Effects of luteinizing hormone receptor signaling in prostate cancer cells.

BACKGROUND: The importance of androgen signaling in prostate cancer (PC) is well described and prostate cancer cells retain the ability to directly synthesize androgens. Luteinizing hormone (LH) can induce expression of steroidogenic enzymes and trigger androgen production, but the regulation of this process is not well-described. Here, we explored the impact of silencing LH receptor (LHR) silencing on androgen synthesis and on several relevant signaling pathways in PC. METHODS: LHR mRNA and protein expression was evaluated in LNCaP PC cells treated with LHR-siRNA. MTS assay was used to measure the effect of LHR-siRNA on proliferation in LNCaP and 22RV1 PC cells. Treated LNCaP and LAPC-3 cells were also assayed for differences in androgen synthesis and expression of steroidogenic enzymes, PSA, AR, and critical signaling molecules including PKA, ERK1/2, PI3K, AKT2, and HER2. RESULTS: We confirmed that functional LHR is expressed in both androgen-sensitive and castrate-resistant PC specimens. Treatment with LHR-siRNA effectively silenced LHR gene and protein expression and prevented LH-mediated proliferation and androgen synthesis in prostate cancer cells. LHR silencing also downregulated expression of AR, PSA, PKA, ERK1/2, PI3K, AKT2, and HER2. CONCLUSION: Collectively, these data demonstrate that silencing LHR expression suppresses androgen synthesis and signaling and the LH-LHR pathway may represent a viable therapeutic strategy in PC.

Effect of Tryptophan Restriction in the Therapy of Irritable Bowel Syndrome: a Systematic Review.

Background & Aims: The metabolic pathways of tryptophan (TRP) have been implicated in the pathophysiology of irritable bowel syndrome (IBS), positing that the strategic modulation of TRP consumption may exert regulatory effects on serotonin levels, consequently altering the clinical manifestation of IBS. This systematic review was meticulously orchestrated to evaluate the effect of TRP restriction on IBS. Methods: A comprehensive search of the MEDLINE/PubMed, Cochrane Library, and Embase databases was conducted. Controlled trials that compared the efficacy of TRP restriction in IBS patients were scrutinized. The primary outcomes were gastrointestinal symptoms, quality of life, and pain, whereas the secondary outcomes included anxiety, mood, and safety. The risk of bias was meticulously assessed according to the guidelines recommended by the Cochrane Collaboration. Results: A total of five trials, enrolling 135 participants, were incorporated into the qualitative synthesis. Low-TRP intake attenuated gastrointestinal discomfort and enhanced psychological well-being in IBS patients, while the effects of acute TRP depletion were controversial. Safety data from one randomized controlled trial reported no occurrence of adverse events. Conclusion: This systematic review suggests that moderating, rather than depleting, TRP intake may potentially be a feasible and safe adjunctive treatment for patients with IBS. Future research incorporating a high-quality study design and consensus on clinical outcome measurements for IBS is warranted.

Analysis of the causes and clinical characteristics of jejunoileal hemorrhage in China: a multicenter 10 year retrospective survey.

BACKGROUND: A retrospective study was performed to assess the causes, diagnostic methods for, and clinical features of, jejunoileal hemorrhage in Shandong province, China and to derive recommendations for management of this condition from these data. METHODS: We performed a retrospective systematic collection of data from between January 1999 and December 2008 in seven cities in Shandong province, China, identified 72 patients with jejunoileal hemorrhage and analyzed the relevant clinical data. RESULTS: Overall, tumors were the most common cause of jejunoileal hemorrhage (42 patients, 58.3%). The causes of this condition were significantly different (P < 0.05) in male and female patients. In male patients, the commonest factors were tumor (52.2%), enteritis (17.4%) and angiopathy (15.2%). However, in female patients, tumors accounted for a greater proportion of cases (18/26, 69.2%). In 38 cases (52.8%) the diagnosis was made by intraoperative enteroscopy or laparotomy, in 14 by capsule endoscopy and in the remainder by radiological methods. The most frequent presentation was melena (62.7%), followed by maroon stools (26.9%) and hematochezia (9.0%). Of the 72 patients,laparotomy is the main treatment method. CONCLUSION: Tumor, enteritis and angiopathy and diverticular disease are the most common causes of jejunoileal hemorrhage in Shandong province, China. The main clinical manifestations are bloody stools, most commonly in the form of melena, with or without abdominal pain. We recommend that female patients over the age of 40 with jejunoileal hemorrhage accompanied by abdominal pain should undergo urgent further assessment because of the strong probability of jejunoileal tumor.

Circ-CCS enhances autophagy during imatinib resistance of gastrointestinal stromal tumor by regulating miR-197-3p/ATG10 signaling.

Context: Drug resistance in gastrointestinal stromal tumors (GISTs) is connected with autophagy activation. Accumulating data demonstrates the critical role of circular RNAs (circRNAs) dysregulation in this development. Aim: To explore the possible function of hsa_circ_0092306 (circ-CCS) in GIST imatinib resistance. Materials and Methods: Quantitative real-time reverse transcription PCR (RT-qPCR) was used to determine the expression levels of circ-CCS and miR-197-3p. The vitality and apoptosis of cells were determined using the Cell Counting Kit-8 and TUNEL assays, respectively. Western blot analysis was used to evaluate the relative protein expression. A dual-luciferase reporter assay was used to validate the link between circ-CCS, miR-197-3p, and ATG10. Statistical Analysis Used: Comparisons of two groups were analyzed using Student's t tests, and analysis of variance (ANOVA) with Tukey's post hoc test was used to compare three or more groups. Results: Circulating-CCS expression was considerably increased in the serum of imatinib-resistant GIST patients (P < 0.001). Circulating-CCS deficiency decreased cell proliferation and autophagy in GIST-882 and GIST-T1 cells, but promoted apoptosis (P < 0.05). Additionally, circ-CCS was predominantly found in the cytoplasm. Mechanically, circ-CCS targeted miR-197-3p, which may influence autophagy by downregulating ATG10, in order to modulate GIST cells' malignant tendencies. Moreover, silencing miR-197-3p reversed the effect of circ-CCS knockdown on apoptosis and autophagy in GIST cells. Conclusions: By modulating the miR-197-3p/ATG10 axis, circ-CCS increased imatinib resistance in GIST cells, establishing a potential target for reversing medication resistance in such patients.

Effect of luteinizing hormone on the steroidogenic pathway in prostate cancer.

BACKGROUND: Recent data has shown that prostate cancer (PCA) cells are capable of producing testosterone directly from cholesterol, which may contribute to the development of castration resistance. While up-regulation of steroidogenic enzymes has been previously described during castration-resistant prostate cancer (CRPC) progression, regulation of this process is poorly defined. These data examine the role of luteinizing hormone (LH) in the regulation of steroidogenic machinery in PCA cells. METHODS: PCA cell lines LNCaP, C4-2B, and 22RV1 were exposed to LH. Gene expression was quantified using real-time PCR and protein expression was characterized with standard Western blot analysis. Steroid analysis was performed using radioimmunoassay (RIA). Cell viability was measured using an MTS viability assay. RESULTS: Androgen-sensitive (LNCaP) and -independent PCA cells (C4-2B and 22RV1) express both mRNA and protein for LH and LH receptor (LHR). Exposure of these cells to LH for 4 hr increased the expression of several steroidogenic genes. Exposure for 10 days resulted in the increase of additional genes. At both time points, the upregulation of these genes was dose-dependent. This was mirrored by an increase in the expression of several key steroidogenic enzymes, including StAR, CYB5B, CYP11A, and 3ßHSD. LH stimulated the production of progesterone and testosterone in LNCaP cells as measured by RIA. We have also demonstrated that treatment of LNCaP cells with LH enhanced their viability. CONCLUSIONS: Our data show that LH-mediated activation of LHR significantly up-regulates the expression of genes and enzymes required for steroidogenesis and increases steroid production in PCA cells.

Correlation between distribution of Helicobacter pylori in oral cavity and chronic stomach conditions.

In this study, the colonization and distribution of Helicobacter pylori (Hp) in patients with chronic gastric diseases were investigated and the relationship between the periodontal initial treatment and presence of Hp in oral cavity was examined to better understand the connection between Hp infection and chronic diseases. Primers for PCR amplification were designed according to ureC gene and cagA genes of Hp. Specimens were harvested from different sites of 96 patients with chronic gastric diseases and the specimens of dental plaques, gargles and dorsal mucosa were tested for Hp. The 96 patients were treated by bismuth triple therapy and among them, 52 subjects were additionally given periodontal initial therapy. The eradication rate of gastric Hp and oral Hp detection rate were determined 4 weeks and 1 year after the treatment. The results showed that the detection rates of oral specimens were in the order of dental plaques (82.3%), gargles (51.1%) and scrapings of dorsal mucosa of tongue (37.5%). One year after bismuth triple therapy or the triple therapy in combination with periodontal initial treatment, the eradication rate of gastric Hp was significantly higher in the combination treatment group than in group treated by the triple therapy alone (62.8% vs. 32.4%, P<0. 05). Moreover, the Hp detection rate was significantly lower in the combination group than in the group treated only with the triple therapy. We are led to conclude that Hp is present at various parts of oral cavity, oral Hp might be an important source of gastric Hp and the triple therapy plus periodontal initial treatment can enhance the long-term eradication rate of gastric Hp in patient with both chronic gastric diseases and chronic periodontitis.

The disintegrin contortrostatin in combination with docetaxel is a potent inhibitor of prostate cancer in vitro and in vivo.

BACKGROUND: There are few available treatments for hormone refractory prostate cancer. Through the inhibition of integrins, contortrostatin (CN) effects tumor cell growth directly as well as through the inhibition of angiogenesis. The effect of CN in combination with docetaxel on prostate cancer cell lines in vitro and in vivo is evaluated in the present study. METHODS: FACS analysis of integrin expression, assessment of CN and docetaxel exposure on viability of plated cancer cells, and scratch test migration analysis were performed on PC-3 prostate cancer cells. CN and docetaxel inhibition of both PC-3 and CWR-22 prostate cancer cell lines were evaluated in a mouse xenograft bone model. Angiogenic activity in tumors were assessed using IHC with antibodies to CD31. RESULTS: Cell culture experiments indicate that the combination of docetaxel and CN inhibits growth in an additive fashion. FACS analysis of PC-3 cells shows expression of alpha5beta1 and alphavbeta5 integrins, but little expression of the alphavbeta3. CN showed complete inhibition of PC-3 migration in cultures grown on matrigel plates. In mice xenograft bone models, CN with docetaxel showed increased inhibition of both PC-3 and CWR-22 derived tumors. Analysis of treated xenograft tumors showed significantly decreased expression of CD31 indicating suppression of angiogenesis.

Gene profiling and pathway analysis of neuroendocrine transdifferentiated prostate cancer cells.

BACKGROUND: Neuroendocrine (NE) cells are present in both normal prostate and prostate cancer. In addition, NE differentiation can be induced by various factors, such as IL-6, in vitro and in vivo. However, the mechanism of this differentiation and the role of NE cells in prostate cancer are not well understood. In this study, we evaluated the gene expression and analyzed the pathways in prostate cancer cells exposed to various NE differentiation inducing factors in vitro. METHODS: Gene expression signatures between control LNCaP cells and each treatment induced NE cell line were compared using Affymetrix GeneChip with network and pathway analysis. RESULTS: All treatments were able to transdifferentiate LNCaP cells into NE phenotype as shown by morphology changes and NE marker measurements. Of the 54,675 oligonucleotide-based probe sets in microarray, 44,975 were mapped into the Ingenuity Pathway Analysis database and were filtered according to the t-test P value. At P < 0.002, the number of genes that were differentially expressed included 302 of the IL-6 treated cells, 201 of genistein, 233 of epinephrine, and 191 of the charcoal stripped serum ones. A pooled data approach also showed 346 differentially expressed genes at the same P value. Gene ontology analysis showed that cancer-related function had the highest significance. CONCLUSIONS: Despite some overlap, each NE transdifferentiation inducing treatment was associated with a changed expression of a unique set of genes, and such gene profiling may help to elucidate the molecular mechanisms involved in NE transdifferentiation of prostate cancer cells.

Locus-wide chromatin remodeling and enhanced androgen receptor-mediated transcription in recurrent prostate tumor cells.

Prostate cancers (PCas) become resistant to hormone withdrawal through increased androgen receptor (AR) signaling. Here we show increased AR-mediated transcription efficiency in PCa cells that have acquired the ability to grow in low concentrations of androgen. Compared to androgen-dependent PCa cells, these cells showed increased activity of transiently transfected reporters and increased mRNA synthesis relative to levels of AR occupancy of the prostate-specific antigen (PSA) gene. The locus also displayed up to 10-fold-higher levels of histone H3-K9/K14 acetylation and H3-K4 methylation across the entire body of the gene. Although similar increased mRNA expression and locus-wide histone acetylation were also observed at another kallikrein locus (KLK2), at a third AR target locus (TMPRSS2) increased gene expression and locus-wide histone acetylation were not seen in the absence of ligand. Androgen-independent PCa cells have thus evolved three distinctive alterations in AR-mediated transcription. First, increased RNA polymerase initiation and processivity contributed to increased gene expression. Second, AR signaling was more sensitive to ligand. Third, locus-wide chromatin remodeling conducive to the increased gene expression in the absence of ligand was apparent and depended on sustained AR activity. Therefore, increased AR ligand sensitivity as well as locus-specific chromatin alterations contribute to basal gene expression of a subpopulation of specific AR target genes in androgen-independent PCa cells. These features contribute to the androgen-independent phenotype of these cells.

Both beta-actin and GAPDH are useful reference genes for normalization of quantitative RT-PCR in human FFPE tissue samples of prostate cancer.

BACKGROUND: Beta-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been frequently considered as constitutive house keeping genes for RT-PCR and used to normalize changes in specific gene expressions. However, these expressions have been shown to be affected by the sample type and experimental conditions. We investigated which housekeeping gene is useful to study gene expression of paraffin embedded human tissue samples of prostate cancer. METHODS: Fifteen pairs of cancer and corresponding normal tissue were obtained from patients with prostate cancer. We evaluated gene expression of beta-actin, GAPDH, androgen receptor (AR), and heat-shock 70-kd protein 5 (HSPA5) using laser captured microdissection and quantitative RT-PCR. AR and HSPA5 gene expression were normalized to each of these reference genes using the 2(-DeltaDeltaCt) method of relative quantification. The quantity 2(Ct(normal)-Ct(cancer)) divided by ratio of cDNA(cancer)/cDNA (normal) was used for comparing differences between cancer and normal tissue in GAPDH and beta-actin expression. RESULTS: Ct value of beta-actin was significantly correlated with that of GAPDH (r = 0.443, P = 0.014). AR and HSPA5 gene expression levels using beta-actin for normalization were significantly correlated with these gene expression levels using GAPDH (AR; r = 0.689, P = 0.004, HSPA5; r = 0.879, P < 0.001). Both reference genes were expressed more highly in cancer tissue than in normal tissue, with that of GAPDH being significantly different between cancer tissue and normal tissue (P = 0.029). CONCLUSIONS: The good correlation between gene expression values obtained when using beta-actin and GAPDH as reference genes suggests that either gene is a valid denominator for gene expression studies in prostate cancer.

Prognostic value of the androgen receptor and its coactivators in patients with D1 prostate cancer.

BACKGROUND: Prostate cancer treated with androgen ablation eventually becomes resistant. Because the androgen receptor (AR) signaling axis affects disease progression, AR coactivator molecules could provide clinical prognostic value. This study investigates the association between AR coactivator molecules and clinical outcome measures in patients with prostate cancer. PATIENTS AND METHODS: Expression levels of AR and its coactivators, SRC1, TIF2, and Her2/neu were determined by quantitative RT-PCR in 148 prostatectomy specimens. AR protein expression was determined by immunohistochemistry. The prognostic value of these expression levels on clinical outcomes was examined. RESULTS: Increased gene and protein AR expression was not correlated with any of the clinical outcome measures. A non-monotonic correlation was observed between SRC1 and overall survival, as well as Her2/neu and time to prostate-specific PSA recurrence. CONCLUSION: Although no statistically significant relationships were found, the weak association between some clinical outcomes and two AR coactivators may help improve the current predictive nomogram for patients with prostate cancer.

[Preliminary study of biweekly regimen of docetaxel, oxaliplatin, 5-fluorouracil and leucovorin for advanced gastric cancer].

OBJECTIVE: To evaluate the efficacy and toxicity of a biweekly DOF regimen consisting of docetaxel, oxaliplatin, 5-fluorouracil and leucovorin for advanced gastric cancer. METHODS: The biweekly DOF regimen was administered in 37 advanced gastric cancer patients. Docetaxel, oxaliplatin and leucovorin were given intravenously at a dose of 35 mg/m2, 85 mg/m2 and 200 mg/m2 for 1 h, 2 h and 2 h on D1, respectively, and 5-Fu was administered as continuous intravenous infusion for 48 h at a dose of 1500 mg/m2 on D1 and D2. This regimen was repeated every 2 weeks. The efficacy and toxicity were evaluated after completion of 3 cycles at least. RESULTS: The overall response rate (RR) of this series was 67.6%, complete response rate and partial response rate were 27.0% and 40.5%, respectively. The time to progression (TTP) was 9.2 months, and median survival time (MST) was 13.7 months. The RRs of 11 chemotherpy-naïve patients and 26 patients pre-treated with chemotherapy were 81.8% and 61.5%, respectively. CONCLUSION: Our preliminary results showed that this biweekly combination regimen of docetaxel, oxaliplatin, 5-fluorouracil and leucovorin is effective and tolerable for advanced gastric cancer. However, further investigation of this regimen is mandatory.

CHINESE HERBAL DECOCTION AS A COMPLEMENTARY THERAPY FOR ATROPHIC GASTRITIS: A SYSTEMATIC REVIEW AND META-ANALYSIS.

BACKGROUND: Chinese herbal decoction (CHD) has been extensively used in the treatment of atrophic gastritis (AG) in China and other Far Eastern countries. We conducted a systematic review and meta-analysis to estimate the efficacy and safety of CHD in AG. MATERIALS AND METHODS: Pubmed, Embase, Cochrane central register of controlled trials (central), VIP, China National Knowledge Infrastructure, Sinomed, Wanfang data were searched (up to December 2015). Randomized controlled trials recruiting patients with AG comparing CHD (alone or with western medicine (WM)) with WM were eligible. Dichotomous data were pooled to obtain relative risk (RR), with a 95% confidence interval (CI). RESULTS: Forty-two articles including 3,874 patients were identified. CHD, used alone or with WM, had beneficial effect over WM in the improvement of clinical manifestations (RR=1.28; 95% CI 1.22-1.34) and pathological change (RR=1.42; 95% CI 1.30-1.54) for AG patients. However, the H. pylori eradication effect of CHD was not supported by the existing clinical evidence, because of the significant study heterogeneity (I2>50%) and inconsistency between the primary results and sensitivity analysis. CONCLUSIONS: CHD, if prescribed as a complementary therapy to WM, may improve the clinical manifestations and pathological change for AG patients. But its monotherapy for H. pylori eradication is not supported by enough clinical evidence.

Discovery and preclinical evaluation of a novel class of small-molecule compounds in hormone-dependent and -independent cancer cell lines.

We discovered a series of salicylhydrazide class of compounds with remarkable anticancer activity against a panel of hormone receptor-positive and -negative cell lines. In the present study, we evaluated the in vitro activity of SC21 and SC23 against a range of human tumor cell types and the in vivo efficacy of compound SC21 in a PC3 human prostate cancer xenograft model in mice. We also determined the effects of SC21 on cell cycle regulation and apoptosis. Our in vitro results show that salicylhydrazides are highly potent compounds effective in both hormone receptor-positive and -negative cancer cells. SC21 induced apoptosis and blocked the cell cycle in G(0)/G(1) or S phase, depending on the cell lines used and irrespective of p53, p21, pRb, and p16 status. SC21 effectively reduced the tumor growth in mice without apparent toxicity. Although the mechanism of action of SC21 is not completely elucidated, the effect on cell cycle, the induction of apoptosis and the activity against a panel of tumor cell lines of different origins prompted us to carry out an in-depth preclinical evaluation of SC21.

Prevalence of depression and anxiety in patients with chronic digestive system diseases: A multicenter epidemiological study.

AIM: To investigate the prevalence of depression and anxiety in patients with chronic digestive system diseases. METHODS: A total of 1736 patients with chronic digestive system diseases were included in this cross-sectional study, including 871 outpatients and 865 in-patients. A self-designed General Information for Patients of the Department of Gastroenterology of General Hospitals questionnaire was used to collect each patient's general information, which included demographic data (including age, sex, marital status, and education) and disease characteristics (including major diseases, disease duration, principal symptoms, chronic pain, sleep disorder, and limited daily activities). RESULTS: The overall detection rate was 31.11% (540/1736) for depression symptoms alone, 27.02% (469/1736) for anxiety symptoms alone, 20.68% (359/1736) for both depression and anxiety symptoms, and 37.44% (650/1736) for either depression or anxiety symptoms. Subjects aged 70 years or above had the highest detection rate of depression (44.06%) and anxiety symptoms (33.33%). χ2 trend test showed: the higher the body mass index (BMI), the lower the detection rate of depression and anxiety symptoms (χ2trend = 13.697, P < 0.001; χ2trend = 9.082, P = 0.003); the more severe the limited daily activities, the higher the detection rate of depression and anxiety symptoms (χ2trend = 130.455, P < 0.001, χ2trend = 108.528, P < 0.001); and the poorer the sleep quality, the higher the detection rate of depression and anxiety symptoms (χ2trend = 85.759, P < 0.001; χ2trend = 51.969, P < 0.001). Patients with digestive system tumors had the highest detection rate of depression (57.55%) and anxiety (55.19%), followed by patients with liver cirrhosis (41.35% and 48.08%). Depression and anxiety symptoms were also high in subjects with comorbid hypertension and coronary heart disease. CONCLUSION: Depression and anxiety occur in patients with tumors, liver cirrhosis, functional dyspepsia, and chronic viral hepatitis. Elderly, divorced/widowed, poor sleep quality, and lower BMI are associated with higher risk of depression and anxiety.

Abnormal Na channel gating in murine cardiac myocytes deficient in myotonic dystrophy protein kinase.

DMPK is a serine/threonine kinase implicated in the human disease myotonic muscular dystrophy (DM). Skeletal muscle Na channels exhibit late reopenings in Dmpk-deficient mice and peak current density is reduced, implicating DMPK in regulation of membrane excitability. Since complete heart block and sudden cardiac death occur in the disease, we tested the hypothesis that cardiac Na channels also exhibit abnormal gating in Dmpk-deficient mice. We made whole cell and cell-attached patch clamp recordings of ventricular cardiomyocytes enzymatically isolated from wild-type, Dmpk+/-, and Dmpk-/- mice. Recordings from membrane patches containing one or a few Na channels revealed multiple Na channel reopenings occurring after the macroscopic Na current had subsided in both Dmpk+/- and Dmpk-/- muscle, but only rare reopenings in wild-type muscle (>3-fold difference, P < 0.05). This resulted in a plateau of non-inactivating Na current in Dmpk-deficient muscle. The magnitude of this plateau current was independent on the magnitude of the test potential from -40 to 0 mV and was also independent of gene dose. Macroscopic Na current density was similar in wild-type and Dmpk-deficient muscle, as was steady-state Na channel gating. Decay of macroscopic currents was slowed in Dmpk-/- muscle, but not in Dmpk+/- or wild-type muscle. Entry into, and recovery from, inactivation were similar at multiple test potentials in wild-type and Dmpk-deficient muscle. Resting membrane potential was depolarized, and action potential duration was significantly prolonged in Dmpk-deficient muscle. Thus in cardiac muscle, Dmpk deficiency results in multiple late reopenings of Na channels similar to those seen in Dmpk-deficient skeletal muscle. This is reflected in a plateau of non-inactivating macroscopic Na current and prolongation of cardiac action potentials.

A new screening system for proliferation-independent anti-cancer agents.

An in vitro screen for identification of novel anti-cancer agents, which can induce proliferation-independent apoptosis of prostate cancer (PCA) cells, is required, since the proliferative growth fraction of human prostate cancers in patients is usually <10%. This is possible using the PCA cell line LNCaP, which can be permanently transdifferentiated into a quiescent neuroendocrine (NE) phenotype without undergoing apoptosis by the cytokine interleukine-6 (IL-6). Transdifferentiation of LNCaP cells into a NE phenotype was documented using western blot analysis and immunohistochemistry for the NE markers, neuron-specific enolase (NSE) and beta III tubulin. Accumulation of NE cells in the G0 phase of the cell cycle was demonstrated by Ki-67 immunohistochemistry. The effects of paclitaxel, vinblastine and thapsigargin (TG) on viability and apoptosis of NE and LNCaP cells were assessed by trypan blue exclusion and 4', 6-diamidino-2-phenylindole nuclear staining assays. In proliferating LNCaP cells, there was a significant decrease in viable cells after 48 h exposure to paclitaxel and vinblastine and a dramatic increase of apoptosis as compared with the controls. On the other hand, treatment with paclitaxel or vinblastine decreased the viability of NE cells only slightly without markedly increasing their rate of apoptosis compared to controls. In contrast, both LNCaP and NE cells showed a significant and comparable decrease in cell viability and similar high levels of apoptosis when treated with TG. These results demonstrate that terminally transdifferentiated NE cells represent a useful in vitro screening system for identification of novel anti-cancer agents, like TG, that can induce apoptosis without requiring proliferation.

Distal hinge of plasminogen activator inhibitor-1 involves its latency transition and specificities toward serine proteases.

BACKGROUND: The plasminogen activator inhibitor-1 (PAI-1) spontaneously converts from an inhibitory into a latent form. Specificity of PAI-1 is mainly determined by its reactive site (Arg346-Met347), which interacts with serine residue of tissue-type plasminogen activator (tPA) with concomitant formation of SDS-stable complex. Other sites may also play roles in determining the specificity of PAI-1 toward serine proteases. RESULTS: To understand more about the role of distal hinge for PAI-1 specificities towards serine proteases and for its conformational transition, wild type PAI-1 and its mutants were expressed in baculovirus system. WtPAI-1 was found to be about 12 fold more active than the fibrosarcoma PAI-1. Single site mutants within the Asp355-Arg356-Pro357 segment of PAI-1 yield guanidine activatable inhibitors (a) that can still form SDS stable complexes with tPA and urokinase plasminogen activator (uPA), and (b) that have inhibition rate constants towards plasminogen activators which resemble those of the fibrosarcoma inhibitor. More importantly, latency conversion rate of these mutants was found to be approximately 3-4 fold faster than that of wtPAI-1. We also tested if Glu351 is important for serine protease specificity. The functional stability of wtPAI-1, Glu351Ala, Glu351Arg was about 18 +/- 5, 90 +/- 8 and 14 +/- 3 minutes, respectively, which correlated well with both their corresponding specific activities (84 +/- 15 U/ug, 112 +/- 18 U/ug and 68 +/- 9 U/ug, respectively) and amount of SDS-stable complex formed with tPA after denatured by Guanidine-HCl and dialyzed against 50 mM sodium acetate at 4 degrees C. The second-order rate constants of inhibition for uPA, plasmin and thrombin by Glu351Ala and Glu351Arg were increased about 2-10 folds compared to wtPAI-1, but there was no change for tPA. CONCLUSION: The Asp355-Pro357 segment and Glu351 in distal hinge are involved in maintaining the inhibitory conformation of PAI-1. Glu351 is a specificity determinant of PAI-1 toward uPA, plasmin and thrombin, but not for tPA.

Endoscopic submucosal dissection of gastric fundus subepithelial tumors originating from the muscularis propria.

Endoscopic resection of gastric subepithelial tumors (SETs) carries a high risk of perforation, particularly for tumors located at the gastric fundus and originating from the muscularis propria. Based on our experience with endoscopic submucosal dissection (ESD) and a novel endoscopic device, namely the 'Resolution clip' for the endoscopic closure of iatrogenic upper gastrointestinal (upper GI) perforations, we evaluated the clinical feasibility and safety of ESD for gastric fundus subepithelial tumors originating from the muscularis propria. In this prospective study, 11 consecutive patients who presented with gastric SETs ≤3 cm in diameter were enrolled. Regardless of whether perforation occurred, the gastric wall defect was closed with clips. The patients were followed up after the surgery. Endoscopic resection was successfully performed in 10 patients; however, in one patient a pure endoscopic approach was impossible as the lesion was severely adhered to surrounding tissue, and a switch to laparoscopic wedge resection was necessary. The mean resected tumor size was 18.8×17.2 mm and the mean surgery time of the 10 patients with ESD was 81 min (range 45-130 min). Histological diagnosis was gastrointestinal stromal tumor (GIST) in eight lesions [very low risk according to the National Institutes of Health (NIH) risk classification] and leiomyoma in three lesions. Perforation occurred in 3/10 patients. Gastric closure with the Resolution clips was performed successfully in all cases. Early post-ESD bleeding (EPEB) occurred in one patient. Basic ferric sulfate solution was sprayed during the upper GI endoscopy examination and the bleeding stopped. No complications occurred and the follow-up was unremarkable. In this early study, ESD using the Resolution clip was demonstrated to be a feasible and minimally invasive treatment for gastric fundus subepithelial tumors originating from the muscularis propria.

Expression and significance of RKIP and E-cadherin in lung squamous cell carcinoma.

The purpose of this study was to investigate the expression of Raf kinase inhibitor protein (RKIP) and epithelial cadherin (E-cadherin) in lung squamous cell carcinoma tissue and its correlation with the clinical pathology of lung squamous cell carcinoma. RKIP and E-cadherin mRNA (by RT-PCR) and protein (by western blotting) levels were monitored in carcinoma tissues and surrounding normal tissues from 86 lung squamous cell carcinoma cases, and their positive rates were calculated. The rates of positive RKIP and E-cadherin mRNA expression were significantly lower in lung squamous cell carcinoma than in the surrounding normal tissues (P < 0.05). The positive expression rates were significantly lower in those with lymph node metastasis than in those without (P < 0.05). The lower the degree of tumor differentiation, the lower the E-cadherin mRNA positive expression rate (P < 0.05). The rates of positive RKIP and E-cadherin mRNA expression were significantly lower in patients at advanced (III, IV) stages than in patients at early (I, II) stages (p < 0.05); this rate, however, was independent of gender, age, and tumor size (P > 0.05). The protein levels of RKIP and E-cadherin were significantly lower in lung squamous cell carcinoma than in the surrounding normal tissues (P < 0.05). The levels were significantly lower in patients with lymph node metastasis than in those without it (P < 0.05). The lower the degree of tumor differentiation, the lower the protein level of E-cadherin (P < 0.05). Both RKIP and E-cadherin are tumor suppressors, their low expression levels may be associated with initiation, invasion and/or metastasis, as well as with the inhibition of lung squamous cell carcinoma differentiation.