Indomethacin can induce cell death in rat gastric parietal cells through alteration of some apoptosis- and autophagy-associated molecules.

In clinical medicine, indomethacin (IND, a non-steroidal anti-inflammatory drug) is used variously in the treatment of severe osteoarthritis, rheumatoid arthritis, gouty arthritis or ankylosing spondylitis. A common complication found alongside the therapeutic characteristics is gastric mucosal damage. This complication is mediated through apoptosis and autophagy of the gastrointestinal mucosal epithelium. Apoptosis and autophagy are critical homeostatic pathways catalysed by caspases downstream of the gastrointestinal mucosal epithelial injury. Both act through molecular signalling pathways characterized by the initiation, mediation, execution and regulation of the cell regulatory cycle. In this study we hypothesized that dysregulated apoptosis and autophagy are associated with IND-induced gastric damage. We examined the spectra of in vivo experimental gastric ulcers in male Sprague-Dawley rats through gastric gavage of IND. Following an 18-hour fast, IND was administered to experimental rats. They were sacrificed at 3-, 6- and 12-hour intervals. Parietal cells (H+ , K+ -ATPase ß-subunit assay) and apoptosis (TUNEL assay) were determined. The expression of apoptosis-signalling caspase (caspases 3, 8, 9 and 12), DNA damage (anti-phospho-histone H2A.X) and autophagy (MAP-LC3, LAMP-1 and cathepsin B)-related molecules in gastric mucosal cells was examined. The administration of IND was associated with gastric mucosal erosions and ulcerations mainly involving the gastric parietal cells (PCs) of the isthmic and upper neck regions and a time-dependent gradual increase in the number of apoptotic PCs with the induction of both apoptotic (upregulation of caspases 3 and 8) cell death and autophagic (MAP-LC3-II, LAMP-1 and cathepsin B) cell death. Autophagy induced by fasting and IND 3 hours initially prompted the degradation of caspase 8. After 6 and 12 hours, damping down of autophagic activity occurred, resulting in the upregulation of active caspase 8 and its nuclear translocation. In conclusion we report that IND can induce time-dependent apoptotic and autophagic cell death of PCs. Our study provides the first indication of the interactions between these two homeostatic pathways in this context.

Prolonged severe hemorrhagic shock at a mean arterial pressure of 40 mmHg does not lead to brain damage in rats.

AIM: To examine whether prolonged hemorrhagic shock (HS) at a mean arterial pressure (MAP) of 40 mmHg leads to brain damage. METHODS: Rats were anesthetized with sevoflurane. The HS model consisted of the following phases: I, pressure-controlled HS at a MAP of 40 mmHg; II, fluid resuscitation to normalize blood pressure; III, observations with outcome evaluations in terms of survival, overall performance categories, and neurological deficit scores, as well as evaluation of apoptosis in the hippocampus at 96 h. Each group of six rats was randomized into 60 min (group 1) or 75 min (group 2) each of phases I and II. Three sham rats were anesthetized for 150 min, and then awakened during phase III. RESULTS: The three sham rats as well as five and two of the six rats in groups 1 and 2 (P < 0.05), respectively, survived for up to 96 h. All survivors were functionally normal with overall performance category = 1 and neurological deficit score = 0 at 96 h. Apoptotic neurons were not found in the hippocampus. CONCLUSIONS: The higher mortality in group 2 suggested a more profound effect of HS compared with group 1. However, prolonged HS for 60 or 75 min did not cause functional damage or apoptosis in the hippocampus. These findings suggest that prolonged HS at a MAP of 40 mmHg, as a level at which cerebral blood flow seems preserved by autoregulatory mechanisms, does not lead to brain damage.

Expression of delta-like 3 is downregulated by aberrant DNA methylation and histone modification in hepatocellular carcinoma.

Delta-like 3 (DLL3) is a member of the Delta/Serrate/Lag-2 family of ligands for the Notch receptor and plays a role in Notch signaling. We have previously revealed that the expression of DLL3 is silenced by aberrant DNA methylation and that overexpression of DLL3 in the HuH2 hepatocellular carcinoma (HCC) cell line induced apoptosis. In the present study, we first confirmed the methylation of DLL3 in HuH2 cells and analyzed the methylation status of the DLL3 promoter region by bisulfite sequencing. Furthermore, we investigated whether other epigenetic modifications, such as histone acetylation and histone methylation, affected the expression of DLL3. Treatment with the DNA methylation inhibitor, 5-azadeoxycytidine (5-Aza-dC) slightly reactivated DLL3 mRNA expression and bisulfite sequencing revealed that CpG sites in the DLL3 promoter region of the HuH2 cells were densely-methylated. In addition, a significant increase in the expression of DLL3 was observed when the cells were treated with 5-Aza-dC in combination with the histone deacetylase inhibitor trichostatin A. However, an inhibitor of the dimethylation of histone H3 lysine 9 (H3K9me2) or the trimethylation of histone H3 lysine 27 (H3K27me3), modifications that are associated with gene silencing, had no effect on DLL3 reactivation. In combination with the findings from our previous study, these results indicated that DLL3 expression was silenced in HCC cells by DNA methylation and was more readily affected by histone acetylation than histone methylation (H3K9me2 or H3K27me3).

Synthetic α-mangostin dilaurate strongly suppresses wide-spectrum organ metastasis in a mouse model of mammary cancer.

We previously reported that, in a mouse model of mammary cancer, α-mangostin alone exhibits anti-metastatic properties. To enhance this anti-metastatic effect, we examined the efficacy of synthetic α-mangostin dilaurate (MGD), prepared by adding lauric acid to α-mangostin, in the same experimental system wherein mice bearing mammary tumors are exposed to dietary MGD at 0, 2000 and 4000 ppm. Lauric acid has a high propensity for lymphatic absorption, which is the most common pathway of initial dissemination of many solid malignancies. Both mammary tumor volumes and wide-spectrum organ metastasis were markedly reduced at 2000 and 4000 ppm: furthermore, survival in the 4000-ppm group was significantly greater than in control mice. Apoptosis in mammary carcinomas was also significantly increased in the 4000-ppm group, whereas blood microvessel density and lymphatic vessel invasion were markedly reduced. In real-time PCR analyses of tumor samples, increased p21 and decreased Pcna expression were observed with 4000 ppm but values were not statistically significant when compared to expression in control tumors. However, exposure to 4000 ppm significantly decreased expression of phospho-Akt (Ser473/Thr308) as compared to the control, indicating a role in the anti-tumorigenic effects of MGD. These findings suggest that MGD may be useful for adjuvant therapy and chemoprevention and that conjugated medium-chain fatty acids may enhance the efficacy of certain chemotherapeutic agents.

Delta-like 3 is silenced by HBx via histone acetylation in HBV-associated HCCs.

Hepatocellular carcinoma (HCC) is a common malignant tumor with poor prognosis. We previously showed that expression of Delta-like 3 (DLL3), a member of the family of Delta/Serrate/Lag2 ligands for the Notch receptor, is silenced by aberrant DNA methylation and that overexpression of DLL3 in an HCC cell line induces cellular apoptosis. However, how DLL3 expression is regulated during hepatocarcinogenesis is still unclear. Here, we show that silencing of DLL3 during hepatocarcinogenesis is closely related to viral infection, especially hepatitis B virus (HBV) infection (p = 0.005). HepG2.2.15 cells, which are stably transformed with the HBV genome, showed lower DLL3 expression than the parent cell line, HepG2 cells. Treatment with Hepatitis B virus X protein (HBx) small interfering RNA upregulated DLL3 expression in HepG2.2.15 cells, and overexpression of HBx in HepG2 cells downregulated DLL3 expression. Treatment of cells with a histone deacetylase inhibitor induced DLL3 expression in HepG2.2.15 cells. These data suggest that DLL3 expression is silenced during hepatocarcinogenesis in association with HBV infection via an epigenetic mechanism.

Quantified kidney echogenicity in mice with renal ischemia reperfusion injury: evaluation as a noninvasive biomarker of acute kidney injury.

The purpose is to evaluate quantified kidney echogenicity as a biomarker for the early diagnosis of acute kidney injury (AKI) and predicting progression to chronic kidney disease (CKD) in a mouse model of ischemia-reperfusion injury (IRI). Two separate protocols of murine models of IRI were used: (1) 10, 30, and 40 min of bilateral ischemia duration and (2) 45 and 60 min of unilateral ischemia duration. Renal echogenicity was measured with ultrasound and compared with serum creatinine or urine neutrophil gelatinase-associated lipocalin (NGAL) at various timepoints after IRI. In mice subjected to 10, 30, and 40 min of bilateral ischemia, renal echogenicity increased about 2 h after IRI for all ischemia times, earlier than serum creatinine or urine NGAL. In those subjected to 45 and 60 min of unilateral ischemia, 60 min of unilateral ischemia, which represents atrophic changes 28 days after IRI, resulted in a sustained high level of echogenicity and was significantly different 24 h after IRI, while 45 min of unilateral ischemia resulted in trivial levels of histological damage 28 days after IRI. Renal echogenicity might have the potential to be a biomarker for the early diagnosis of AKI and the prognosis of CKD.

Pathological and molecular analyses of atherosclerotic lesions in ApoE-knockout mice.

The establishment of consistent and reliable methods for the analysis of atherosclerosis molecular pathways and for testing the efficiency of new therapeutics is of utmost importance. Here, we fed ApoE-knockout (KO) mice with high-fat diet to for 16 weeks to induce atherosclerosis. Atherosclerotic lesions in mice were methodically investigated using pathologic analyses and molecular biology tools. These lesions were histopathologically classified into three categories: early, progressive, and combined lesions. Immunohistochemical analyses showed that both F4/80 (macrophage marker) and tenascin-C are expressed in these lesions. Real-time PCR analysis conducted using formalin-fixed paraffin-embedded tissues with atherosclerotic lesions demonstrated an increase in the levels of many inflammatory chemokines, including Cxcl16, while antibody arrays performed using frozen atherosclerotic tissue samples showed elevated TIMP-1 expression. Subsequent immunohistochemical analyses showed that the expression of CXCL16, TIMP-1, MMP-9, MMP-8, and LOX-1 is localized in the atherosclerotic lesions. We confirmed that the expression of these proteins is localized to atherosclerotic lesion, which suggests their roles in the development of the lesions in ApoE-KO mice. Therefore, this mouse model represents an appropriate tool for elucidating molecular mechanisms underlying the development of atherosclerosis, and a model for the evaluation of therapeutic efficiency of novel drugs.

Recombinant human soluble thrombomodulin improved lipopolysaccharide/d-galactosamine-induced acute liver failure in mice.

The effect of recombinant human soluble thrombomodulin (TM-α) on acute liver failure (ALF) is unclear, and we elucidated the effect of TM-α in lipopolysaccharide (LPS)/d-galactosamine (GalN)-induced ALF in mice. Placebo (saline) or TM-α (100 mg/kg) was administered 1 h after LPS/GalN administration. Survival rates were evaluated for 24 h after LPS/GalN administration. Plasma and liver samples were evaluated 1, 3, and 7 h after LPS/GalN administration. Survival rates were significantly higher in the TM-α-treated group than in the placebo group. A significant augmentation of plasma high-mobility group box 1 protein (HMGB1) was observed 7 h after LPS/GalN administration. In the TM-α-treated mice, plasma HMGB1 was significantly lower than in the placebo group. A significant augmentation of hepatic nuclear factor (NF)-κB p65 was observed in the placebo-treated group, whereas a significant reduction, relative to placebo, was observed in the TM-α-treated group. Hepatic expression of tumor necrosis factor (TNF)-α and myeloperoxidase were significantly increased in the placebo group, and were similarly significantly attenuated in the TM-α-treated group. TM-α treatment also produced a significant attenuation of liver neutrophil accumulation after LPS/GalN administration. Thus, TM-α may become a useful treatment strategy for reducing the symptoms of ALF via the attenuation of LPS/GalN-induced HMGB1 levels.

Indomethacin suppresses LAMP-2 expression and induces lipophagy and lipoapoptosis in rat enterocytes via the ER stress pathway.

BACKGROUND: Indomethacin enhances small intestinal epithelial cell apoptosis, which may account for mucosal ulceration. However, the involvement of autophagy in indomethacin-induced enterocyte damage is unreported. METHODS: Using light microscopy and electron microscopy techniques, Western blot analysis, and pharmacological inhibition of autophagy, we investigated the autophagic response of cultured rat enterocytes to indomethacin treatment (200 µM) at various time points. Furthermore, autophagy was examined in enterocytes of rats given indomethacin by gavage (10 mg/kg). RESULTS: Our data indicate that indomethacin induced accumulation of cytoplasmic lipid droplets (LDs) in cultured enterocytes, which was associated with time-dependent autophagic responses. Initially (0-6 h), mediated by endoplasmic reticulum stress and suppression of mammalian target of rapamycin, a predominant cytoprotective lipophagy was activated in indomethacin-treated enterocytes, as evidenced by induction and colocalization of LC3-II with LDs, excessive formation of autophagosomes sequestering LDs (autolipophagosomes; ALPs), and decreased viability of enterocytes on blocking autophagy with 3-methyladenine. On prolonged exposure to indomethacin (6-24 h), there was a decrease of LAMP-2 expression in enterocytes coupled with accumulation of ALPs and LDs with fewer autolysosomes in addition to an elevation of lipoapoptosis. These time-dependent autophagic and apoptotic responses to indomethacin treatment were detected in enterocytes of indomethacin-treated rats, confirming in vitro results. CONCLUSIONS: The findings of this study describe a novel mechanism of enterocyte damage by indomethacin mediated by endoplasmic reticulum stress, accumulation of LDs, and subsequent activation of the early phase of cytoprotective lipophagy. This is followed by a late phase characterized by reduced expression of lysosomal autophagic proteins, accumulation of ALPs, and enhanced lipoapoptosis.

Elevated autophagic sequestration of mitochondria and lipid droplets in steatotic hepatocytes of chronic ethanol-treated rats: an immunohistochemical and electron microscopic study.

Ethanol-induced hepatic steatosis may induce the progression of alcoholic liver disease. The involvement of autophagic clearance of damaged mitochondria (mitophagy) and lipid droplets (LDs) (lipophagy) in chronic ethanol-induced hepatic steatosis is not clearly understood. Adult Wistar rats were fed either 5 % ethanol in Lieber-DeCarli liquid diet or an isocaloric control diet for 10 weeks. Light microscopy showed marked steatosis in hepatocytes of ethanol-treated rats (ETRs), which was further revealed by transmission electron microscopy (TEM), where significant numbers of large LDs and damaged mitochondria were detected in steatotic hepatocytes. Moreover, TEM demonstrated that hepatocyte steatosis was associated with greatly enhanced autophagic vacuole (AV) formation compared to control hepatocytes. Mitochondria and LDs were the predominant contents of AVs in steatotic hepatocytes. Immunohistochemistry of LC3, a specific marker of early AVs (autophagosomes), demonstrated an extensive punctate pattern in hepatocytes of ETRs, while LC3 puncta were much less frequent in control hepatocytes. This was confirmed by immunoelectron microscopy (IEM), which showed localization of LC3 to autophagosomes sequestering damaged mitochondria and LDs. In addition, IEM revealed that PINK1 (a sensor of mitochondrial damage and marker of mitophagy) was overexpressed in mitochondria of ETRs. Enhanced autophagic lysosomal activity was evidenced by increased immunolabeling of LAMP-2, a marker of late AVs (autolysosomes) in hepatocytes of ETRs and colocalization of LC3 and lysosomal cathepsins using double immunofluorescence labeling. Increased AVs in hepatocytes of ETRs reflect ethanol toxicity and could represent a possible protective mechanism via stimulation of mitophagy and lipophagy.

Delta-like 3 is silenced by methylation and induces apoptosis in human hepatocellular carcinoma.

The genetic and epigenetic events of hepato-carcinogenesis are relatively poorly understood. By analyzing genes from human hepatocellular carcinoma (HCC) with restriction landmark genomic scanning, several aberrantly methylated genes, including Delta-like 3 (DLL3), have been isolated. In this study, we investigated the function of DLL3 in hepatocarcinogenesis. Methylation of the DLL3 gene in HCC cell lines was investigated with methylation-specific PCR and expression of DLL3 mRNA in HCC cells was examined by RT-PCR. Reactivation of DLL3 expression by treatment with a demethylating agent was examined in methylation-silenced HuH2 cells. Human DLL3 cDNA was cloned and DLL3 function was examined by restoring DLL3 expression in HuH2 cells. The effects of DLL3 on cell growth were evaluated by colony formation assay. Induction of cell death by overexpression of DLL3 was examined by flow cytometric assay using Annexin V and PI. Apoptotic cells were detected by TUNEL staining and the amount of single-stranded DNA was measured by ELISA. As a result, the promoter region of the DLL3 gene was methylated in four of ten HCC cell lines. This aberrant methyl-ation correlated well with the suppression of RNA expression and a demethylating agent reactivated DLL3 expression in methylation-silenced HCC cells. Interestingly, the restoration of DLL3 in the methylation-silenced HuH2 cells led to growth suppression on colony formation assay. Flow cytometric assay with Annexin V and PI showed that this growth suppression by DLL3 expression is associated with the induction of apoptosis. Furthermore, these apoptotic effects were confirmed by TUNEL staining and measurement of single-stranded DNA. These results suggest that DLL3 was silenced by methylation in human HCC and that it negatively regulates the growth of HCC cells.

R-etodolac induces E-cadherin and suppresses colitis-related mouse colon tumorigenesis.

Colorectal cancer is one of the most serious complications of ulcerative colitis (UC), and the risk of UC-associated neoplasia increases as the region and duration of the disease increase. Selective cyclooxygenase (COX)-2 inhibitors effectively diminish carcinogenesis in a murine UC model. However, this may exacerbate colitis. The selective COX-2 inhibitor etodolac is marketed as a racemic mixture of the R- and S-enantiomers. The biochemical and pharmacological effects of etodolac are caused by the S-enantiomer, while the R-enantiomer lacks COX-inhibitory activity. In this study, we evaluated the effect of R-etodolac on colitis-related mouse colon tumorigenesis. The mice received 1,2-dimethlhydrazine (DMH), and then chronic colitis was induced by administration of two cycles of DSS (each cycle: 3% DSS for 7 days followed by distilled water for 14 days). The mice were sacrificed 28 days after the completion of both cycles. Mice were divided into the following groups: group A served as a disease control; group B received a low (2-mg/kg) dose of R-etodolac every 3 days during the entire period; group C received a high (10-mg/kg) dose of R-etodolac on the same schedule as group B; and group D served as a normal control. Administration of R-etodolac decreased the disease activity index during the DSS administration cycle. The mean number of tumors was 17.8, 15.2, 6.0, and 0 in groups A-D, respectively. In group C, R-etodolac significantly suppressed the occurrence of neoplasia (p<0.05). Although R-etodolac treatment did not affect COX-2 expression, it significantly enhanced expression of E-cadherin in both neoplastic lesions and background mucosa (i.e., lesion-free colon). Thus, administration of R-etodolac exerts a suppressive effect on the development of neoplasia in a murine model of DSS-induced colitis without exacerbation of the colitis. These results suggest that R-etodolac could be useful in the prevention of UC-associated neoplasia.

Proliferative effects of gamma-aminobutyric acid on the gastric cancer cell line are associated with extracellular signal-regulated kinase 1/2 activation.

BACKGROUND AND AIM: Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the adult mammalian brain. However, GABA is found not only in peripheral neuronal tissue, but also in many peripheral non-neuronal tissues, and is thought to have important physiological functions in addition to neurotransmission. We previously reported that GABA participates in chondrocyte proliferation. In the present study, we investigated the effects of GABA on the proliferation of a gastric cancer cell line, KATO III. METHODS: Reverse transcription polymerase chain reaction and immunohistochemical analyses were performed to examine the expression of the GABA synthesis enzyme, glutamate decarboxylase (GAD), and that of the GABA(A) and GABA(B) receptor subunits. The production of GABA was confirmed by immunohistochemistry. The proliferative effect of GABA on KATO III cells was analyzed by bromodeoxyuridine incorporation assay, and the activation status of mitogen-activated protein (MAP) kinases (extracellular signal-regulated kinase [ERK]-1/2, Jun-N-terminal kinase, and p38) and the expression of cyclin D1 were analyzed by western blotting. RESULTS: KATO III cells expressed GAD and GABA. More than five GABA(A) receptor subunits, including the pi subunit, were expressed in KATO III cells; however, GABA(B) receptor subunits were not seen. The addition of GABA to the medium promoted KATO III proliferation, and maximum proliferative effects were observed in the presence of 10 or 1 microM GABA. The addition of 1 microM GABA predominantly activated ERK-1/2 among the three MAP kinases in addition to increasing cyclin D1 expression. CONCLUSION: GABA is able to promote KATO III cell proliferation in an autocrine or a paracrine fashion through GABA(A) receptors followed by MAP kinase activation.

Aire controls the differentiation program of thymic epithelial cells in the medulla for the establishment of self-tolerance.

The roles of autoimmune regulator (Aire) in the expression of the diverse arrays of tissue-restricted antigen (TRA) genes from thymic epithelial cells in the medulla (medullary thymic epithelial cells [mTECs]) and in organization of the thymic microenvironment are enigmatic. We approached this issue by creating a mouse strain in which the coding sequence of green fluorescent protein (GFP) was inserted into the Aire locus in a manner allowing concomitant disruption of functional Aire protein expression. We found that Aire(+) (i.e., GFP(+)) mTECs were the major cell types responsible for the expression of Aire-dependent TRA genes such as insulin 2 and salivary protein 1, whereas Aire-independent TRA genes such as C-reactive protein and glutamate decarboxylase 67 were expressed from both Aire(+) and Aire(-) mTECs. Remarkably, absence of Aire from mTECs caused morphological changes together with altered distribution of mTECs committed to Aire expression. Furthermore, we found that the numbers of mTECs that express involucrin, a marker for terminal epidermal differentiation, were reduced in Aire-deficient mouse thymus, which was associated with nearly an absence of Hassall's corpuscle-like structures in the medulla. Our results suggest that Aire controls the differentiation program of mTECs, thereby organizing the global mTEC integrity that enables TRA expression from terminally differentiated mTECs in the thymic microenvironment.

International and inter-observer differences in macroscopic classification of superficial early colorectal cancer.

BACKGROUND/AIMS: The aim of this research is to investigate the international and intra-observer differences in the macroscopic classification of early colorectal cancer between Japan and China. METHODOLOGY: Color pictures of 9 cases of early colorectal cancer were distributed to 6 Japanese and 5 Chinese endoscopists. After reviewing the pictures, the doctors made their classificatory diagnoses independently and indicated their findings on which the diagnoses were based. RESULTS: There was some consistency in the classification of distinctly elevated lesions among all the Japanese and Chinese endoscopists. However, some elevated lesions classified as type II in Japan might be diagnosed as type I by Chinese endoscopists. For superficial lesions consisting of elevation plus central depression, IIa+IIc or IIc+IIa, were classified according to the ratio of elevation and depression. Although international difference is not significant, inter-observer differences still exist in classifying these lesions. In addition, the differences in laterally spreading tumor were mainly due to terminology. CONCLUSIONS: Japanese and Chinese doctors share a lot of similarities in the classification of flat elevated lesions; however, both international and inter-observer differences still exist in the macroscopic classification for early CRC.

Expression of GABAergic system in pulmonary neuroendocrine cells and airway epithelial cells in GAD67-GFP knock-in mice.

Gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter in the brain, is also located in many peripheral nonneuronal tissues. The glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mouse is a useful model for studying the distribution of GABAergic cells in many tissues and organs. The lungs of these mice contain cells with an intense GFP signal exclusively in the airway epithelium. We aimed to characterize the GFP-positive cells and to clarify their relationship with the GABAergic system. We identified the GFP-positive cells as pulmonary neuroendocrine cells (PNECs) by immunohistochemistry for the protein gene product 9.5 and calcitonin gene-related peptide and by ultrastructural analysis. Immunohistochemistry for GADs and GABA revealed GAD65/67 and GABA in GFP-positive PNECs. Reverse transcription-polymerase chain reaction analyses revealed mRNAs encoding the GABA(B) receptor subunits necessary for the assembly of functional receptors, R1 and R2, in the lung. GABA(B) receptor subunit R1 and R2 proteins were expressed in many airway epithelial cells including alveolar epithelial cells other than GFP-positive PNECs. The present findings demonstrated that PNECs in the airway epithelium have a GABA production system and indicated that GABA plays functional roles in airway epithelial cells through GABA(B) receptors.

Increased proliferation of middle to distal colonic cells during colorectal carcinogenesis in experimental murine ulcerative colitis.

Patients with ulcerative colitis (UC) exhibit an increased risk for the development of cancer of the colon and rectum. This association is widely attributed to colonic inflammation. However, the severity of colonic inflammation necessary for the development of dysplasia and/or cancer remains unknown. In this study, we investigated the pattern of cell proliferation in colorectal carcinogenesis in an experimental murine model of UC. Chronic colitis was induced by administration of four cycles of dextran sulfate sodium (DSS) (each cycle: 5% or 2% DSS for 7 days and then distilled water for 14 days). Mice were sacrificed after every cycle and at 120 days following the completion of the fourth cycle. Colonic cell proliferation was immunohistochemically evaluated using the thymidine analogue bromodeoxyuridine and the labeling index (LI) was determined. The incidence of dysplasia and/or cancer was 28%, 6.7%, and 0% in the 5% DSS, 2% DSS, and normal control groups respectively. All gross lesions were present in the middle to distal colon. Disease activity index and total LI after four cycles of DSS were significantly higher in the 5% DSS group compared to the 2% DSS group. In the 5% DSS group, the LI was significantly higher in the middle colon than in the proximal colon. Simple repeated administration of the non-genotoxic colon carcinogen DSS induced dysplasia and/or cancer. In addition, we have demonstrated the presence of regional differences in proliferation pattern between the middle and the proximal colon during carcinogenesis in experimental murine UC. These findings may provide insight into the development of colorectal cancer in humans with long-standing UC.

Therapeutic effect of nimesulide on colorectal carcinogenesis in experimental murine ulcerative colitis.

BACKGROUND: Patients with ulcerative colitis (UC) exhibit an increased risk for the development of cancer of the colon and rectum. Cyclooxygenase (COX)-2 inhibitors are known to suppress sporadic colorectal cancer, but it is unknown whether selective COX-2 inhibitors exhibit a preventive effect in UC-associated neoplasia. This study investigated the preventive effect of nimesulide, a selective COX-2 inhibitor, on colorectal carcinogenesis in an experimental model of murine UC. METHODS: Chronic colitis was induced in mice by administration of four cycles of dextran sulfate sodium (DSS) (each cycle: 5% DSS for 7 days and then distilled water for 14 days). The mice were killed 120 days after the completion of the fourth cycle. The mice were divided into the following five groups: group A served as a disease control; group B received a diet mixed with 400 p.p.m. of nimesulide during the whole period; group C received nimesulide during the four cycles of DSS administration (active phase); group D received nimesulide for 120 days from the end of the fourth cycle (remission phase); group E received no agents including DSS and served as a normal control. RESULTS: The incidence of dysplasia and/or cancer was 28%, 15%, 11.8%, 6.7% and 0% in groups A-E, respectively. In group D, nimesulide significantly suppressed the occurrence of dysplasia and/or cancer (P < 0.05). Strong COX-2 expression was detected by immunohistochemistry in cancer and dysplastic lesions while diffusely weak COX-2 expression was also found in the residual colon (i.e. lesion-free colon). The mucosal concentration of prostaglandin E(2) was significantly lower in groups B and D than in group A. CONCLUSIONS: The administration of the selective COX-2 inhibitor nimesulide (especially during the remission phase) exerts a suppressive effect on the development of dysplasia and/or cancer in a murine model of DSS-induced colitis. These findings may have relevance to long-standing UC in humans.

Histopathological and genetic differences between polypoid and non-polypoid submucosal colorectal carcinoma.

AIM: To investigate the histopathological and genetic differences between polypoid growth (PG) and non-polypoid growth (NPG) submucosal invasive colorectal carcinoma (CRC). METHODS: A total of 96 cases of submucosal CRC were divided into two groups according to their growth type; 60 cases of PG and 36 cases of NPG. The size, histological degree of dysplasia, depth of submucosal invasion and lymph node metastasis were compared between the two groups. Furthermore, expression of p53 was detected by immunohistochemical staining, and K-ras gene mutation was examined by polymerase chain reaction based single-strand conformation polymorphism (SSCP). RESULTS: The average size of the lesions in the NPG group was significantly smaller than those in the PG group (7.5 mm vs 13.8 mm, P<0.001). The histological degree of dysplasia tended to be more severe in NPG group, while the incidence of submucosal massive invasion and the lymph node metastasis were both significantly higher in the NPG type than in the PG group (64.3% vs 43.3%, P=0.004; 43% vs 7%, P=0.008, respectively). In addition, K-ras gene mutations were detected in 67% of lesions in the PG group, but none in the NPG group, while no difference in p53 immunohistochemical expression was found between the two groups. CONCLUSION: Compared with PG submucosal CRC, NPG type demonstrates more frequent submucosal massive invasion, more lymph node metastasis and a higher degree dysplasia. Genetically, NPG type shows much less frequent K-ras mutation.

Expression of gamma-aminobutyric acid and glutamic acid decarboxylases in rat descending colon and their relation to epithelial differentiation.

OBJECTIVE: To detect the expression of gamma-aminobutyric acid (GABA) and glutamic acid decarboxylases (GADs; including two isoforms GAD65 and GAD67) in the epithelial growth zones of the descending colon in rats, and to investigate their relation to epithelial differentiation and proliferation. METHODS: The expression of GABA and GADs in rat descending colon was investigated by immunofluorescent staining and confocal laser scanning techniques, and goblet cells were further investigated by wheat-germ agglutinin histochemistry. In addition, GAD65 and GAD67 mRNAs were also detected by reverse transcription-polymerase chain reaction. Furthermore, evaluation of cell kinetics in colonic epithelia was conducted by ABC immunostaining using a monoclonal antibody against proliferating cell nuclear antigen (PCNA). RESULTS: Immunoreactive GABA and GADs were distributed in the upper third of the crypts and at the luminal surface in the rat descending colon. Strong staining for GABA and GADs was localized mainly in the cytoplasm of epithelial cells near the neck of the crypts and along the luminal surface. In addition, GABA and GAD65 were also detected at the lamina propria in colonic mucosa. No staining for GABA or GADs was found in goblet cells. GAD65 and GAD67 mRNAs were identified in homogenates of rat descending colon. PCNA labeled nuclei were found in the lower two-thirds of the crypts. CONCLUSIONS: The expression of GABA and GADs in the maturation and function zones of rat descending colon suggests that GABA may be involved in the differentiation of colonic epithelial cells.