Effect of staurosporine on the mobility and invasiveness of lung adenocarcinoma A549 cells: an in vitro study

<p>Abstract</p> <p>Background</p> <p>Lung cancer is one of the most malignant tumors, representing a significant threat to human health. Lung cancer patients often exhibit tumor cell invasion and metastasis before diagnosis which often render current treatments ineffective. Here, we investigated the effect of staurosporine, a potent protein kinase C (PKC) inhibitor on the mobility and invasiveness of human lung adenocarcinoma A549 cells.</p> <p>Methods</p> <p>All experiments were conducted using human lung adenocarcinoma A549 cells that were either untreated or treated with 1 nmol/L, 10 nmol/L, or 100 nmol/L staurosporine. Electron microscopy analyses were performed to study ultrastructural differences between untreated A549 cells and A549 cells treated with staurosporine. The effect of staurosporine on the mobility and invasiveness of A549 was tested using Transwell chambers. Western blot analyses were performed to study the effect of staurosporine on the levels of PKC-α, integrin β1, E-cadherin, and LnR. Changes in MMP-9 and uPA levels were identified by fluorescence microscopy.</p> <p>Results</p> <p>We demonstrated that treatment of A549 cells with staurosporine caused alterations in the cell shape and morphology. Untreated cells were primarily short spindle- and triangle-shaped in contrast to staurosporine treated cells which were retracted and round-shaped. The latter showed signs of apoptosis, including vacuole fragmentation, chromatin degeneration, and a decrease in the number of microvilli at the surface of the cells. The A549 cell adhesion, mobility, and invasiveness significantly decreased with higher staurosporine concentrations. E-cadherin, integrin β1, and LnR levels changed by a factor of 1.5, 0.74, and 0.73, respectively compared to untreated cells. In addition, the levels of MMP-9 and uPA decreased in cells treated with staurosporine.</p> <p>Conclusion</p> <p>In summary, this study demonstrates that staurosporine inhibits cell adhesion, mobility, and invasion of A549 cells. The staurosporine-mediated inhibition of PKC-α, induction of E-Cad expression, and decreased integrin β1, LnR, MMP-9, and uPA levels could all possibly contribute to this biological process. These results represent a significant step forward in the ongoing effort to understand the development of lung carcinoma and to design novel strategies to inhibit metastasis of the tumor by targeting the cell-adhesion, mobility and invasion of tumor cells.</p

Translational Stroke Research Using a Rabbit Embolic Stroke Model: A Correlative Analysis Hypothesis for Novel Therapy Development

Alteplase (tissue plasminogen activator, tPA) is currently the only FDA-approved treatment that can be given to acute ischemic stroke (AIS) patients if patients present within 3 h of an ischemic stroke. After 14 years of alteplase clinical research, evidence now suggests that the therapeutic treatment window can be expanded 4.5 h, but this is not formally approved by the FDA. Even though there remains a significant risk of intracerebral hemorrhage associated with alteplase administration, there is an increased chance of favorable outcome with tPA treatment. Over the last 30 years, the use of preclinical models has assisted with the search for new effective treatments for stroke, but there has been difficulty with the translation of efficacy from animals to humans. Current research focuses on the development of new and potentially useful thrombolytics, neuroprotective agents, and devices which are also being tested for efficacy in preclinical and clinical trials. One model in particular, the rabbit small clot embolic stroke model (RSCEM) which was developed to test tPA for efficacy, remains the only preclinical model used to gain FDA approval of a therapeutic for stroke. Correlative analyses from existing preclinical translational studies and clinical trials indicate that there is a therapeutic window ratio (ARR) of 2.43-3 between the RSCEM and AIS patients. In conclusion, the RSCEM can be used as an effective translational tool to gauge the clinical potential of new treatments

Nicotine-induced membrane perturbation of intact human granulocytes spin-labeled with 5-doxylstearic acid. Correlation with chemotaxis.

The effects of nicotine on intact human granulocytes were examined, using 5-doxylstearic acid as a spin probe. At micromolar concentrations, (-)-nitocine produces a membrane perturbation in granulocytes not observable with oriented lipid bilayers. The effect, which is stereoselective for the (-)-isomer, occurs at concentrations of nicotine that bind to noncholinergic nicotine receptors on granulocytes and which are present in the blood after smoking. At comparable concentrations, (-)-nicotine modulates granulocyte chemotaxis towards a chemotactic peptide in a stereospecific and dose-dependent manner. Cotinine, the major metabolite of nicotine, does not bind to the receptor, does not produce the membrane perturbation observed with nicotine, and has no effect on chemotaxis. These results suggest that (-)-nicotine present in the blood after smoking binds to a receptor on granulocytes, perturbs granulocyte membranes and modulates chemotaxis

Validation of a Total Score for the Critical Care Famuly Satisfaction Survey.

Objective: To establish the validity of a total score for the Critical Care Family Satisfaction Survey (CCFSS). Design: Instrument validation study. Setting and participants: 2494 family members of patients admitted to 10 critical care units over a 3-year period. Methods: A total score was computed by summing the 5 subscales of the survey, providing a score in the range of 5 to 25. Total score validation techniques included comparison of a model developed from the recently collected survey data with the model developed for initial validation of the survey (n = 145). Goodness-of-fit analyses included calculation and comparison of internal consistency estimates (Cronbach’s alpha) from both models using Fisher’s r-to-z transformations as well as construct validation using r-to-z transformation and comparisons of standardized regression weights from both models. A scree plot was generated to demonstrate the similarity between the model’s eigenstructures, and a chi-square goodness-of-fit analysis was performed to compare the percentage of variance explained by each model. Results: Internal consistency estimates from the 2 models were not significantly different (z score P values \u3e 0.10). Statistical differences between standardized regression weights were observed, and these were caused by the large sample size in this study. However, the patterns of differences between these weights across the 4- and 5-construct model were nearly identical. Goodness-of-fit analysis of the percentage of variance explained by each model demonstrated agreement of the underlying eigenstructure of the models (P = 0.80). Conclusion: Comparison of the models showed significant agreement and no meaningful differences, demonstrating the validity of a total score for the CCFSS. Evidence of a single latent construct being estimated from both models provides additional support for the computation of a total score

The Effect of Body Mass Index on Patient Outcomes in a Medical ICU.

STUDY OBJECTIVES: To examine the effect of patient body mass index (BMI) on outcome in intensive care. DESIGN: In a prospective study, the patients were classified into groups based on the calculated BMI, as follows: BMI \u3c 19.0 (n = 350), \u3e or = 19.0 and \u3c 25.0 (n = 663), \u3e or = 25.0 and \u3c 29.9 (n = 585), \u3e or = 30.0 and \u3c 40.0 (n = 396), and \u3e or = 40.0 (n = 154). Groups were compared by age, APACHE (acute physiology and chronic health evaluation) II score, mortality, ICU length of stay (LOS), hospital LOS, number receiving ventilation, and ventilator-days. Adverse events including nosocomial pneumonia, ventilator-days per patient, failed extubations, and line-related complications were recorded. SETTING: The study was conducted in a 9-bed medical ICU of a 650-bed tertiary care hospital. MEASUREMENTS: Height and weight were prospectively recorded for the first ICU admission during a hospital stay. RESULTS: Between January 1, 1997, and August 1, 2001, 2,148 of 2,806 patients admitted to the ICU had height and weight recorded. There were no differences in APACHE II score, mortality, ICU LOS, hospital LOS, number receiving ventilation, ventilator-days, average total cost, or average variable cost among the five groups. However, the severely obese patients were more frequently female and younger than those who were overweight and obese (p \u3c 0.001). Adverse events were infrequent, but there were no differences between the obese/very obese compared with others. CONCLUSION: BMI has minimal effects on ICU outcome after patients are admitted to a critical care unit

Characterization of noncholinergic nicotine receptors on human granulocytes.

The noncholinergic nicotine receptor on leukocytes identified earlier [Davies et al., Molec. Cell. Biochem. 44, 23 (1982)] was further characterized. Structure-activity relationships showed that a pyrrolidine ring containing a basic N atom is an important structural feature for ligands that bind to the receptor. Accordingly, the carcinogenic component of tobacco smoke, N-nitrosonornicotine, does not bind to the receptor. The stereoselectivity for the d-isomer, which was confirmed using [3H]d-nicotine as a ligand, together with the absolute configurational relationship between d-nicotine and L-proline, suggested that basic peptides containing proline as the N-terminal amino acid would bind to the receptor. The finding that Pro-Lys-Pro-Arg, which has been reported to inhibit granulocyte phagocytosis, bound to the receptor with an IC50 value of 3.5 microM is compatible with this idea. An increase in receptor binding, which was observed in the presence of plasma, could be ascribed to bicarbonate. The presence of bicarbonate in the binding assay, even when the pH of the buffer was carefully controlled, resulted in an increase (approximately 2-fold) in the apparent number of receptors without affecting the Kd value significantly. Increasing the pH of the buffer in the absence of bicarbonate also increased receptor binding, suggesting that bicarbonate may increase receptor binding by its known ability to increase intracellular pH at constant extracellular pH. Preincubation of cells with d-nicotine under certain conditions reduced the subsequent binding of [3H]d-nicotine to the receptor

Predicting mortality after intracerebral hemorrhage: comparison of scoring systems and influence of withdrawal of care.

OBJECTIVE: The Broderick system and the intracerebral hemorrhage (ICH) score are two systems for predicting 30-day mortality in patients with spontaneous ICH. No previous study has compared two ICH scoring systems in an independent patient cohort. Our purpose was to externally validate and directly compare these two systems and evaluate the effect of withdrawal of care on system performance. METHODS: In all, 307 consecutive patients admitted with ICH between 1998 and 2002 were evaluated. Broderick exclusion criteria were used, resulting in a cohort of 241 patients. Admission Glasgow Coma Scale score, ICH volume, 30-day mortality, and day-30 location were collected. The sensitivity, specificity, receiver operator characteristic curves, and model explained variance (R2) of the two systems were directly compared. The statistical performances of both systems were then compared in subsets that included or excluded patients from whom care was withdrawn. RESULTS: Overall mortality was 76 of 241 (31.5%). The ICH score had significantly higher sensitivity (66% v 45%, P = .001) and higher receiver operator characteristic curves (0.814 v 0.773, P \u3c .001) for predicting 30-day mortality. The model R2 and specificity were not significantly different between systems. Both systems were significantly associated with 30-day location stratified as home, acute rehabilitation, skilled nursing facility, or death (ICH score Chi square = 79.28, P \u3c .001; Broderick Chi square = 60.63, P \u3c .001). Inclusion or exclusion of patients who had supportive care withdrawn did not significantly affect overall statistical performance. CONCLUSIONS: The ICH score performed significantly better than the Broderick system. Overall statistical performance of both systems was not influenced by withdrawal of care

Establishing reliability and validity of the critical care family satisfaction survey.

OBJECTIVE: To develop and validate the Critical Care Family Satisfaction Survey as a proxy for patient satisfaction. DESIGN: Instrument validation study. SETTING AND TIME FRAME: The Medical Intensive Care, Shock Trauma, Acute Coronary Care, Central Nervous System, Surgical Intensive Care, and Special Care units of Lehigh Valley Hospital (Allentown, PA), for the period December 1997 through September 1998. PATIENTS/PARTICIPANTS: One family member for each of 237 critical care patients. INTERVENTION(S): Content and construct validity were examined on 37 items and 6 constructs thought to measure family satisfaction with the quality of critical care in hospitals. Initially, 14 items and 1 construct were removed from the questionnaire based on this analysis. It was then administered to 237 family members. MEASUREMENTS AND MAIN RESULTS: Factor analysis and confirmatory factor analysis using path models were performed. Internal consistency using Pearson correlations and Cronbach\u27s alpha, and discriminant validation were also calculated. Factor analysis yielded a single eigenvalue \u3e1 (3.712), whereas confirmatory factor analysis led to the final instrument being reduced to 20 items and 5 subscale constructs. One subscale ( Comfort ) performed poorly, indicating the possible need for a four-factor model. Subsequently, internal consistency assessed by Cronbach\u27s alpha was 0.9101 for the five-factor model and 0.9327 for the four-factor model. Subscale correlations were no lower than 0.750 for the five-factor model and 0.856 for the four-factor model. CONCLUSIONS: This study provides support that the Critical Care Family Satisfaction Survey-which yields five subscales, Assurance, Information, Proximity, Support, and Comfort --is reliable and valid. Using five constructs rather than four is recommended because of the following: a) the internal consistency loss of 0.0226 for the Comfort subscale is not enough to warrant its removal, b) a four-factor questionnaire can be administered and totaled independently of this subscale, c) the need for the fifth construct is indicated by this study\u27s results, and d) including the extra data may allow for more detailed analysis