Endothelial lincRNA-p21 alleviates cerebral ischemia/reperfusion injury by maintaining blood-brain barrier integrity.

Blood-brain barrier (BBB) disruption is increasingly recognized as an early contributor to the pathophysiology of cerebral ischemia/reperfusion (I/R) injury, and is also a key event in triggering secondary damage to the central nervous system. Recently, long non-coding RNA (lncRNA) have been found to be associated with ischemic stroke. However, the roles of lncRNA in BBB homeostasis remain largely unknown. Here, we report that long intergenic non-coding RNA-p21 (lincRNA-p21) was the most significantly down-regulated lncRNA in human brain microvascular endothelial cells (HBMECs) after oxygen and glucose deprivation/reoxygenation (OGD/R) treatment among candidate lncRNA, which were both sensitive to hypoxia and involved in atherosclerosis. Exogenous brain-endothelium-specific overexpression of lincRNA-p21 could alleviate BBB disruption, diminish infarction volume and attenuate motor function deficits in middle cerebral artery occlusion/reperfusion (MCAO/R) mice. Further results showed that lincRNA-p21 was critical to maintain BBB integrity by inhibiting the degradation of junction proteins under MCAO/R and OGD/R conditions. Specifically, lincRNA-p21 could inhibit autophagy-dependent degradation of occludin by activating PI3K/AKT/mTOR signaling pathway. Besides, lincRNA-p21 could inhibit VE-cadherin degradation by binding with miR-101-3p. Together, we identify that lincRNA-p21 is critical for BBB integrity maintenance, and endothelial lincRNA-p21 overexpression could alleviate cerebral I/R injury in mice, pointing to a potential strategy to treat cerebral I/R injury.

Effect of an Online Mindfulness-Based Stress Reduction Intervention on Postpandemic Era Nurses' Subjective Well-being, Job Burnout, and Psychological Adaptation.

This study explores the effects of an online mindfulness-based stress reduction intervention on postpandemic era nurses' subjective well-being, job burnout, and psychological adaptation. Previous studies on the psychological adaptability of nurses mainly focused on investigation rather than intervention. Ninety nurses were randomly classified into an intervention or control group. The intervention group received weekly online mindfulness-based stress reduction training for 8 weeks. The Subjective Well-being, Job Burnout, and Psychological Use scales were administered pre- and postintervention. Postintervention, nurses' positive emotions and life satisfaction significantly improved. Nurses' psychological adaptation was significantly higher postintervention than preintervention. The total scores for negative emotion, low personal accomplishment, and job burnout were significantly lower postintervention than preintervention. The scores for positive emotion and life satisfaction in the intervention group were significantly higher than those in the control group, and the scores for low personal accomplishment in the intervention group were significantly lower than those in the control group. Online mindfulness-based stress reduction interventions can improve nurses' subjective well-being, reduce job burnout, and improve their level of psychological adaptability. Moreover, it could promote nurses' ability to communicate mindfully with patients and their families. This intervention could help promote the development of mindfulness in the nursing field.

Plant endophytic bacteria: A potential resource pool of electroactive micro-organisms.

AIMS: Electroactive micro-organisms play a significant role in microbial fuel cells. It is necessary to discover potential resources in plant endophytes. In this study, plant tissues were selected to isolate endophytic bacteria, and the electrochemical activity potential was evaluated. METHODS AND RESULTS: The microbial fuel cell (MFC) is used to evaluate the electricity-producing activity of endophytic bacteria in plant tissues, and the species distribution of micro-organisms in the anode of the MFC after inoculation of plant tissues is determined by high-throughput sequencing. Twenty-six strains of bacteria were isolated from plant tissues belonging to Angelica and Sweet Potato, of which 17 strains from six genera had electrochemical activity, including Bacillus sp., Pleomorphomonas sp., Rahnella sp., Shinella sp., Paenibacillus sp. and Staphylococcus sp. Moreover, the electricity-producing micro-organisms in the plant tissue are enriched. Pseudomonas and Clostridioides are the dominant genera of MFC anode inoculated with angelica tissue. Staphylococcus and Lachnoclostridium are the dominant genera in MFC anode inoculated with sweet potato tissue. And the most representative Gram-positive strain Staphylococcus succinus subsp. succinus H6 and plant tissue were further analysed for electrochemical activity. And a strain numbered H6 and plant tissue had a good electrogenerating activity. CONCLUSION: This study is of great significance for expanding the resource pool of electricity-producing micro-organisms and tapping the potential of plant endophytes for electricity-producing. SIGNIFICANCE AND IMPACT OF STUDY: This is the first study to apply plant endophytes to MFC to explore the characteristics of electricity production. It is of great significance for exploring the diversity of plant endophytes and the relationship between electricity producing bacteria and plants.

Fungal pathogens causing postharvest fruit rot of wolfberry and inhibitory effect of 2,3-butanedione.

Fungal pathogen contamination is one of the most important factors affecting the postharvest quality and shelf life of wolfberry fruits. Therefore, the prevention and control of fungal pathogens that cause fruit rot has become particularly important. Volatile antifungal agents of biological origin have broad application prospects. They may be safer and more efficient than traditional physical and chemical methods. Four pathogenic fungi were isolated and purified from rotting wolfberry. These pathogenic fungi were determined to be Mucor circinelloides LB1, Fusarium arcuatisporum LB5, Alternaria iridiaustralis LB7, and Colletotrichum fioriniae LB8. In vitro fumigation experiments showed that 2,3-butanedione can effectively inhibit the mycelial growth, spore germination, and sporulation ability of pathogenic fungi. The scanning electron microscope (SEM) showed morphological changes in hyphae. Propidium iodide (PI) Staining and leakage of 260 and 280 nm-absorbing increased, suggesting damage to cell membranes. Furthermore, 2,3-butanedione was found to significantly improve fruit firmness, soluble solid, total phenol, flavonoid, and soluble sugar content, as well as higher SOD enzyme activity and lower PPO and POD enzyme activity in the treated fruit, indicating that 2,3-butanedione can effectively reduce the adverse effects of pathogenic fungi in wolfberry. Based on these results, we conclude that 2,3-butanedione is effective against infection by pathogenic fungi in post-harvest wolfberry. 2,3-butanedione should be considered a viable substitute for conventional fungicides that are currently used to control rot in wolfberry.

Tripartite Motif-Containing 44 is Involved in the Tumorigenesis of Laryngeal Squamous Cell Carcinoma, and its Expression is Downregulated by Nuciferine.

Tripartite motif-containing 44 (TRIM44) was reported to be involved in the tumorigenesis of several tumors, but its function in laryngeal squamous cell carcinoma has not been investigated yet. In the present study, we aimed to elucidate the function of TRIM44 in laryngeal squamous cell carcinoma, and identify the compounds which could inhibit TRIM44 expression. Our results showed that TRIM44 was upregulated in tumor tissues and cell lines of laryngeal squamous cell carcinoma. Knockdown of TRIM44 significantly inhibited cell growth of laryngeal squamous cell carcinoma by suppressing TLR4, phosphorylated AKT and phosphorylated NF-κB p65 expression in vitro. Moreover, TRIM44 knockdown inhibited tumor growth in nude mice, which further suggested that TRIM44 exerted oncogenic activity in laryngeal squamous cell carcinoma. Interestingly, it was found that nuciferine significantly inhibited the mRNA levels of TRIM44 after screening a small natural compound library. Our further studies showed nuciferine markedly downregulated the protein levels of TRIM44 and its substrate TLR4 in a concentration-dependent manner in laryngeal squamous cell carcinoma cells. Moreover, the activation of downstream kinases of TLR4 such as AKT signaling pathway was also inhibited by nuciferine. Additionally, nuciferine markedly inhibited cell survival of laryngeal squamous cell carcinoma in a concentration-dependent manner. In contrast, TRIM44 overexpression significantly reduced the cytotoxicity of nuciferine in laryngeal squamous cell carcinoma cells. In conclusion, this study indicated that inhibiting TRIM44 would be a useful strategy for the treatment of laryngeal squamous cell carcinoma, and nuciferine could be a potential chemical applicated in the therapy of laryngeal squamous cell carcinoma.

The inhibitory effect of volatile organic compounds produced by Bacillus subtilis CL2 on pathogenic fungi of wolfberry.

Bacillus subtilis strain CL2 is antagonistic to wolfberry postharvest pathogenic fungi. In this study, we isolated and screened this strain for in vitro experiments. The result of the two-sealed-base-plates method revealed that volatile organic compounds (VOCs) emitted from the strain CL2 inhibited the hyphal growth of four pathogenic fungi Mucor circinelloides LB1, Fusarium arcuatisporum LB5, Alternaria iridiaustralis LB7, and Colletotrichum fioriniae LB8. After exposure to VOCs for 5 days, the hyphal growth of the pathogen C. fioriniae LB8 was inhibited by 73%. Scanning electron microscopy revealed that the VOCs produced by B. subtilis CL2 caused the mycelium morphology of the pathogenic fungi to deform, twist, fold, and shrink. In the in vivo experiments, we noticed that VOCs could significantly reduce the weight loss rate of wolfberry fruits caused by the pathogenic fungus M. circinelloides LB1 and that the decay incidence rate were caused by the pathogenic fungi F. arcuatisporum LB5, A. iridiaustralis LB7, and C. fioriniae LB8. On the basis of the headspace-gas chromatography-ion mobility spectrometry analysis, seven VOCs produced by strain CL2 were identified. Among them, 2,3-butanedione and 3-methylbutyric acid are the main antifungal active substances. This study investigated the antifungal properties of VOCs produced by the strain CL2 on postharvest pathogenic fungi isolated from wolfberry fruits both in vivo and in vitro, thereby providing the theoretical basis for its future applications.

Effects of Xingnaojing on serum high-sensitivity C-reactive protein and neuron-specific enolase in patients with acute cerebral hemorrhage: A protocol of systematic review and meta-analysis.

BACKGROUND: This study will systematically explore the effects of Xingnaojing (XNJ) on serum high-sensitivity C-reactive protein (hs-CRP) and neuron-specific enolase (NSE) in patients with acute cerebral hemorrhage (ACH). METHODS: We will comprehensively search the following electronic databases (MEDLINE, EMBASE, Cochrane Library, Allied and Complementary Medicine Database, and China National Knowledge Infrastructure) from inception to the March 1, 2020. There are no limitations related to the language and publication status. Two authors will independently perform all citation identification, information extraction, and study quality. All potential conflicts will be solved through discussion with the help of a third author. RevMan 5.3 software will be used for data synthesis and statistical analysis. RESULTS: This study will summarize the present evidence to investigate the effects of XNJ on serum hs-CRP and NSE in patients with ACH. CONCLUSION: This study may provide an impressive understanding of perspective from scientific basis for effects of XNJ on serum hs-CRP and NSE in patients with ACH. STUDY REGISTRATION: PROSPERO CRD42020171648.

A potentially important resource: endophytic yeasts.

Recent advancements in the research on endophytes isolated from plants and crops have greatly broadened its application in various fields. Endophytic bacteria and endophytic fungi are known to promote the growth of various plants. Besides, the secondary metabolites such as alcohol and xylitol secreted by the endophytic yeast also help their hosts to resist microbial invasion. This makes them a potential substitute for chemical-based control methods. Moreover, the plant hosts can also provide nutrients for the growth of endophytic yeasts. To achieve the symbiotic relationship, yeasts must colonize most parts of the plant tissues, including intercellular spaces, cytoplasm, stomata of seeds, roots, stems, leaves, and fruits as well. Conventionally, isolation of endophytic yeasts from different plant tissues and understanding their interior plants colonization mechanism have remainedkey strategies to exploit their key potentials. In this review, we will elaborate on the diversity, characteristics of colonization, and the factors that influence the distribution of endophytic yeasts. This review also lays a theoretical foundation for the application of endophytic yeasts in various industrial and agricultural practices.

Multiple Mechanisms Drive Calcium Signal Dynamics around Laser-Induced Epithelial Wounds.

Epithelial wound healing is an evolutionarily conserved process that requires coordination across a field of cells. Studies in many organisms have shown that cytosolic calcium levels rise within a field of cells around the wound and spread to neighboring cells, within seconds of wounding. Although calcium is a known potent second messenger and master regulator of wound-healing programs, it is unknown what initiates the rise of cytosolic calcium across the wound field. Here we use laser ablation, a commonly used technique for the precision removal of cells or subcellular components, as a tool to investigate mechanisms of calcium entry upon wounding. Despite its precise ablation capabilities, we find that this technique damages cells outside the primary wound via a laser-induced cavitation bubble, which forms and collapses within microseconds of ablation. This cavitation bubble damages the plasma membranes of cells it contacts, tens of microns away from the wound, allowing direct calcium entry from extracellular fluid into damaged cells. Approximately 45 s after this rapid influx of calcium, we observe a second influx of calcium that spreads to neighboring cells beyond the footprint of cavitation. The occurrence of this second, delayed calcium expansion event is predicted by wound size, indicating that a separate mechanism of calcium entry exists, corresponding to cell loss at the primary wound. Our research demonstrates that the damage profile of laser ablation is more similar to a crush injury than the precision removal of individual cells. The generation of membrane microtears upon ablation is consistent with studies in the field of optoporation, which investigate ablation-induced cellular permeability. We conclude that multiple types of damage, including microtears and cell loss, result in multiple mechanisms of calcium influx around epithelial wounds.

BIBLIOMETRIC ANALYSIS OF ACUPUNCTURE RESEARCH FRONTS AND THEIR WORLDWIDE DISTRIBUTION OVER THREE DECADES.

BACKGROUND: Considerable research has been conducted on acupuncture worldwide. This study chronologically examined the changing features and research fronts of acupuncture and elucidated the differences among the six most productive countries. METHODS: Bibliographic coupling is a powerful tool for identifying the research fronts of a field. Acupuncture-related publications worldwide and from the six most productive countries during 1983-2012 were retrieved from the Science Citation Index Expanded and Social Science Citation Index. To form the research fronts, the 100 most highly cited papers (HCPs) were clustered in terms of references shared. RESULTS: The United States had the highest proportion of HCPs. The effectiveness of acupuncture in areas such as relieving neck and back pain, migraines and headaches, and knee osteoarthritis symptoms was a predominant topic. Initially, the endogenous opioid peptide system was the primary research focus in the acupuncture mechanism research; however, during 1993-2012, researchers focused more on the functional magnetic resonance imaging of brain activity. In addition, acupuncture use and prevalence, the attitudes of health practitioners, and the effects of expectancy and belief were also major topics. Researches from Western countries, including the United States, England, and Germany, showed more interest in clinical trials and economic- and ethics-related studies, whereas those from East Asian countries including China, Japan, and South Korea focused more on mechanism research. CONCLUSION: Western countries dominated the research fronts of acupuncture. The patterns of the research fronts varied worldwide, indicating continuity and innovation in research in each country.

Serine deprivation enhances antineoplastic activity of biguanides.

Metformin, a biguanide widely used in the treatment of type II diabetes, clearly exhibits antineoplastic activity in experimental models and has been reported to reduce cancer incidence in diabetics. There are ongoing clinical trials to evaluate its antitumor properties, which may relate to its fundamental activity as an inhibitor of oxidative phosphorylation. Here, we show that serine withdrawal increases the antineoplastic effects of phenformin (a potent biguanide structurally related to metformin). Serine synthesis was not inhibited by biguanides. Instead, metabolic studies indicated a requirement for serine to allow cells to compensate for biguanide-induced decrease in oxidative phosphorylation by upregulating glycolysis. Furthermore, serine deprivation modified the impact of metformin on the relative abundance of metabolites within the citric acid cycle. In mice, a serine-deficient diet reduced serine levels in tumors and significantly enhanced the tumor growth-inhibitory actions of biguanide treatment. Our results define a dietary manipulation that can enhance the efficacy of biguanides as antineoplastic agents that target cancer cell energy metabolism.

Use of acupuncture to treat cerebral infarction in the last 10 years: A Scopus-based literature analysis.

OBJECTIVE: To identify global research trends in the use of acupuncture to treat cerebral infarction. DATA RETRIEVAL: We performed a bibliometric analysis of studies on the use of acupuncture to treat cerebral infarction published during 2002-2011, retrieved from Scopus, using the key words of acupuncture and cerebral infarction or ischemic stroke. INCLUSION CRITERIA: peer-reviewed articles on the use of acupuncture to treat cerebral infarction indexed in Scopus and published between 2002 and 2011; types of publications were original research articles, reviews, meeting abstracts, proceedings papers, book chapters, editorial material, and news items. EXCLUSION CRITERIA: articles that required manual searching or telephone access; documents that were not published in the public domain; and corrected papers. MAIN OUTCOME MEASURES: (a) Annual publication output; (b) language of publication; (c) type of publication; (d) key words of publication; (e) publication by research field; (f) publication by journal; (g) publication by country and institution; (h) publication by author; (i) most-cited papers between 2002 and 2006; and (j) most-cited papers between 2007 and 2011. RESULTS: A total of 160 publications on the use of acupuncture to treat cerebral infarction from 2002-2011 were retrieved from Scopus. The number of publications increased gradually over the 10-year study period; most were written in Chinese or English. Articles and reviews constituted the major types. The most frequent key word used was acupuncture. The most prolific journals in this area were Zhongguo Zhen Jiu and the Chinese Journal of Clinical Rehabilitation. Of the 160 publications retrieved, half came from Chinese authors and institutions. Tianjin University of Traditional Chinese Medicine was the most prolific research institute. Two papers were cited 30 times; they were published in 2002 and 2009, respectively. CONCLUSION: In the field of neuroscience, there is little literature on acupuncture for cerebral infarction. The most-cited papers were cited 30 times in the past 3 years. We believe that, with advances in the study of mechanisms in neurobiology, research on acupuncture will also advance and will become the concern of more scholars.

[Analysis on the clinical characteristics and related risk factors of patients with hemorrhagic transformation after cerebral infarction].

OBJECTIVE: To analyze and summarize the clinical characteristics and risk factors for patients with hemorrhagic transformation (HT) after cerebral infarction to provide guidance for its clinical treatment and prevention. METHODS: In this study, data from 49 hospitalized patients with HT in the First Department of Neurology, China-Japan Union Hospital of Jilin University from October 2009 to March 2012, were reviewed retrospectively and 106 cases with acute cerebral infarction only during the same period, were chosen randomly as controls. Gender and age of the patients were comparable. Relevant information was collected. SPSS 17.0 statistical package was applied for data processing. Qualitative data were processed with χ(2) test, and measurable data was processed with t-test. Each index was analyzed with uni-variate analysis while statistically significant risk factors were included in the logistic review model to conduct the multivariate regression analysis. RESULTS: (1) Clinical symptoms deteriorating after hemorrhage in 21 cases accounted for 42.9% of the HT group, among which the cases on degree of disturbance to consciousness had an aggravation in 8 cases and hemiplegia increase in another 7 cases. Headaches and dizziness were found in 5 cases. (2) Number of infarction within 15 days after the occurrence of HT accounted for 87.0%. (3) HT-related factors increased the risk of HT in cerebral infarction such as cortical infarction, large area of infarction, atrial fibrillation, cerebral embolism, diabetes and high level of low-density lipoprotein cholesterol (P < 0.05). The most important factors were atrial fibrillation and cerebral embolism. (4) PH-2 seemed more unlikely to link with clinical symptoms than other subtypes of HT. CONCLUSION: Cerebral infarction after occlusion of the main artery trunk was prone to HT, especially when it appeared within 15 days. Short-term prognosis seemed to be related to the subtypes of HT, with risk factors as cortical infarct, massive cerebral infarction, atrial fibrillation, cerebral embolism, diabetes, high low-density lipoprotein cholesterol etc. on HT.

IGF1/insulin receptor kinase inhibition by BMS-536924 is better tolerated than alloxan-induced hypoinsulinemia and more effective than metformin in the treatment of experimental insulin-responsive breast cancer.

Epidemiologic and experimental evidence suggest that a subset of breast cancer is insulin responsive, but it is unclear whether safe and effective therapies that target the insulin receptor (IR), which is homologous to oncogenes of the tyrosine kinase class, can be developed. We demonstrate that both pharmacologic inhibition of IR family tyrosine kinase activity and insulin deficiency have anti-neoplastic activity in a model of insulin-responsive breast cancer. Unexpectedly, in contrast to insulin deficiency, pharmacologic IR family inhibition does not lead to significant hyperglycemia and is well tolerated. We show that pharmacokinetic factors explain the tolerability of receptor inhibition relative to insulin deficiency, as the small molecule receptor kinase inhibitor BMS-536924 does not accumulate in muscle at levels sufficient to block insulin-stimulated glucose uptake. Metformin, which lowers insulin levels only in settings of hyperinsulinemia, had minimal activity in this normoinsulinemic model. These findings highlight the importance of tissue-specific drug accumulation as a determinant of efficacy and toxicity of tyrosine kinase inhibitors and suggest that therapeutic targeting of the IR family for cancer treatment is practical.

Metformin abolishes increased tumor (18)F-2-fluoro-2-deoxy-D-glucose uptake associated with a high energy diet.

Insulin regulates glucose uptake by normal tissues. Although there is evidence that certain cancers are growth-stimulated by insulin, the possibility that insulin influences tumor glucose uptake as assessed by ( 18) F-2-Fluoro-2-Deoxy-d-Glucose Positron Emission Tomography (FDG-PET) has not been studied in detail. We present a model of diet-induced hyperinsulinemia associated with increased insulin receptor activation in neoplastic tissue and with increased tumor FDG-PET image intensity. Metformin abolished the diet-induced increases in serum insulin level, tumor insulin receptor activation and tumor FDG uptake associated with the high energy diet but had no effect on these measurements in mice on a control diet. These findings provide the first functional imaging correlate of the well-known adverse effect of caloric excess on cancer outcome. They demonstrate that, for a subset of neoplasms, diet and insulin are variables that affect tumor FDG uptake and have implications for design of clinical trials of metformin as an antineoplastic agent.

Loss of function of PTEN alters the relationship between glucose concentration and cell proliferation, increases glycolysis, and sensitizes cells to 2-deoxyglucose.

PTEN loss of function enhances proliferation, but effects on cellular energy metabolism are less well characterized. We used an inducible PTEN expression vector in a PTEN-null glioma cell line to examine this issue. While proliferation of PTEN-positive cells was insensitive to increases in glucose concentration beyond 2.5mM, PTEN-null cells significantly increased proliferation with increasing glucose concentration across the normal physiologic range to approximately 10mM, coinciding with a shift to glycolysis and "glucose addiction". This demonstrates that the impact of loss of function of PTEN is modified by glucose concentration, and may be relevant to epidemiologic results linking hyperglycemia to cancer risk and cancer mortality.

The effects of varying dietary carbohydrate and fat content on survival in a murine LNCaP prostate cancer xenograft model.

PURPOSE: Numerous dietary factors elevate serum levels of insulin and insulin-like growth factor I (IGF-I), both potent prostate cancer mitogens. We tested whether varying dietary carbohydrate and fat, without energy restriction relative to comparison diets, would slow tumor growth and reduce serum insulin, IGF-I, and other molecular mediators of prostate cancer in a xenograft model. EXPERIMENTAL DESIGN: Individually caged male severe combined immunodeficient mice (n = 130) were randomly assigned to one of three diets (described as percent total calories): very high-fat/no-carbohydrate ketogenic diet (NCKD: 83% fat, 0% carbohydrate, 17% protein), low-fat/high-carbohydrate diet (LFD: 12% fat, 71% carbohydrate, 17% protein), or high-fat/moderate-carbohydrate diet (MCD: 40% fat, 43% carbohydrate, 17% protein). Mice were fed to maintain similar average body weights among groups. Following a preliminary feeding period, mice were injected with 1 x 10(6) LNCaP cells (day 0) and sacrificed when tumors were >or=1,000 mm(3). RESULTS: Two days before tumor injection, median NCKD body weight was 2.4 g (10%) and 2.1 g (8%) greater than the LFD and MCD groups, respectively (P < 0.0001). Diet was significantly associated with overall survival (log-rank P = 0.004). Relative to MCD, survival was significantly prolonged for the LFD (hazard ratio, 0.49; 95% confidence interval, 0.29-0.79; P = 0.004) and NCKD groups (hazard ratio, 0.59; 95% confidence interval, 0.37-0.93; P = 0.02). Median serum insulin, IGF-I, IGF-I/IGF binding protein-1 ratio, and IGF-I/IGF binding protein-3 ratio were significantly reduced in NCKD relative to MCD mice. Phospho-AKT/total AKT ratio and pathways associated with antiapoptosis, inflammation, insulin resistance, and obesity were also significantly reduced in NCKD relative to MCD tumors. CONCLUSIONS: These results support further preclinical exploration of carbohydrate restriction in prostate cancer and possibly warrant pilot or feasibility testing in humans.

The hedgehog pathway inhibitor cyclopamine increases levels of p27, and decreases both expression of IGF-II and activation of Akt in PC-3 prostate cancer cells.

The hedgehog signalling inhibitor cyclopamine has been shown to induce growth inhibition and cell cycle arrest in prostate cancer cell lines, but the mechanism of action has not been clearly defined, and observations between laboratories have not always been consistent. We first observed that albumin can protect PC-3 prostate cancer cells from cyclopamine-induced growth inhibition, suggesting that cyclopamine binds to albumin, and that only free cyclopamine is active. We then conducted a phospho-site protein kinase screen to elucidate the mechanism of cyclopamine-induced growth inhibition. Treatment of PC-3 cells with 5 or 10 microM cyclopamine for 72h resulted in a decrease in cell viability of approximately 50% and approximately 75%, respectively. A phospho-site protein kinase screen showed that cyclopamine decreased levels of phospho-Thr(187)-p27 by 71%. This phospho-site on p27 positively regulates its ubiquitin degradation; therefore a decrease in phospho-Thr(187)-p27 should correlate with increased levels of p27. Consistent with this hypothesis, treatment of PC-3 cells with cyclopamine resulted in a approximately 3-fold increase in p27 protein levels. Cdk-2 phosphorylates Thr(187)-p27, and immunoblotting demonstrated that cyclopamine treatment of PC-3 cells reduces the expression of cdk-2. Furthermore, cyclopamine decreased the levels of phosphorylated (activated) Akt, which is known to increase p27 degradation via Skp-2-induced ubiquitination. The mechanism by which cyclopamine decreases phosphorylated Akt is currently under investigation, but it may involve our observed cyclopamine-induced reduction in IRS-1 and IGF-II expression. These results demonstrate novel molecular correlates of cyclopamine-induced growth inhibition of prostate cancer cells.

Insulin-like growth factor receptor I targeting in epithelial ovarian cancer.

OBJECTIVES: Preclinical evaluation of the anti-neoplastic activity of an insulin-like growth factor I receptor (IGF-IR) kinase inhibitor in ovarian cancer. METHODS: The OVCAR-3 and OVCAR-4 cell lines were investigated under serum-free tissue culture conditions. IGF-I and IGF-II production were evaluated by standard ELISA and immunohistochemistry. IGF-IR expression and protein levels were evaluated by Western blotting. Cytotoxicity assays were performed in triplicates using the Alamar colorimetric assay. Apoptosis was evaluated by flow cytometry and by Western blotting for PARP. RESULTS: The OVCAR-3 and OVCAR-4 cell lines produce IGF-I and IGF-II, and express IGF-IR, detectable by Western blotting, supporting the existence of an autocrine loop. The existence of this loop justified studies of NVP-AEW541, a small molecular weight inhibitor of the IGF-IR kinase. We observed growth inhibition of the ovarian cancer cell lines, with IC50 between 5 and 15 microM. We also observed that NVP-AEW541 sensitized cells to cisplatin in vitro. Western blotting demonstrated that NVP-AEW541 induced apoptosis at the concentrations that were used in the cytotoxicity assays, and decreased the concentration of the phosphorylated AKT signaling protein downstream of the IGF-IR. CONCLUSIONS: IGF-IR is a potential new molecular target in ovarian cancer. The anti-neoplastic activity of NVP-AEW541 in ovarian cancer was observed at concentrations higher than those previously reported for multiple myeloma, suggesting the possibility that a portion of the observed anti-neoplastic activity could involve targets other than the IGF-IR. Experiments are being conducted to investigate the cytotoxicity profile in vivo and the clinical relevance of NVP-AEW541 in ovarian cancer treatment.

Overexpression of ErbB2 receptor inhibits IGF-I-induced Shc-MAPK signaling pathway in breast cancer cells.

Overexpression of the ErbB2 receptor in one-third of human breast cancers contributes to the transformation of epithelial cells and predicts poor prognosis for breast cancer patients. We report that the overexpression of ErbB2 inhibits IGF-I-induced MAPK signaling. IGF-I-induced MAPK phosphorylation and MAPK kinase activity are reduced in ErbB2 overexpressing MCF-7/HER2-18 cells relative to control MCF-7/neo cells. In SKBR3/IGF-IR cells, reduction of ErbB2 by antisense methodology restores the IGF-I-induced MAPK activation. The inhibition of IGF-I-induced MAP kinase activation in ErbB2 overexpressing breast cancer cells is correlated with decreased IGF-I-induced Shc tyrosine-phosphorylation, leading to a decreased association of Grb2 with Shc and decreased Raf phosphorylation. However, IGF-I-induced tyrosine-phosphorylation of IGF-I receptor and IRS-I and AKT phosphorylation were unaffected by ErbB2 overexpression. Consistent with these results, we observed that the proportion of IGF-I-stimulated proliferation blocked by the MAPK inhibitor PD98059 fell from 82.6% in MCF-7/neo cells to 41.2% in MCF-7/HER2-18 cells. These data provide evidence for interplay between the IGF-IR and ErbB2 signaling pathways. They are consistent with the view that the IGF-IR mediated attenuation of trastuzumab-induced growth inhibition we recently described is dependent on IGF-I-induced PI3K signaling rather than IGF-I-induced MAPK signaling.