Melatonin inhibits cell proliferation in a rat model of breast hyperplasia by mediating the PTEN/AKT pathway.

In the present study, a rat model of breast hyperplasia was established via the administration of estradiol benzoate and progesterone. Subsequent changes associated with breast hyperplasia were then investigated by measuring the diameter and height of the nipples and by staining breast tissue with hematoxylin and eosin. The proliferation and apoptosis of hyperplastic cells in the breast tissue were then determined by analyzing the expression of proliferating cell nuclear antigen (PCNA) and cleaved­caspase­3 by immunohistochemistry and TUNEL staining. We also determined the expression of proteins associated with the phosphatase and tensin homolog (PTEN)/protein kinase B (AKT) signaling pathway by western blotting. Melatonin treatment led to a significant reduction in the degree of breast hyperplasia (P<0.05), a significant reduction in PCNA, a significant increase in the level of apoptosis (P<0.05), a significant increase in PTEN (P<0.05), and a significant reduction in AKT/p­AKT (P<0.05). Furthermore, melatonin significantly decreased the aggravation of breast hyperplasia induced by application of a PTEN inhibitor. Melatonin reduced the degree of breast hyperplasia, reduced the proliferation of hyperplastic breast tissue cells, and promoted cell apoptosis in hyperplastic tissue. These effects were achieved by the specific regulation of proteins in the PTEN/PI3K/AKT axis.

MT1-MMP evaluation in neointimal hyperplasia in the late follow-up after prosthesis implantation.

Vascular surgical interventions are often burdened with late complications, including thrombosis or restenosis. The latter is generally caused by neointimal hyperplasia. Although extracellular matrix (ECM) remodelling is an important part of neointima formation, this process is not clearly understood. The aim of the study was to assess the content and activity of membrane-type 1 matrix metalloproteinase in human neointima in the late stages of its development. Matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase-2 were also evaluated. The research was performed on neointima samples collected during secondary vascular interventions from patients with chronic limb ischaemia who developed vascular occlusion at 6-18 months after aorto/ilio-femoral bypass grafting. The control material consisted of segments of femoral arteries collected from organ donors. Western blot and/or ELISA were used for the determination of MT1-MMP and TIMP-2 expression. The activity of MT1-MMP was measured by fluorometric assay and that of MMP-2 by zymography. We demonstrated significantly increased MT1-MMP protein content in neointima when compared to normal arteries. However, the activity of MT1-MMP was significantly lower in neointima than in control samples. The decreased MT1-MMP activity was concomitant with reduced activity of MMP-2. The TIMP-2 protein levels in neointima and normal arteries were not significantly different. The results of our study suggest that the reduced activity of MT1-MMP and consequently MMP-2 in human neointima may play a role in decreased degradation of ECM components and thus promote neointimal overgrowth.

GZMB gene silencing confers protection against synovial tissue hyperplasia and articular cartilage tissue injury in rheumatoid arthritis through the MAPK signaling pathway.

INTRODUCTION: Rheumatoid arthritis (RA) represents the most commonly occurring inflammatory type of arthritis and is a major cause of disability. Reports have placed emphasis on the potential of, granzyme B (GZMB) as a potentially valuable prognostic marker in early RA, the mechanism of which still remains largely unclear. Thus, the aim of the current study was to investigate the effects GZMB gene silencing influences synovial tissue hyperplasia and articular cartilage tissue injury of RA through the regulation of the MAPK signaling pathway. METHODS: Following the successful establishment of the collagen-induced animal model of RA in rats, a five-grade scoring method was applied to evaluate the swelling degree measurement of the rats for model identification. The various rat responses to GZMB shRNA and U-46619 (activator of the MAPK signaling pathway) were subsequently detected. The general status of rats was observed and recorded, with their weight and ankle diameter kept accurate record of. ELISA was employed to detect the levels of inflammatory cytokines, while RT-qPCR and Western blotting techniques were applied to determine the expressions of GZMB and pathway-related genes and proteins. RESULTS: GZMB gene silencing was observed to aid in the maintenance of rat weight increases, while acting to reduce the degree of ankle swelling, while hypertrophy of the synovial tissue and the injury of the articular cartilage tissue were not obvious. GZMB gene silencing was shown to decrease inflammatory cytokine levels, as well as decreased bcl-2, Cyclin D1, VEGF and bFGF while increasing caspase 3. Notably, GZMB gene silencing suppressed the activation of the MAPK signaling pathway by reducing the phosphorylation extent of ERK and MEK. CONCLUSION: Taken together, the key findings of the present study ultimately suggest that GZMB gene silencing acts to inhibit MAPK signaling pathway through regulating the expressions of inflammatory factors, factors correlated with apoptosis (bcl-2 and caspase), as well as factors associated with angiogenesis (VEGF and bFGF), thus relieving synovial tissue hyperplasia and articular cartilage tissue injury brought about by RA. The GZMB gene could well be a new therapeutic target for RA treatment.

Genetic Disruption of 21-Hydroxylase in Zebrafish Causes Interrenal Hyperplasia.

Congenital adrenal hyperplasia is a group of common inherited disorders leading to glucocorticoid deficiency. Most cases are caused by 21-hydroxylase deficiency (21OHD). The systemic consequences of imbalanced steroid hormone biosynthesis due to severe 21OHD remains poorly understood. Therefore, we developed a zebrafish model for 21OHD, which focuses on the impairment of glucocorticoid biosynthesis. A single 21-hydroxylase gene (cyp21a2) is annotated in the zebrafish genome based on sequence homology. Our in silico analysis of the 21-hydroxylase (Cyp21a2) protein sequence suggests a sufficient degree of similarity for the usage of zebrafish cyp21a2 to model aspects of human 21OHD in vivo. We determined the spatiotemporal expression patterns of cyp21a2 by whole-mount in situ hybridization and reverse transcription polymerase chain reaction throughout early development. Early cyp21a2 expression is restricted to the interrenal gland (zebrafish adrenal counterpart) and the brain. To further explore the in vivo consequences of 21OHD we created several cyp21a2 null-allele zebrafish lines by using a transcription activator-like effector nuclease genomic engineering strategy. Homozygous mutant zebrafish larvae showed an upregulation of the hypothalamic-pituitary-interrenal (HPI) axis and interrenal hyperplasia. Furthermore, Cyp21a2-deficient larvae had a typical steroid profile, with reduced concentrations of cortisol and increased concentrations of 17-hydroxyprogesterone and 21-deoxycortisol. Affected larvae showed an upregulation of the HPI axis and interrenal hyperplasia. Downregulation of the glucocorticoid-responsive genes pck1 and fkbp5 indicated systemic glucocorticoid deficiency. Our work demonstrates the crucial role of Cyp21a2 in glucocorticoid biosynthesis in zebrafish larvae and establishes an in vivo model allowing studies of systemic consequences of altered steroid hormone synthesis.

TdT expression in normal and neoplastic sebaceous cells.

AIMS: Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase expressed in immature, normal and neoplastic, lymphoid or haematopoietic cells and in neuroendocrine carcinomas, such as Merkel cell carcinoma and small-cell carcinoma. It has not yet been described in cells of epithelial origin. After observing TdT immunoreactivity in normal sebaceous glands, we analysed its spectrum of expression in cases of sebaceous cell hyperplasia (SGH) and sebaceous cell neoplasm. METHODS AND RESULTS: Twelve cases of SGH and three cases of other benign lesions, namely sebaceoma, sebaceous adenoma, and sebaceous naevus, along with four archived cases of sebaceous cell carcinoma (SC) were collected and stained with TdT antibody. In addition, tissue microarrays were constructed from 11 cases of basal cell carcinoma (BCC) and 10 cases of squamous cell carcinoma (SCC), which had nine evaluable cases each, and, after carcinoma type confirmation with immunostaining for epithelial membrane antigen, TdT immunohistochemistry was performed. All cases of SGH and sebaceous cell neoplasm were positive for TdT. The staining intensity was variable, being often weak to moderate in a significant proportion of cells, apart from one case of SC and the case of sebaceous naevus, which were only focally positive. No BCCs and only one SCC showed immunoreactivity. CONCLUSIONS: TdT protein can be found in cells of epithelial origin and specifically sebaceous cells, both benign and malignant. It can be hypothesized that this expression is due to sebaceous cell differentiation as a prelude to apoptosis and holocrine secretion. Additional studies are needed to further elucidate its biological role.

A first immunohistochemistry study of transketolase and transketolase-like 1 expression in canine hyperplastic and neoplastic mammary lesions.

BACKGROUND: Canine mammary tumors represent the most common neoplasm in female dogs, and the discovery of cancer biomarkers and their translation to clinical relevant assays is a key requirement in the war on cancer. Since the description of the 'Warburg effect', the reprogramming of metabolic pathways is considered a hallmark of pathological changes in cancer cells. In this study, we investigate the expression of two cancer-related metabolic enzymes, transketolase (TKT) and transketolase-like 1 (TKTL1), involved in the pentose phosphate pathway (PPP), an alternative metabolic pathway for glucose breakdown that could promote cancer by providing the precursors and energy required for rapidly growing cells. RESULTS: TKT and TKTL1 protein expression was investigated by immunohistochemistry in canine normal (N = 6) and hyperplastic glands (N = 3), as well as in benign (N = 11) and malignant mammary tumors (N = 17). TKT expression was higher in hyperplastic lesions and in both benign and malignant tumors compared to the normal mammary gland, while TKTL1 levels were remarkably higher in hyperplastic lesions, simple adenomas and simple carcinomas than in the normal mammary glands (P < 0.05). CONCLUSIONS: This study reveals that the expression of a key PPP enzyme varies along the evolution of canine mammary neoplastic lesions, and supports a role of metabolic changes in the development of canine mammary tumors.

Immunoexpression of cleaved caspase-3 shows lower apoptotic area indices in lip carcinomas than in intraoral cancer.

OBJECTIVE: This study aimed to evaluate apoptosis by assessing cleaved caspase-3 immunoexpression in hyperplastic, potentially malignant disorder (PMD), and malignant tumors in intraoral and lower lip sites. MATERIAL AND METHODS: A retrospective study using paraffin blocks with tissues from patients with inflammatory fibrous hyperplasia (IFH), actinic cheilitis, oral leukoplakia, lower lip and intraoral squamous cell carcinoma (SCC) was performed. The tissues were evaluated by immunohistochemical analysis with anti-cleaved caspase-3 antibody. Apoptotic area index was then correlated with lesion type. RESULTS: From 120 lesions assessed, 55 (46%) were cleaved caspase-3-positive. The SCC samples (n=40) had the highest apoptotic area indices (n=35; 87.5%). Significant differences were detected between SCCs and PMDs (p=0.0003), as well as SCCs and IFHs (p=0.001), regarding caspase-3 immunopositivity. Carcinomas of the lower lip had lower apoptotic area indices than intraoral cancer (p=0.0015). CONCLUSIONS: Cleaved caspase-3 immunoexpression showed differences in oral SCCs and PMDs and demonstrated a distinct role of apoptosis in carcinogenesis of intraoral and lower lip cancer. In future, the expression of cleaved caspase-3 with other target molecules in oral cancer may be helpful in delineating the prognosis and treatment of these tumors.

Immunoexpression of cleaved caspase-3 shows lower apoptotic area indices in lip carcinomas than in intraoral cancer

ABSTRACT Objective This study aimed to evaluate apoptosis by assessing cleaved caspase-3 immunoexpression in hyperplastic, potentially malignant disorder (PMD), and malignant tumors in intraoral and lower lip sites. Material and Methods A retrospective study using paraffin blocks with tissues from patients with inflammatory fibrous hyperplasia (IFH), actinic cheilitis, oral leukoplakia, lower lip and intraoral squamous cell carcinoma (SCC) was performed. The tissues were evaluated by immunohistochemical analysis with anti-cleaved caspase-3 antibody. Apoptotic area index was then correlated with lesion type. Results From 120 lesions assessed, 55 (46%) were cleaved caspase-3-positive. The SCC samples (n=40) had the highest apoptotic area indices (n=35; 87.5%). Significant differences were detected between SCCs and PMDs (p=0.0003), as well as SCCs and IFHs (p=0.001), regarding caspase-3 immunopositivity. Carcinomas of the lower lip had lower apoptotic area indices than intraoral cancer (p=0.0015). Conclusions Cleaved caspase-3 immunoexpression showed differences in oral SCCs and PMDs and demonstrated a distinct role of apoptosis in carcinogenesis of intraoral and lower lip cancer. In future, the expression of cleaved caspase-3 with other target molecules in oral cancer may be helpful in delineating the prognosis and treatment of these tumors.

The expression and prognostic relevance of indoleamine 2,3-dioxygenase in tongue squamous cell carcinoma.

Conclusion IDO might be useful for predicting progression of primary tumor stage T2 and T3 in tongue squamous cell carcinoma (TSCC), but does not seem like a specific biomarker for diagnosing TSCC and predicting patient survival. Objectives Indoleamine 2,3-dioxygenase (IDO) is expressed in many cells and it catabolises the essential amino acid tryptophan to kynurenine. IDO acts as an immune modulator through suppression of T-cell immunity and other pathways. In cancer cells, IDO has been proposed to promote tumor progression by enabling malignant cells to escape from the immune system. The aim of this study was to evaluate the association and prognostic relevance of IDO expression in TSCC. Method One hundred and eight retrospective tongue and lymph node specimens were stained immunohistochemically with monoclonal antibody anti-indoleamine 2,3-dioxygenase. The relative abundance of IDO positive epithelial cells, IDO staining intensity, and inflammation were assessed semi-quantitatively with light microscopy. Results IDO was expressed stronger in tongue hyperplasia than in TSCC. However, IDO expression associated with poor survival in the sub-groups with primary tumor stage T2-T4 and in the sub-group with strong inflammation in tumors' invasive front.

Targeting thiamine-dependent enzymes for metabolic therapies in oral squamous cell carcinoma?

Purpose. Thiamine-dependent enzymes (TDEs) linking glycolysis with the tricarboxylic acid cycle (TCA) pyruvate dehydrogenase (PDH), of the pentose phosphate pathway transketolases (TKTs), the TCA alpha-ketoglutarate deydrogenase (KGDH)/2-oxoglutarate dehydrogenase (OGDH) complex, and the amino acid catabolism branched-chain alpha-ketoacid dehydrogenase (BCKDH) complex are crucial factors for tumor metabolism. The expression of these enzymes has not been analyzed for carcinogenesis of oral squamous cell carcinoma (OSCC) with special focus on new targeted metabolic therapies as yet. Methods. TDEs PDH, KGDH (OGDH), and BCKDH were analyzed in normal oral mucosa (n = 14), oral precursor lesions (simple hyperplasia, n = 21; squamous intraepithelial neoplasia, SIN I-III, n = 35), and OSCC specimen (n = 46) by immunohistochemistry and western blot (WB) analysis in OSCC tumor cell lines. Results. Although the total numbers of PDH and KGDH (OGDH) positive samples decreased in OSCC, both enzymes were significantly overexpressed in the carcinogenesis of OSCC compared with normal tissue. BCKDH has been demonstrated to be significantly overexpressed in the carcinogenesis of OSCC. Specificity of the antibodies was confirmed by WB analysis. Conclusions. This is the first study showing increased expression of TDEs in OSCC. Metabolic targeting of TDEs (including TKTs) by antagonistic compounds like oxythiamine or oxybenfothiamine may be a useful strategy to sensitize cancer cells to common OSCC cancer therapies (AU)

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Gingerol Inhibits Serum-Induced Vascular Smooth Muscle Cell Proliferation and Injury-Induced Neointimal Hyperplasia by Suppressing p38 MAPK Activation.

PURPOSE: Gingerol inhibits growth of cancerous cells; however, its role in vascular smooth muscle cell (VSMC) proliferation is not known. The present study investigated the effect of gingerol on VSMC proliferation in cell culture and during neointima formation after balloon injury. METHOD AND RESULTS: Rat VSMCs or carotid arteries were harvested at 15 minutes, 30 minutes, 1, 6, 12, and 24 hours of fetal bovine serum (FBS; 10%) stimulation or balloon injury, respectively. Gingerol prevented FBS (10%)-induced proliferation of VSMCs in a dose-dependent manner (50 µmol/L-400 µmol/L). The FBS-induced proliferating cell nuclear antigen (PCNA) upregulation and p27(Kip1) downregulation were also attenuated in gingerol (200 µmol/L) pretreated cells. Fetal bovine serum-induced p38 mitogen-activated protein kinase (MAPK) activation, PCNA upregulation, and p27(Kip1) downregulation were abrogated in gingerol (200 µmol/L) and p38 MAPK inhibitor (SB203580, 10 µmol/L) pretreated cells. Balloon injury induced time-dependent p38 MAPK activation in the carotid artery. Pretreatment with gingerol (200 µmol/L) significantly attenuated injury-induced p38 MAPK activation, PCNA upregulation, and p27(Kip1) downregulation. After 14 days of balloon injury, intimal thickening, neointimal proliferation, and endothelial dysfunction were significantly prevented in gingerol pretreated arteries. In isolated organ bath studies, gingerol (30 nmol/L-300 µmol/L) inhibited phenylephrine-induced contractions and induced dose-dependent relaxation of rat thoracic aortic rings in a partially endothelium-dependent manner. CONCLUSION: Gingerol prevented FBS-induced VSMC proliferation and balloon injury-induced neointima formation by regulating p38 MAPK. Vasodilator effect of gingerol observed in the thoracic aorta was partially endothelium dependent. Gingerol is thus proposed as an attractive agent for modulating VSMC proliferation, vascular reactivity, and progression of vascular proliferative diseases.

Hyaluronidases and hyaluronan synthases expression is inversely correlated with malignancy in lung/bronchial pre-neoplastic and neoplastic lesions, affecting prognosis

We collected a series of 136 lung/bronchial and 56 matched lung parenchyma tissue samples from patients who underwent lung/bronchial biopsies and presented invasive carcinoma after lung surgery. The lung/bronchial samples included basal cell hyperplasia, squamous metaplasia, moderate dysplasia, adenomatous hyperplasia, severe dysplasia, squamous cell carcinoma and adenocarcinoma. Matched lung parenchyma tissue samples included 25 squamous cell carcinomas and 31 adenocarcinomas. Immunohistochemistry was performed to analyze for the distribution of hyaluronidase (Hyal)-1 and −3, and hyaluronan synthases (HAS)-1, −2, and −3. Hyal-1 showed significantly higher expression in basal cell hyperplasia than in moderate dysplasia (P=0.01), atypical adenomatous hyperplasia (P=0.0001), or severe dysplasia (P=0.03). Lower expression of Hyal-3 was found in atypical adenomatous hyperplasia than in basal cell hyperplasia (P=0.01) or moderate dysplasia (P=0.02). HAS-2 was significantly higher in severe dysplasia (P=0.002) and in squamous metaplasia (P=0.04) compared with basal cell hyperplasia. HAS-3 was significantly expressed in basal cell hyperplasia compared with atypical adenomatous hyperplasia (P=0.05) and severe dysplasia (P=0.02). Lower expression of HAS-3 was found in severe dysplasia compared with squamous metaplasia (P=0.01) and moderate dysplasia (P=0.01). Epithelial Hyal-1 and −3 and HAS-1, −2, and −3 expressions were significantly higher in pre-neoplastic lesions than in neoplastic lesions. Comparative Cox multivariate analysis controlled by N stage and histologic tumor type showed that patients with high HAS-3 expression in pre-neoplastic cells obtained by lung/bronchial biopsy presented a significantly higher risk of death (HR=1.19; P=0.04). We concluded that localization of Hyal and HAS in lung/bronchial pre-neoplastic and neoplastic lesions was inversely related to malignancy, which implied that visualizing these factors could be a useful diagnostic procedure for suspected lung cancer. Finalizing this conclusion will require a wider study in a randomized and prospective trial.

Hyaluronidases and hyaluronan synthases expression is inversely correlated with malignancy in lung/bronchial pre-neoplastic and neoplastic lesions, affecting prognosis.

We collected a series of 136 lung/bronchial and 56 matched lung parenchyma tissue samples from patients who underwent lung/bronchial biopsies and presented invasive carcinoma after lung surgery. The lung/bronchial samples included basal cell hyperplasia, squamous metaplasia, moderate dysplasia, adenomatous hyperplasia, severe dysplasia, squamous cell carcinoma and adenocarcinoma. Matched lung parenchyma tissue samples included 25 squamous cell carcinomas and 31 adenocarcinomas. Immunohistochemistry was performed to analyze for the distribution of hyaluronidase (Hyal)-1 and -3, and hyaluronan synthases (HAS)-1, -2, and -3. Hyal-1 showed significantly higher expression in basal cell hyperplasia than in moderate dysplasia (P=0.01), atypical adenomatous hyperplasia (P=0.0001), or severe dysplasia (P=0.03). Lower expression of Hyal-3 was found in atypical adenomatous hyperplasia than in basal cell hyperplasia (P=0.01) or moderate dysplasia (P=0.02). HAS-2 was significantly higher in severe dysplasia (P=0.002) and in squamous metaplasia (P=0.04) compared with basal cell hyperplasia. HAS-3 was significantly expressed in basal cell hyperplasia compared with atypical adenomatous hyperplasia (P=0.05) and severe dysplasia (P=0.02). Lower expression of HAS-3 was found in severe dysplasia compared with squamous metaplasia (P=0.01) and moderate dysplasia (P=0.01). Epithelial Hyal-1 and -3 and HAS-1, -2, and -3 expressions were significantly higher in pre-neoplastic lesions than in neoplastic lesions. Comparative Cox multivariate analysis controlled by N stage and histologic tumor type showed that patients with high HAS-3 expression in pre-neoplastic cells obtained by lung/bronchial biopsy presented a significantly higher risk of death (HR=1.19; P=0.04). We concluded that localization of Hyal and HAS in lung/bronchial pre-neoplastic and neoplastic lesions was inversely related to malignancy, which implied that visualizing these factors could be a useful diagnostic procedure for suspected lung cancer. Finalizing this conclusion will require a wider study in a randomized and prospective trial.

Improving arteriovenous fistula patency: Transdermal delivery of diclofenac reduces cannulation-dependent neointimal hyperplasia via AMPK activation.

Creation of an autologous arteriovenous fistula (AVF) for vascular access in haemodialysis is the modality of choice. However neointimal hyperplasia and loss of the luminal compartment result in AVF patency rates of ~60% at 12months. The exact cause of neointimal hyperplasia in the AVF is poorly understood. Vascular trauma has long been associated with hyperplasia. With this in mind in our rabbit model of AVF we simulated cannulation autologous to that undertaken in vascular access procedures and observed significant neointimal hyperplasia as a direct consequence of cannulation. The neointimal hyperplasia was completely inhibited by topical transdermal delivery of the non-steroidal anti-inflammatory (NSAID) diclofenac. In addition to the well documented anti-inflammatory properties we have identified novel anti-proliferative mechanisms demonstrating diclofenac increases AMPK-dependent signalling and reduced expression of the cell cycle protein cyclin D1. In summary prophylactic transdermal delivery of diclofenac to the sight of AVF cannulation prevents adverse neointimal hyperplasic remodelling and potentially offers a novel treatment option that may help prolong AVF patency and flow rates.

Knockout of histidine decarboxylase decreases bile duct ligation-induced biliary hyperplasia via downregulation of the histidine decarboxylase/VEGF axis through PKA-ERK1/2 signaling.

Histidine is converted to histamine by histidine decarboxylase (HDC). We have shown that cholangiocytes 1) express HDC, 2) secrete histamine, and 3) proliferate after histamine treatment via ERK1/2 signaling. In bile duct-ligated (BDL) rodents, there is enhanced biliary hyperplasia, HDC expression, and histamine secretion. This studied aimed to demonstrate that knockdown of HDC inhibits biliary proliferation via downregulation of PKA/ERK1/2 signaling. HDC(-/-) mice and matching wild-type (WT) were subjected to sham or BDL. After 1 wk, serum, liver blocks, and cholangiocytes were collected. Immunohistochemistry was performed for 1) hematoxylin and eosin, 2) intrahepatic bile duct mass (IBDM) by cytokeratin-19, and 3) HDC biliary expression. We measured serum and cholangiocyte histamine levels by enzyme immunoassay. In total liver or cholangiocytes, we studied: 1) HDC and VEGF/HIF-1α expression and 2) PCNA and PKA/ERK1/2 protein expression. In vitro, cholangiocytes were stably transfected with shRNA-HDC plasmids (or control). After transfection we evaluated pPKA, pERK1/2, and cholangiocyte proliferation by immunoblots and MTT assay. In BDL HDC(-/-) mice, there was decreased IBDM, PCNA, VEGF, and HDC expression compared with BDL WT mice. Histamine levels were decreased in BDL HDC(-/-). BDL HDC(-/-) livers were void of necrosis and inflammation compared with BDL WT. PKA/ERK1/2 protein expression (increased in WT BDL) was lower in BDL HDC(-/-) cholangiocytes. In vitro, knockdown of HDC decreased proliferation and protein expression of PKA/ERK1/2 compared with control. In conclusion, loss of HDC decreases BDL-induced biliary mass and VEGF/HIF-1α expression via PKA/ERK1/2 signaling. Our data suggest that HDC is a key regulator of biliary proliferation.

Suppression of stent-induced tissue hyperplasia in rats by using small interfering RNA to target matrix metalloproteinase-9.

BACKGROUND AND STUDY AIMS: We evaluated the efficacy of small interfering RNA (siRNA) in targeting matrix metalloproteinase (MMP-9) to suppress stent-induced tissue hyperplasia in a rat esophageal model. METHODS: The silencing effect of the candidate siRNA (termed (MMP-9 siRNA) was evaluated in 9 L rat glial cells. Four groups of rats (n = 10, each group) were used: Eso-S, stent insertion only, comparison; Eso-R, stent insertion plus treatment with MMP-9 siRNA complexed with Chol-R9 for delivery, experimental; Eso-P, stent insertion plus treatment with pCMV-luc complexed with Chol-R9, for confirmation of Chol-R9 delivery effect; and Eso-N, no stent insertion and no treatment, controls. All rats were sacrificed at 3 weeks. The therapeutic efficacy of the MMP-9 siRNA/Chol-R9 complex was assessed. RESULTS: The most potent MMP-9 siRNA was selected. Compared with the Eso-S group, the Eso-R group showed significantly less tissue hyperplasia with a lower percentage of granulation tissue and smaller granulation tissue area, and also significantly lower MMP-9 level. CONCLUSIONS: MMP-9 siRNA/Chol-R9 is effective for inhibiting stent-induced tissue hyperplasia in a rat esophageal model.

Carbonic anhydrase type IX expression in lobular endocervical glandular hyperplasia and gastric-type adenocarcinoma of the uterine cervix.

The significance of carbonic anhydrase (CA-IX) expression in endocervical glandular lesions was determined to assess the utility of CA-IX immunohistochemistry for the identification of gastric-type endocervical adenocarcinoma and its related lesions. In this study, lobular endocervical glandular hyperplasia (LEGH) (n=5), atypical LEGH (ALEGH) (n=6), and endocervical adenocarcinomas, including usual-type (UEA) (n=6) and gastric-type (GAS) (n=7), were examined by immunohistochemistry for CA-IX expression. The proportion of positive staining and intensity was evaluated as between 0 and 3+. All 13 UEAs and GASs were positive for CA-IX, with 9 cases showing 3+ staining, and 8 cases showing positive areas exceeding 50%. All 11 LEGHs were positive for CA-IX, with all cases showing 50% or more positive areas, although only one case was 3+ positive. Five of 6 ALEGHs were positive for CA-IX, with 4 cases showing 3+ positivity in 100% of the lesion. Non-neoplastic glands were negative for CA-IX, although reserve cell populations and tubal metaplasias were positive for CA-IX. Although CA-IX appears to be involved in tumor progression and may be diagnostically useful in cases of endocervical adenocarcinoma and its precursors, LEGH can be a pitfall, and staining results should be combined with morphology.

Endocervical glandular neoplasia associated with lobular endocervical glandular hyperplasia is HPV-independent and correlates with carbonic anhydrase-IX expression: a Gynaecological Oncology Group Study.

BACKGROUND: Lobular endocervical glandular hyperplasia (LEGH) is a rare lesion of the uterine cervix. It has been proposed that LEGH may represent a precursor lesion to a group of mucinous adenocarcinoma with gastric phenotype (GA) that is independent of high-risk human papillomavirus (H-HPV) infection. Carbonic anhydrase-IX (CA-IX) is highly expressed in conventional glandular lesions (CGLs). However, expression of CA-IX in LEGH or GA has not been studied. METHODS: In all, 12 CGLs, 7 LEGHs, 6 LEGHs with coexisting adenocarcinoma in situ (AIS, 3) and GA (3) were identified from Japanese women with a cytological diagnosis of atypical glandular cells of undetermined significance. Immunostaining was used to detect CA-IX and p16(INK)4(a) (hereafter termed p16) protein expression in the tissues and CA-IX protein expression in the Papanicolaou smears (PSs). Polymerase chain reaction was used to detect H-HPV DNA in liquid-based cytology. RESULTS: Out of 12 (83%) CGLs, 10 were positive with H-HPV and high levels of CA-IX expression were seen in all (100%) cases. P16 protein expression was observed in 11 out of 12 (92%) cases. None of the LEGHs, LEGHs with AIS or GA were positive for H-HPV and only 8 out of 13 (62%) showed focal weak (1+) p16 expression. In contrast, all cases (100%) exhibited strong CA-IX protein expression. CONCLUSION: Our study suggests that there are different molecular mechanisms of carcinogenesis resulting in CGLs vs LEGHs associated with AIS or GA. There is also a possible link between LEGHs and GAs. Furthermore, CA-IX expression may serve as a useful biomarker for the detection of GAs in the absence of H-HPV infection.

Altered peptidase activities in thyroid neoplasia and hyperplasia.

BACKGROUND: Papillary thyroid carcinoma (PTC), follicular thyroid adenoma (FTA), and thyroid nodular hyperplasia (TNH) are the most frequent diseases of the thyroid gland. Previous studies described the involvement of dipeptidyl-peptidase IV (DPPIV/CD26) in the development of thyroid neoplasia and proposed it as an additional tool in the diagnosis/prognosis of these diseases. However, very little is known about the involvement of other peptidases in neoplastic and hyperplastic processes of this gland. METHODS: The catalytic activity of 10 peptidases in a series of 30 PTC, 10 FTA, and 14 TNH was measured fluorimetrically in tumour and nontumour adjacent tissues. RESULTS: The activity of DPPIV/CD26 was markedly higher in PTC than in FTA, TNH, and nontumour tissues. Aspartyl aminopeptidase (AspAP), alanyl aminopeptidase (AlaAP), prolyl endopeptidase, pyroglutamyl peptidase I, and aminopeptidase B activities were significantly increased in thyroid neoplasms when compared to nontumour tissues. AspAP and AlaAP activities were also significantly higher in PTC than in FTA and TNH. CONCLUSIONS: These data suggest the involvement of DPPIV/CD26 and some cytosolic peptidases in the neoplastic development of PTC and FTA. Further studies will help to define the possible clinical usefulness of AlaAP and AspAP in the diagnosis/prognosis of thyroid neoplasms.