Aryl Hydrocarbon Receptor Suppresses the Prostate Cancer LNCaP Cell Growth and Invasion by Promoting DNA Damage Response Under Oxidative Stress.

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that interacts with multiple signaling pathways during prostate development. In the present study, LNCaP cells were knocked down of AhR by siRNA, or treated with the AhR agonist 3-methylcholanthrene (3MC). The effects of AhR on LNCaP cells and the associated mechanisms were studied both under normal condition and under hydrogen peroxide (H2O2)-induced oxidative stress. MTT, transwell chamber assays and flow cytometry were employed to investigate cell proliferation, invasion, and apoptosis, respectively, whereas the DNA damage response (DDR) signaling (phosphorylation of ataxia-telangiectasia mutated [ATM], check-point kinase 2 [Chk2], histone H2AX, p53, and cleaved poly-ADP-ribose polymerase [PARP]) was detected by western blotting. Exposure of LNCaP cells to H2O2 inhibited their viability and migration, and induced apoptosis, at a greater extent compared with the culture under normal conditions. In addition, the oxidative stress increased p-ATM, p-Chk2, p-p53, and p-H2AX expression levels significantly. Knockdown of AhR attenuated the aforementioned effects caused by H2O2-induced oxidative stress. Activation of AhR by 3MC treatment, further aggravated these changes of LNCaP cells on oxidative stress. The findings indicated that AhR suppresses the viability and migration of LNCaP cells notably under oxidative stress, and this process is associated with positive regulation of the responses to oxidative DNA damage.

Reduction of α-dystroglycan expression is correlated with poor prognosis in glioma.

Dystroglycan (DG), a multifunctional protein dimer of non-covalently linked α and ß subunits, is best known as an adhesion and transduction molecule linking the cytoskeleton and intracellular signaling pathways to extracellular matrix proteins. Loss of DG binding, possibly by degradation or disturbed glycosylation, has been reported in a variety of cancers. DG is abundant at astroglial endfeet forming the blood-brain barrier (BBB) and glia limitans; so, we examined if loss of expression is associated with glioma. Expression levels of α-DG and ß-DG were assessed by immunohistochemistry in a series of 78 glioma specimens to determine the relationship with tumor grade and possible prognostic significance. α-DG immunostaining was undetectable in 44 of 49 high-grade specimens (89.8%) compared to 15 of 29 low-grade specimens (51.72%) (P<0.05). Moreover, loss of α-DG expression was an independent predictor of shorter disease-free survival (DFS) (hazards ratio (HR) = 0.142, 95% confidence interval (CI) 0.033-0.611, P=0.0088). Reduced expression of both α-DG and ß-DG was also a powerful negative prognostic factor for DFS (HR=2.556, 95% CI 1.403-4.654, P=0.0022) and overall survival (OS) (HR=2.193, 95% CI 1.031-4.666, P=0.0414). Lack of α-DG immunoreactivity is more frequent in high-grade glioma and is an independent predictor of poor clinical outcome. Similarly, lack of both α-DG and ß-DG immunoreactivity is a strong independent predictor of clinical outcome.

Overexpression of focal adhesion kinase correlates with increased lymph node metastasis and poor prognosis in non-small-cell lung cancer.

BACKGROUND: The aim of this study was to investigate whether focal adhesion kinase (FAK) overexpression correlates with lymph node metastases and prognosis. METHODS: The protein expression of FAK was investigated in 153 paraffin-embedded tissues by immunohistochemical analysis and then correlated with various clinicopathologic parameters. FAK mRNA level was detected with quantitative RT-PCR in 57 NSCLC frozen tissues and 20 normal matched tissues. RESULTS: Immunohistochemistry showed FAK overexpression was significantly associated with positive lymph node metastasis and more advanced disease stage of NSCLCs and adenocarcinoma subtype; real-time PCR also indicated a statistically significant correlation between increased FAK mRNA level and the presence of nodal metastases. Moreover, in survival analysis, FAK overexpression was significantly associated with worse overall survival. CONCLUSIONS: FAK overexpression is a promising pathological factor to predict aggressive behavior and prognosis in patients with NSCLC, particularly in the adenocarcinoma subtype.

Expression of the androgen receptor and its correlation with molecular subtypes in 980 chinese breast cancer patients.

BACKGROUND: Recent studies have shown that androgen displays an inhibitory effect on breast cancer cell lines that express androgen receptor (AR) but not estrogen receptor (ER) and progesterone receptor (PR). We have previously reported that approximately 1/3 of ER negative high grade invasive ductal carcinomas express AR. Thus, AR can serve as a potential therapeutic target for this group of patients. AIM: Here we investigated AR expression patterns in 980 consecutive breast carcinomas. RESULTS: We found that (1) AR was expressed more frequently (77%) than ER (61%) and PR (60%) in breast carcinomas; (2) AR expression was associated with ER and PR expression (P < 0.0001), small tumor size (P = 0.0324) and lower Ki-67 expression (P = 0.0013); (3) AR expression was found in 65% of ER negative tumors; (4) AR expression was associated with PR and Ki-67 in ER negative tumors, but not in ER positive tumors; (5) AR expression was higher in ER positive subtypes (Luminal A, Luminal B and Luminal HER2 subtypes, 80%-86%) and lower in ER negative subtypes [HER2, triple negative (TN), and TN EFGR positive subtypes; 52%-66%], with over 50% of TN tumors expressing AR. CONCLUSION: More breast carcinomas express AR than ER and PR, including significant numbers of ER negative and TN tumors, for which AR could serve as a potential therapeutic target.

[Expression and function of vascular endothelial growth factor and angiopoietins in rat brain after cerebral ischemia].

OBJECTIVE: To examine the temporal and spatial expression of vascular endothelial growth factor (VEGF) and angiopoietins (Ang) in rat brain after cerebral ischemia, and to elucidate the roles they played in angiogenesis and vascular permeability. METHODS: Rats were subjected to either middle cerebral artery occlusion (MCAO) or sham operation. Reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry were used to detect the expression of VEGF, Ang-1 and Ang-2 at different time points after ischemia. CD31 was used to label endothelial cells after MCAO. Vascular permeability was determined by Evans blue. RESULTS: VEGF was markedly increased at 2 h, had an initial peak at 12 h (0.7249 ± 0.1933, P < 0.01), and a second peak at 7 days (0.5264 ± 0.1519, P < 0.01). Ang-2 mRNA and protein significantly increased after MCAO, both of them peaked at 12 h (0.6747 ± 0.2416, P < 0.01; 1.1197 ± 0.1780, P < 0.01). In contrast, Ang-1 mRNA and protein gradually decreased after MCAO, respectively reaching a minimum at 3 d (0.3220 ± 0.1427, P < 0.01) and 1 d (0.1298 ± 0.0293, P < 0.01). Changes in the expression of these factors correlated with the progress of angiogenesis and vascular permeability. Evans blue test revealed that the vascular permeability gradually increased, and peaked at day 1 after ischemia [(6.219 ± 0.887) µg/g, P < 0.01]. CONCLUSION: Dynamic temporal changes in VEGF, Ang-1 and Ang-2 expression stimulate the cerebral angiogenesis after focal cerebral ischemia.

Hyalinizing trabecular tumor and papillary carcinoma of the thyroid.

BACKGROUND: Hyalinizing trabecular tumor (HTT) is a rare thyroid neoplasm, which shares some histologic features with thyroid papillary carcinoma (TPC). Clinically, it is frequently misdiagnosed as papillary carcinoma, even for some experienced pathologists. The aim of this study was to investigate whether HTT is variant of TPC or HTT is an independent entity of thyroid neoplasm. METHODS: The expression of CK19, galectin-3, HBME-1 and MIB-1 was detected by immunohistochemical staining in 12 cases of hyalinizing trabecular tumor and 20 cases of thyroid papillary carcinoma. RESULTS: Two of the 12 HTT samples were positive or focally positive for CK19. Four of the 12 samples of HTT presented positive to galectin-3; 3 were stained strongly and the other one was focally positive. None of the 12 samples of HTT was positive for HBME-1. Five in 12 HTT samples were stained in nucleus for MIB-1. Almost all the 20 cases of thyroid papillary carcinoma were intensely stained for CK19, galectin-3 and HBME-1. Fifteen in 20 cases of thyroid papillary carcinoma showed nuclear staining for MIB-1. CONCLUSIONS: HTT is an independent thyroid neoplasm, not a variant of TPC. This study could help in the differential diagnosis of HTT from TPC. CK19, galectin-3 and HBME-1 are adequate to identify HTT and TPC, but MIB-1 does not play an important role in discrimination between HTT and TPC.

Expression of thrombin and its associated protein in cerebellum of human and rat after intracerebral hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) can cause brain damage through a number of pathways. The purpose of the study was to explore the effect of thrombin, protease nexin-1 (PN-1) and protease activated receptor-1 (PAR-1) in rat and human cerebellum after ICH. METHODS: A model of ICH was produced in adult Sprague-Dawley rats by direct injection of autologous blood (50 microl) into caudate nucleus. Patients with injured hemorrhage were also enrolled in this study. Different expressions of thrombin, PAR-1, PN-1 were detected in rat and human cerebellum by immunohistochemistry and in situ hybridization. RESULTS: In rat cerebellum, thrombin protein significantly increased at 6 hours and reached the maximum 2 days after ICH. The expression of PAR-1 protein reached the maximum at 24 - 48 hours, and then began to decrease. The expression of PN-1 protein reached the maximum at 3 hours, decreased somewhat after that and increased a little at 5 days after ICH. While in human cerebellum, the changing tendency of thrombin, PAR-1 and PN-1 was almost conform to the rat. CONCLUSION: In cerebellum, thrombin can activate PAR-1 expression after ICH, and PN-1 appears quickly after ICH in order to control the deleterious effect of thrombin.

Reduced expression and novel splice variants of ING4 in human gastric adenocarcinoma.

ING4, a new member of the ING (inhibitor of growth) family of tumour suppressor genes, has been found to be deleted or down-regulated in gliomas, breast tumours, and head and neck squamous cell carcinomas. The goal of the present study was to investigate whether the expression and alternative splicing of ING4 transcripts are involved in the initiation and progression of stomach adenocarcinoma. ING4 mRNA and protein expression was examined in gastric adenocarcinoma tissues and human gastric adenocarcinoma cell lines by RT-PCR, real-time RT-PCR, tissue microarray immunohistochemistry, and western blot analysis. Alterations in ING4 transcripts were determined through sequence analysis of ING4 cDNA. Our data showed that ING4 mRNA and protein were dramatically reduced in stomach adenocarcinoma cell lines and tissues, and significantly less in female than in male patients. We also found that reduced ING4 mRNA expression correlated with the stage of the tumour. Interestingly, by sequence analysis, we discovered five novel aberrantly spliced variant forms of ING4_v1 and ING4_v2. These variants cause a codon frame-shift and, eventually, deletion of the NLS or PHD domain contributing to the mislocalization of p53 and/or HAT/HDAC complexes and, subsequently, altered gene expression in gastric adenocarcinoma. These results suggest that attenuated and aberrant ING4 expression may be involved in the initiation and progression of stomach adenocarcinoma.

[Influences of bFGF and EGF on the proliferation and differentiation of endogenous neural stem cells after cerebral infarction in human].

OBJECTIVE: To investigate the relationship of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) and proliferation of endogenous neural stem cells (NSCs) after human cerebral infarction. METHODS: Paraffin-embedded brain tissues of 22 human fatal cases of CI from the brain tissues around subventricular zone and subgranular layer zone were stained with HE and immunohistochemistry stain. The endogenous neural stem cells were marked by nestin. The expression changes of EGF, bFGF and nestin in the perihematomal tissues were analysed with the SPSS 13.0 system. RESULTS: (1) Compared with the controls, the number of nestin-positive cells increased at 24 - 72 h (14 +/- 6)/HP in the ipsilateral SVZ and began to rise at 4.5 - 10 h (11 +/- 5)/HP in the ipsilateral SGZ, reached maximum at 120 - 144 h ((38 +/- 7)/HP in the SVZ, (54 +/- 17)/HP in the SGZ, and decreased markedly at 216 - 336 h, but it was still elevated compared with the controls (P < 0.05). (2) The number of bFGF-positive cells increased at 4.5 - 10 h (8.1 +/- 2.9)/HP in the SVZ, (19.0 +/- 8.2)/HP in the SGZ, reached maximum at 24 - 70 h (15.6 +/- 3.5)/HP in the SVZ, (32.0 +/- 5.7)/HP in the SGZ and decreased at 72 - 96 h, but it was still elevated compared with the controls (P < 0.05). (3) The number of EGF-positive cells increased at 4.5 - 10 h (4.3 +/- 1.6)/HP in the SVZ, (7.0 +/- 3.7)/HP in the SGZ, reached maximum at 120 - 144 h (27.0 +/- 1.4)/HP in the SVZ, (51.5 +/- 4.9)/HP in the SGZ and decreased at 216 - 336 h, but it was still elevated compared with the controls (P < 0.05). CONCLUSIONS: Perhaps the increased expression of EGF and bFGF after CI was a reaction of endogenous reparation and it correlated with the proliferation and endogenous of neural stem cells in human.

[Expressions of heme oxygenase-1 and apoptosis-modulating proteins in peri-hematoma cortex after intracerebral hemorrhage in human being].

OBJECTIVE: To observe the expression of heme oxygenase-1 (HO-1) and Bcl-2, an apoptosis-modulating protein, in the neurons surrounding the hematoma in human being. METHODS: Specimens of cerebral cortex tissue 1 - 3 cm around the hemorrhagic focus with the size of 2.0 cm x 1.5 cm x 0.3 cm were collected during autopsy from 39 patients, 17 males and 22 females, aged 62.8 (36 - 84), who died from intracerebral hemorrhage 2 - 10 h, 17 - 30 h, 36 - 96 h, 120 - 216 h, or 240 - 408 h before. Specimens of brain tissue of the same size at the opposite side were collected as controls. Immunohistochemistry was used to examine the expression of HO-1 and Bcl-2 protein. RESULTS: (1) Expression of HO-1 could be detected in the specimens of the 2 h group, increased in the specimens of the 2 - 10 h group [(5.1 +/- 2.0)/HP], reached the peak in the 17 - 30 h group [(11.3 +/- 0.9)/HP], then began to decrease in the specimens of the 240 - 408 h group [(6.4 +/- 0.6)/HP] (F = 42.80, P < 0.001). The HO-1 expression of the control group remained negative at any time-point. (2) Expression of Bcl-2 could be detected in the specimens of the 2 - 10 h group [(4.2 +/- 1.7)/HP], was increased in the 17 - 30 h group [(6.6 +/- 0.5)/HP], reached the peak in the 36 approximately 96 h group [(8.9 +/- 1.1)/HP], then began to decrease, and was (4.7 +/- 0.6)/HP in the 240 approximately 408 h group (F = 29.59, P < 0.001). The Bcl-2 expression remained negative at any time point in the control group. (3) The expressions of HO-1 was positively correlated with the expression of Bcl-2 (r = 0.66, P < 0.001). CONCLUSIONS: Over-expression of HO-1 and Bcl-2 in the neurons provide a potential protection or destruction mechanism after intracerebral hemorrhage in human.

Cerebral ischemia and Alzheimer's disease: the expression of amyloid-beta and apolipoprotein E in human hippocampus.

Growing evidence suggests a synergistic and perhaps etiological relationship between vascular disease and Alzheimer's disease (AD), which is characterized by the progressive accumulation of amyloid-beta peptide (Abeta). Moreover, apolipoprotein E (ApoE) has also been shown to be associated with AD and cerebral ischemia. It seems that cerebral ischemia may play an important, both direct and indirect, role in the pathogenesis of AD. We investigated the expression and distribution of Abeta1-40, beta1-42 and ApoE in human hippocampus after cerebral ischemia in this study to determine the role of cerebral ischemia in Alzheimer's disease. Our study has demonstrated that the accumulation of both Abeta1-40 and beta1-42 were increased dramatically and consistently after cerebral ischemia. Neuronal ApoE immunoreactivity was also significantly increased in all ischemic groups compared with controls. The most likely stimulus for the increased Abeta1-40, Abeta1-42 and ApoE immunoreactivity in the CA1 and CA3 neurons is the ischemic conditions, and their upregulation, in turn, may partly explain the contribution of cerebral ischemia to the pathogenesis of AD. Therefore our observations provide a basis for establishing therapeutic strategies aimed at preventing ischemic insults and subsequent neurodegeneration in AD.

[Clinicopathologic study of ischemic intestinal disease due to mesenteric venous lesions].

OBJECTIVE: To study the clinical and pathologic features of ischemic intestinal disease due to mesenteric phlebitis. METHOD: The clinical and pathologic features of the mesenteric venous lesions in 3 patients of ischemic intestinal disease admitted during the period from 2003 to 2004 were studied. RESULTS: All 3 patients had a clinical history of acute abdominal pain accompanying with a diffuse peritonitis. During operation, an infarcted intestinal segment was identified and was resected respectively in each patient. Histologic examination showed a lymphocytic infiltration and fibrinoid necrosis of the small to medium-sized veins, associated with mural thrombosis and infarction of the corresponding intestinal wall and mesentery. The mesenteric arteries were spared. Two-year follow up of one case showed no evidence of local recurrence or systemic vasculitis. CONCLUSIONS: Ischemic intestinal disease due to mesenteric phlebitis is a rare entity with a pathological feature of inflammation of venous wall accompanying with the development of mural thrombosis and subsequent haemorrhagic infarction of intestine. The etiology is unknown and surgical resection of the involved intestinal segment is usually recommended.

Clinicopathological and immunohistochemical analysis of gastrointestinal stromal tumor.

AIM: To investigate the clinicopathological features of gastrointestinal stromal tumor (GIST) and to study the reference indexes for malignancy. METHODS: Fifty-two cases of primary GIST were distinguished from a group of gastrointestinal mesenchymal tumors using a panel of antibodies such as CD117 and CD34 by immunohistochemical SP method. Their biological behaviors were analyzed using the expression of p21WAF1 and Bax in 52 cases of GIST. RESULTS: Grossly, the tumor size was between 1.5 cm and 13 cm (mean: 5.5 cm). Focal areas of hemorrhage, necrosis, or small cyst formation could be seen. Microscopically, the tumor was composed of spindle cells (20 cases), epithelioid cells (20 cases) and mixed cells (12 cases). Immunohistochemically, CD117 and CD34 showed diffuse strong positive expressions, the positive rates were 98.1% and 92.3%. SMA, S-100, NSE, NF and MBP showed focal positive expressions, the positive rates were 48.1%, 28.8%, 25%, 21.2% and 42.3% respectively. Vimentins were all positive desmin and CgA were all negative. In normal adult stomach and intestine, the immunoreactive staining for CD117 and CD34 showed immunoreactive interstitial cells of Cajal in myenteric neuroplexus. Among the 52 cases of GIST, 27 were positive for p21WAF1 (51.9%), 29 for Bax (55.8%). The expression of p21WAF1 and Bax had no significent difference with the localization, size, histological subtype of GIST, but had a significent difference with the histological grade (P = 0.000, respectively). p21WAF1 expression had a positive correlation to Bax expression (r = 0.461, P = 0.001, kappa = 0.459). CONCLUSION: GIST has complicated arrangements and various cell types. Positivity of CD117 and CD34 is the most valuable factor in diagnosing GIST. Expression of p21WAF1 and Bax plays an important role in potential malignancy and malignancy rather than in benign GIST. p21WAF1 and Bax may be used as the markers in the assessment of GIST malignant potential.

[Expression of amyloid beta protein and amyloid precursor protein after focal brain ischemia in human].

OBJECTIVE: To observe the expression of amyloid beta precursor protein and amyloid beta protein in neurons of hippocampal CA1 region after brain ischemia in human. METHODS: Neuronal damage was examined by using HE staining, and expression of APP and Abeta(1-40) was determined by immunohistochemistry in the hippocampal CA1 region of the brain specimens of 43 patients who died 2 h-6 h, 7 h-24 h, 25 h-48 h, 49 h-72 h, 73 h-96 h, 97 h-144 h, or 145 h-168 h after cerebral ischemia and in 2 specimens of patients who died of other diseases as control group. RESULTS: The expression of Abeta(1-40) was 25.07 +/- 2.79 in the specimens 2 h-6 h after cerebral ischemia, peaked 73 h-96 h after cerebral ischemia (33.22 +/- 2.67), then decreased till 145 h-168 h after cerebral ischemia, however, all higher than that in the control group (2.88 +/- 0.18, all P < 0.05). The expression of beta-APP was 33.30 +/- 0.42 2 h-6 h after cerebral ischemia, was 28.11 +/- 2.03 7 h-24 h after cerebral ischemia, increased to peak value (32.32 +/- 1.36) 73 h-96 h after cerebral ischemia, and then decreased to 28.48 +/- 2.01, all higher than that of the control group (25.90 +/- 1.55) with significant differences between those 2 h-6 h and 73 h-96 h after ischemia and that of the control group (both P < 0.01). The increase of beta-APP was positively correlated with the expression of Abeta(1-40) 24 h after ischemia. CONCLUSION: The expression of beta-APP and that of Abeta(1-40) are up-regulated after cerebral ischemia, thus aggravating cerebral ischemia.

Correlation between neuronal injury and Caspase-3 after focal ischemia in human hippocampus.

BACKGROUND: Cerebral ischemia is a significant clinical problem, and cerebral ischemia usually causes neuron injury such as apoptosis in various brain areas, including hippocampus. Cysteinyl aspartate-specific protease (Caspases) are fundamental factors of apoptotic mechanism. Caspase-3 inhibitors show effect in attenuating brain injury after ischemia. But all the results were from animal models in research laboratories. This study aimed at investigating the correlation between the change of ischemic neuronal injury and Caspase-3 post-ischemia in human hippocampus. METHODS: We selected and systematized 48 post-mortem specimens from 48 patients, who died of cerebral infarction. Morphological change was firstly analyzed by observing hematoxyline/eosin-staining hippocampal sections. The expression of Caspase-3 was investigated using the methods of in situ hybridization and immunohistochemistry. Terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick-end labeling (TUNEL) method was used to clarify the involvement of Caspase-3 in neuron death. The loss of MAP 2 (MAP-2) was applied to judging the damaged area and degree of neuronal injury caused by ischemia. RESULTS: In the CA1 sector of hippocampus, Caspase-3 immunostaining modestly increased at 8 hours [8.05/high-power field (hpf)], dramatically increased at 24 hours (24.85/hpf), decreased somewhat after 72 hours. Caspase-3 mRNA was detectable at 4 hours (6.75/hpf), reached a maximum at 16 hours (17.60/hpf), faded at 72 hours. TUNEL-positive cells were detectable at 24 hours (10.76/hpf), markedly increased at 48 - 72 hours. The loss of MAP-2 was obviously detected at 4 hours, progressed significantly between 24 and 72 hours; MAP-2 immunoreactivity was barely detectable at 72 hours. Before 72 hours, the Caspase-3 evolution was related with the upregulation of TUNEL and the loss of MAP-2. The positive correlation between Caspase-3 mRNA and TUNEL was significant at the 0.05 level (correlation coefficient was 0.721); the negative correlation between Caspase-3 mRNA and MAP-2 was significant at the 0.05 level (correlation coefficient is 0.857). In the early stage (before 72 hours), the staining of Caspase-3 mRNA and immunohistochemistry was predominantly present in cytoplasm; the staining of TUNEL was predominantly localized in nucleus. At 4 - 16 hours, most neurons in hippocampal CA1 areas had relatively normal morphology; at 24 - 48 hours, neurons showed apoptotic morphology; at 72 hours, most cells showed significantly pathological morphology. CONCLUSIONS: There exist a time-dependent evolution of neuronal damage after hippocampal ischemia in human brain, which was characterized by its close correspondence to Caspase-3.