also, hypoxia was found to result in a 3- and 2-fold increase in NFκB and HIF-1α mRNA expression by HRPC and a 4- and 6-fold increase in NFκB and HIF-1α protein by co-cultures, whether non-contacting or contacting.Treatment of HRPC cells with hypoxic HUVEC-CM activated and promoted the translocation of NFκB and HIF-1α to the nuclear compartment. This finding was subsequently confirmed by finding that hypoxic HUVEC-CM resulted in higher expression of NFκB and HIF-1α in the nuclear fraction of HRPC and corresponding decrease in cytoplasmic NFκB and HIF-1α. Lastly, hypoxic conditioned media induced a greater formation of capillary-like structures (angiogenic response) compared to control conditioned media. This effect was attenuated by exogenous anti-human VEGF antibody, suggesting that VEGF was the primary factor in the hypoxic conditioned media responsible for the angiogenic response.ConclusionsThese findings suggest that intercellular communications between HRPC and HUVEC lead to the modulation of expression of transcription factors associated with the production of pro-angiogenic factors under hypoxic conditions, which are necessary for an enhanced neovascular response. Our data suggest that the hypoxia treatment results in the up-regulation of both mRNA and protein expression for VEGF and VEGFR-2 through the translocation of NFκB and HIF-1α into the nucleus, and results in enhanced HRPC-induced neovascularization. Hence, a better understanding of the underlying mechanism for these interactions might open perspectives for future retinal neovascularization therapy.

Possible Mechanisms of Salt-Induced Hypertension in Dahl Salt-Sensitive Rats

Genetic factors, diet, and salt sensitivity have all been implicated in hypertension. To further understand the mechanisms involved in salt-induced hypertension, cardiovascular, hemodynamics, and biochemical parameters in Dahl salt-sensitive rats were evaluated in animals on high- and low-sodium diets. During a 4-week treatment period, blood pressure was significantly elevated in the high (8.0%) salt group compared to the low (0.3%) salt group (p< or =0.05 for weeks 2 and 4, respectively). No significant changes were observed in heart rate. The increase in blood pressure was associated with significant increases in lower abdominal aortic and renal vascular resistance, along with a reduction in blood flow. A fourfold increase in arginine vasopressin was observed in animals on the high-salt diet. In contrast, there was no effect on plasma sodium, potassium, or aldosterone levels during the treatment period. As measured in isolated aortic rings, the high-salt diet also caused a significant elevation in stimulated norepinephrine release and a reduction in cyclic GMP levels. These data suggest that salt-induced elevation in blood pressure is due to activation of both the sympathetic and arginine vasopressin systems via mechanisms involving decreased cyclic GMP generation in vascular smooth muscle.

Influence of low density lipoproteins on vascular smooth muscle cell growth and motility: Modulation by extracellular matrix

Low density lipoproteins (LDL) are thought to play a major role in cardiovascular diseases such as atherosclerosis. Much remains to be done to understand the cellular effects of LDL and how the extracellular matrix (ECM) influences these effects. We found that LDL produced a dose dependent increase in vascular smooth muscle cell (SMC) proliferation. The ECM altered the proliferative response of SMC to LDL: on collagen I there was a 66% inhibition, endothelial cell derived-ECM a 2-fold increase, and collagen IV no difference in proliferation compared to paired controls. LDL affected SMC motility (cell area and shape factor) but the extent and direction of the effect depended on whether the cells were cultured on uncoated or coated dishes. LDL treated cultures had a 5-fold lower migration rate but net movement was not different, suggesting that LDL decreased SMC random movement. There was a dose-dependent accumulation of lipid by SMC incubated with LDL and, subsequently, cytoplasmic lipid droplets were observed. Cells cultured on uncoated plates showed an increased cholesterol content as a function of LDL concentration. In contrast, cells cultured on a collagen IV matrix showed no net change in cholesterol content over the range of LDL concentrations studied. Hence, the uptake of LDL cholesterol appears to be completely inhibited by this matrix. These studies indicate that the influence of LDL on several SMC parameters is modulated by ECM components.

Enhanced Nitric Oxide Synthesis Reverses Salt-Induced Alterations in Blood Flow and Cgmp Levels

To understand the role of nitric oxide in salt-induced hypertension, we evaluated cardiovascular, hemodynamic and biochemical parameters in Dahl salt-sensitive rats fed low (0.3%) and high (8.0%) sodium diets. Two high salt groups received 1.25 and 2.5 g/L l-arginine in their drinking water. After three weeks of treatment, blood pressure was greater in the high salt groups. l-arginine did not modify salt-induced hypertension. Eicosapentaenoic acid (EPA) caused a smaller depressor response compared to normotensive rats. The increase in blood pressure was associated with decreases in aortic and renal blood flows. In renal artery, the reduction was counteracted by both l-arginine doses; whereas in the aorta, only the higher l-arginine one restored blood flow. The salt-induced reduction in aortic cyclic GMP level was only overcome by the higher l-arginine treatment. These data suggest that at the dose levels tested, nitric oxide reverses the reduction in cGMP and blood flow, but not the blood pressure changes associated with salt-induced hypertension.

Abstract 5643: Role of leptin-induced Notch-Wnt crosstalk in proliferation/survival of triple negative breast cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Obese African American and Latina women show the high mortality rate linked to triple negative breast cancer, TNBC. The disease is commonly detected at more advance degree and associated with resistance to common therapeutic treatments. Obesity, characterized by high levels of leptin, has consistently been associated with many different cancers including breast cancer. We hypothesize that leptin signaling is instrumental in the increased proliferation and survival of breast cancer cells, which is mediated through a crosstalk between the Notch and Wnt signaling pathways. We further hypothesized that these leptin actions are more evident in TNBC cells. Objective/Methods: The effects of leptin-Notch-Wnt crosstalk on the proliferation and survival (MTT-Vybrant assay and Annexin V-flow cytometry) of breast cancer cells responsive to estrogen (MCF-7 and T47D) and TNBC (MDA-MB231 and HC1806) were investigated. The cells were treated with agonists and inhibitors of leptin-Notch-Wnt axis [i.e., leptin, Wnt1, JAG1/14-mer peptide and leptin peptide receptor antagonist 2 (LPrA2), g-secretase inhibitor and Wif]. The expression of Notch and Wnt signaling components (ligands/receptors) and levels of cyclin D1 and myc were also determined via western blot and real-time RT-PCR. In a series of experiments the cells were treated with chemotherapeutic drugs, doxorubicin and cisplatin plus leptin and the activation of caspase 3, levels of Bcl-2 and, the drug resistance marker (multidrug resistance-associated protein 1, ABCC1/MRP1) were determined using as control the etoposide resistant MCF-7/VP cell line. The relative expression of leptin, Notch and Wnt systems in breast cancer tissue arrays (n=150; Pantomics) was also investigated. Results: Leptin induced significant increase of TNBC cell proliferation, cyclin D1, bcl-2, ABCC1 and Notch/Wnt axis. Leptin also decreased the anti-cancer effects of doxorubicin and cisplatin, which were related to leptin upregulation on Notch-Wnt crosstalk. HScore analyses of breast cancer tissue arrays showed significant correlation of the expression of leptin-targeted proteins (Notch and Wnt) in breast cancer tissues. These results suggest that leptin can induce the crosstalk between Notch and Wnt and breast cancer progression. Conclusions: Targeting leptin-Notch-Wnt signaling axis could be a new strategy to treat and/or prevent breast cancer. The inhibition of leptin signaling could be a promising new way to target the most deadly TBNC, especially in obese patients. Acknowledgments: This work was supported by NIH/NCI 5SC1CA138658-04, Georgia Cancer Coalition Distinguished Cancer Scholar Award (to RRGP), NIH/2G12RR003034-26 and U54 MSM/TU/UAB Cancer Center Partnership, and NIH/NIGMS R25 GM058268. This research was also supported, in part, facilities and support services at Morehouse School of Medicine (NIH 1C06 RR18386). Citation Format: Tanisha Z. McGlothen, Corey Gillespie, Krysta Greenleaf, Movado Long, Laronna S. Colbert, Gary L. Sanford, Ruben R. Gonzalez-Perez. Role of leptin-induced Notch-Wnt crosstalk in proliferation/survival of triple negative breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5643. doi:10.1158/1538-7445.AM2013-5643