Curcumin Sensitizes Silymarin to Exert Synergistic Anticancer Activity in Colon Cancer Cells.

We studied combinatorial interactions of two phytochemicals, curcumin and silymarin, in their action against cancer cell proliferation. Curcumin is the major component of the spice turmeric. Silymarin is a bioactive component of milk thistle used as a protective supplement against liver disease. We studied antiproliferative effects of curcumin alone, silymarin alone and combinations of curcumin and silymarin using colon cancer cell lines (DLD-1, HCT116, LoVo). Curcumin inhibited colon cancer cell proliferation in a concentration-dependent manner, whereas silymarin showed significant inhibition only at the highest concentrations assessed. We found synergistic effects when colon cancer cells were treated with curcumin and silymarin together. The combination treatment led to inhibition of colon cancer cell proliferation and increased apoptosis compared to single compound treated cells. Combination treated cells exhibited marked cell rounding and membrane blebbing of apoptotic cells. Curcumin treated cells showed 3-fold more caspase3/7 activity whereas combination treated cells showed 5-fold more activity compared to control and silymarin treated cells. When DLD-1 cells were pre-exposed to curcumin, followed by treatment with silymarin, the cells underwent a high amount of cell death. The pre-exposure studies indicated curcumin sensitization of silymarin effect. Our results indicate that combinatorial treatments using phytochemicals are effective against colorectal cancer.

Whole beetroot consumption acutely improves running performance.

Nitrate ingestion improves exercise performance; however, it has also been linked to adverse health effects, except when consumed in the form of vegetables. The purpose of this study was to determine, in a double-blind crossover study, whether whole beetroot consumption, as a means for increasing nitrate intake, improves endurance exercise performance. Eleven recreationally fit men and women were studied in a double-blind placebo controlled crossover trial performed in 2010. Participants underwent two 5-km treadmill time trials in random sequence, once 75 minutes after consuming baked beetroot (200 g with ≥500 mg nitrate) and once 75 minutes after consuming cranberry relish as a eucaloric placebo. Based on paired t tests, mean running velocity during the 5-km run tended to be faster after beetroot consumption (12.3±2.7 vs 11.9±2.6 km/hour; P=0.06). During the last 1.1 miles (1.8 km) of the 5-km run, running velocity was 5% faster (12.7±3.0 vs 12.1±2.8 km/hour; P=0.02) in the beetroot trial, with no differences in velocity (P≥0.25) in the earlier portions of the 5-km run. No differences in exercise heart rate were observed between trials; however, at 1.8 km into the 5-km run, rating of perceived exertion was lower with beetroot (13.0±2.1 vs 13.7±1.9; P=0.04). Consumption of nitrate-rich, whole beetroot improves running performance in healthy adults. Because whole vegetables have been shown to have health benefits, whereas nitrates from other sources may have detrimental health effects, it would be prudent for individuals seeking performance benefits to obtain nitrates from whole vegetables, such as beetroot.

Students as vital participants in research projects.

Inclusion of research coursework into a medical technology or clinical laboratory science program is currently viewed as a mark of a good degree program. Examples of this type of coursework are evaluation of scientific papers, techniques of scientific writing, application and performance of statistical analysis and introduction to research ethics (e.g., Institutional Review Board approval process). While many programs have the ability to recruit experienced scientists into research mentorship of medical technology/clinical laboratory science students, it is recognized that not all programs have this ability. It is also recognized that clinical laboratorians are performing critical diagnostic tests and, in this capacity, have the ability to identify research projects that are necessary, evidence-based and timely. It is hereby proposed that clinical laboratorians take advantage of this innate ability and create rich teaching experiences for students by including them in performance of research projects. Because of the fact that students are armed with up-to-date knowledge, have willing and enthusiastic spirits and are highly motivated to learn, they are vital participants in research. The students receive an invaluable active learning experience and possibly a future job; the clinical laboratorians meet and possibly exceed the research and scholarship expectations of their institutions; and the scientific community benefits by the science being shared through publication in scientific journals.

Research in the Medical Laboratory Science curriculum.

Medical Laboratory Science (MLS) is increasing its numbers of advanced degree offerings and is accelerating involvement in evidence-based practice. Expectations of MLS faculty have increased to include research and scholarship. Many MLS programs have undergraduate requirements for research. To assist MLS faculty as they increase research productivity, information is provided to guide professionals and students striving to expand their research achievements.

C-reactive protein levels are elevated in asthma and asthma-like conditions.

OBJECTIVE: Asthma is a chronic disease involving airway hyperresponsiveness. It was proposed that asthma/chronic shortness of breath elicit chronic systemic inflammation even in the absence of episodic events. DESIGN: Volunteers completed questionnaires relevant to asthma and/or dyspnea and consented to C-reactive protein (CRP) quantitation. Subject groups were: control (no reported dyspnea) and asthma/shortness of breath [dyspnea] group. SETTING: Studies were performed in the Saint Louis metropolitan area. PARTICIPANTS: Participants consisted of volunteers aged 18-57. Inclusion criteria were good health, not pregnant, weight > 110 pounds and absence of antiinflammatory medicine use. RESULTS: Serum CRP ranged from undetectable to 22,013 ng/mL. Mean results for asthmatic/dyspnea (n = 22) and control (n = 27) groups were 4,203 +/- 1,323 ng/mL and 1,741 +/- 467 ng/mL (p < 0.05) respectively. CONCLUSION: Individuals with asthma/asthma-like symptoms have chronic low levels of systemic inflammation despite the absence of episodic pulmonary events. Understanding chronic systemic inflammation relevant to asthma/asthma-like conditions may lead to design of targeted therapeutics.

Growth inhibition of human colon cancer cells by plant compounds.

OBJECTIVE: Evidence is accumulating that compounds of plant origin (phytochemicals) exert anti-cancer effects. The purpose of this study was to determine if resveratrol, cinnamaldehyde, and piperine (from red grapes, cinnamon, black pepper respectively) have anti-proliferative effects on colon cancer. DESIGN: Quantitative effects of each phytochemical on concentration responses and time courses of proliferation of cultured human colon cancer cells (DLD-1) were assessed. SETTING: Research was performed at Saint Louis University. MAIN OUTCOMES MEASURES: Responses were measured by spectrophotometry of surviving cells stained by a dye method. RESULTS: Phytochemicals displayed anti-proliferative effects on DLD-1 cells in concentration- and kinetic-dependent manners. Cinnamaldehyde offered statistically significant effects at 24 hours [200 microM], 48 hours [100 - 200 microM], and 72 hours [200 microM]. Piperine displayed a trend towards anti-proliferation at 24 hours and statistically significant inhibition at 48 and 72 hours [100 - 200 microM]. Resveratrol displayed significant anti-proliferative effects at 24 hours [50-200 microM], 48 hours [10-200 microM], and 72 hours [10-200 microM]. CONCLUSION: Cinnamaldehyde, piperine, and resveratrol offer significant in vitro anti-proliferative effects on cultured human colon cancer cells. While each phytochemical exhibited significant anti-proliferative effects, resveratrol results were most impressive in that lower concentrations administered at regular intervals were significantly effective. These results taken together with everyday dietary availability of concentrations used in this study strongly suggest that regular intake of low doses of these phytochemicals offer preventive effects against colon cancer.

Single embryoid body formation in a multi-well plate

Embryoid bodies (EB) formed from murine embryonic stem (ES) cells recapitulate many aspects of a developing embryo. Of specific importance, synchronous differentiation of EB recapitulates organ-specific development and is achieved in culture by formation of uniformly sized EB. The method described here demonstrates a simple and cost-effective way of generating EB from murine ES cells. Single EB are formed in a multi-well plate format and large numbers of EB are generated using a 96-well multi-well plate. Uniform single-sized EB formed in the multi-well are an ideal system for screening compounds and determining differentiation effects. Since EB contain all three germ layers, they are appropriate for studying small molecule effects on differentiation of ES such as is performed in high-throughput screening protocols

Blockade of p38 map kinase inhibits complement-induced acute lung injury in a murine model.

Features of acute lung injury include neutrophil influx and increased vascular permeability with resultant pulmonary edema. Inhibition of p38 mitogen-activated protein kinase (MAPK) in in vivo models of endotoxin-induced inflammation results in reduction of organ injury as well as symptomatic relief. In this study, mice received an oral dose (100 mg/kg) of the p38 MAPK inhibitor, SB203580, followed by intratracheal instillation of an agent of complement origin, C5a des arg, at a concentration (10 microg) that induced acute lung injury. Neutrophil and protein content of bronchoalveolar lavage fluid as indicators of leukocyte influx and vascular permeability respectively were assessed. Animals that received C5a-instillation had a significant influx of neutrophils into the lungs (49+/-8%) while mice receiving C5a-instillation and prior treatment with SB203580 exhibited diminished influx (16+/-5%). Similarly, pretreatment with oral SB203580 resulted in decreased vascular permeability (241+/-34 microg/ml) than the positive control animals (407+/-135 microg/ml). Activity analysis of total lung p38 MAPK revealed that p38 activity was increased at 4 h after C5a-instillation and that SB203580-treated C5a-instilled mouse lungs had lower p38 activity than did the C5a-instilled control. These data indicate that oral administration of an agent inhibitory for p38 MAPK offers a protective effect in the lungs from both neutrophil influx and protein leak associated with acute lung injury.

Native and modified C-reactive protein bind different receptors on human neutrophils.

Native C-reactive protein (CRP) is a planar pentamer of identical subunits expressed at high serum levels during the acute phase of inflammation. At inflammatory sites, an isomeric form termed modified CRP (mCRP) is expressed and reveals neoantigenic epitopes associated with modified monomeric CRP subunits. mCRP cannot assume the native pentameric conformation but rather forms a nonpentameric aggregate of monomers. While native CRP inhibits neutrophil movement in vitro and in vivo, the effect of mCRP on neutrophil movement has not been reported. To model the biological function and biochemical interaction of mCRP on neutrophils, in vitro chemotaxis and binding experiments were performed using mCRP. Reported here, mCRP effectively inhibited fMLP-induced chemotaxis similar to native CRP. Additionally, mCRP increased binding of labeled native CRP to neutrophils. This increased binding occurred by direct protein-protein interaction of the C-terminus thereby implicating the CRP(199-206) sequence. Binding of mCRP to neutrophils was blocked by anti-CD16 monoclonal antibody whereas native CRP was not. These results suggest that modified forms of CRP inhibit chemotaxis, a function similar to native CRP, but that mCRP and native molecule bind different receptors on human neutrophils.

C-reactive protein increases F-actin assembly and cortical distribution with resultant loss of lamellipod formation in human neutrophils.

C-reactive protein (CRP) inhibits neutrophil movement through a p38 MAP kinase pathway. We hypothesized that CRP altered F-actin content and distribution on human neutrophils as a means of inhibiting movement. CRP produced simultaneous increased F-actin and decreased G-actin levels. CRP increased F-actin levels in a concentration-dependent manner once a threshold (>100 microg/ml) was reached, and transiently increased F-actin (peak levels at 2.5 and 10 min) that returned to baseline by 30 min. Confocal microscopy of neutrophils revealed that fMLP provoked acquisition of a migratory phenotype as evidenced by the appearance of F-actin rich lamellipods. In contrast, CRP caused neutrophil rounding, prevented lamellipod formation and shifted F-actin from the cytoskeleton to the cortex. The p38 MAP kinase inhibitor, SB203580, produced a similar effect on neutrophil shape. Concentrations of SB203580 that dramatically decreased p38 activity in neutrophils also caused round cell morphology and cortical F-actin distribution. Since CRP inhibits p38 MAP kinase and p38 blockade leads to actin polymerization and prevention of lamellipod formation, it is concluded that round morphology and loss of lamellipod formation result from CRP inhibition of p38 MAP kinase. Understanding the signal transduction of CRP prevention of lamellipod formation will aid in the development of therapeutic agents against neutrophil-associated inflammatory disease.

Eosinophil peroxidase-derived reactive brominating species target the vinyl ether bond of plasmalogens generating a novel chemoattractant, alpha-bromo fatty aldehyde.

Plasmalogens are a subclass of glycerophospholipids that are enriched in the plasma membrane of many mammalian cells. The vinyl ether bond of plasmalogens renders them susceptible to oxidation. Accordingly, it was hypothesized that reactive brominating species, a unique oxidant formed at the sites of eosinophil activation, such as in asthma, might selectively target plasmalogens for oxidation. Here we show that reactive brominating species produced by the eosinophil peroxidase system of activated eosinophils attack the vinyl ether bond of plasmalogens. Reactive brominating species produced by eosinophil peroxidase target the vinyl ether bond of plasmalogens resulting in the production of a neutral lipid and lysophosphatidylcholine. Chromatographic and mass spectrometric analyses of this neutral lipid demonstrated that it was 2-bromohexadecanal (2-BrHDA). Reactive brominating species produced by eosinophil peroxidase attacked the plasmalogen vinyl ether bond at acidic pH. Bromide was the preferred substrate for eosinophil peroxidase, and chloride was not appreciably used even at a 1000-fold molar excess. Furthermore, 2-BrHDA production elicited by eosinophil peroxidase-derived reactive brominating species in the presence of 100 microM NaBr doubled with the addition of 100 mM NaCl. The potential physiological significance of this pathway was suggested by the demonstration that 2-BrHDA was produced by phorbol myristate acetate-stimulated eosinophils and by the demonstration that 2-BrHDA is a phagocyte chemoattractant. Taken together, the present studies demonstrate the targeting of the vinyl ether bond of plasmalogens by the reactive brominating species produced by eosinophil peroxidase and by activated eosinophils, resulting in the production of brominated fatty aldehydes.