Shi-Bing Su

Expression of Clusterin in Human Pancreatic Cancer

Introduction Clusterin, also known as apolipoprotein J, has been implicated in numerous processes, including active cell death. Clusterin is reported to be overexpressed in breast and prostate cancers. However, its expression in pancreatic cancer is yet to be reported. Aim and Methodology To examine clusterin expression and apoptosis in 52 pancreatic tissues, including specimens from 33 cases of pancreatic cancer, by immunohistochemistry. Results Clusterin was expressed in 49% (16) of 33 cases of pancreatic cancer, 50% (13) of 26 cases of chronic pancreatitis, and 67% (4) of 6 cases of mucinous cystadenoma. It was not expressed in normal pancreas. Clusterin mRNA was also expressed in the pancreatic cancer cell lines. Clusterin was significantly more highly expressed at stage I and II (well-differentiated and moderately differentiated cancers). Clusterin and apoptosis were localized in the same cells in pancreatic cancer. However, clusterin expression was not significantly associated with apoptosis in any pancreatic disease. Clusterin-positive patients with pancreatic cancer survived significantly longer. Conclusion These results suggest that clusterin expression is induced in pancreatic cancer as well as chronic pancreatitis and that downregulation of clusterin may be involved in the progression of pancreatic cancer.

The combination of baicalin and baicalein enhances apoptosis via the ERK/p38 MAPK pathway in human breast cancer cells

AbstractAim:To examine whether the cell growth inhibitory effect of the combination of baicalin and baicalein is related to apoptosis. Moreover, to determine whether the expression of some apoptosis-related proteins is regulated by the ERK/p38 MAPK pathway.Methods:Cell viability was measured using a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was detected by acridine orange (AO) staining, DNA ladder assay and flow cytometric analysis. Apoptosis-related proteins were observed using Western blot analysis.Results:Compared with baicalin or baicalein alone, the combination treatment of baicalin (50 μmol/L) and baicalein (25 μmol/L) had an anti-proliferative effect in a time-dependent manner. Isobologram analysis demonstrated that the combination treatment had a synergistic effect. Moreover, apoptosis in MCF-7 cells was increased by 12% and 20% with the combination treatment at 24 h and 48 h, respectively. With the combination treatment in MCF-7 cells, cleaved caspase-3 and caspase-9 were observed, and the level of bcl-2 expression was decreased approximately 20% and 40% at 24 h and 48 h, respectively. The expression of bax and p53 were increased about 25% and 15% at 48 h, respectively. Moreover, the activation of caspase-3, -9 and the regulation of bcl-2, bax and p53 were related to ERK /p38 MAPK activation.Conclusion:In this study, apoptosis was enhanced by the combination treatment of baicalin and baicalein, which activated caspases-3 and caspase-9, downregulated the level of bcl-2 and upregulated the level of bax or p53 via the ERK/p38 MAPK pathway.

Expression of Tumor Necrosis Factor-α, Interleukin-6, and Interferon-γ in Spontaneous Chronic Pancreatitis in the WBN/Kob Rat

To clarify the pathophysiological significance of cytokines in chronic pancreatitis (CP), we analyzed tissue expressions of various cytokines in the onset and progression of spontaneous CP in the WBN/Kob rat. Four-week-old male WBN/Kob rats were fed a special pellet diet (MB-3) for 20 weeks, and 6 rats were killed every 4 weeks. Pathologically, CP occurred at 12 weeks and progressed thereafter. The inflammation and fibrosis peaked at 12 and 16 weeks, respectively. By semiquantitative reverse transcription-polymerase chain reaction, the expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and interferon (IFN)-gamma mRNAs peaked at 8, 12, and 16 weeks, respectively. Immunohistochemistry showed IL-6 expression in infiltrating inflammatory cells and vascular endothelial cells, whereas TNF-alpha was expressed in both acinar and infiltrating cells. IFN-gamma was localized to acinar, infiltrating and ductal cells, and its expression intensity showed significant correlation with those of fibrosis, type III collagen and alpha-smooth muscle actin. The in situ hybridization results were consistent with the RT-PCR data. These results suggest that tissue expressions of TNF-alpha and IL-6 are involved in the onset of pancreatitis and that IFN-gamma expression is related to the progression of CP.

Expression of Transforming Growth Factor-Beta in Spontaneous Chronic Pancreatitis in the WBN/Kob Rat

Transforming growth factor-beta1 (TGF-β1) is suggested to be a mediator of fibrosis in chronic pancreatitis, but the serial change of TGF-β1 expression in the onset and progression of chronic pancreatitis is still unclear. We investigated the TGF-β1 expression in the spontaneous chronic pancreatitis model. Four-week-old male WBN/Kob rats were fed with special pellet diet (MB-3) for 20 weeks. TGF-β1 mRNA in the pancreas was detected by reverse transcription–polymerase chain reaction assay from four weeks, and its expression peaked at 12 weeks when the pancreatic fibrosis first appeared. The localizations of TGF-β1 mRNA and protein were confirmed in the cytoplasm of pancreatic acinar and ductal cells by in situ hybridization and immunohistochemistry, respectively. Although fibronectin expression peaked at 12 weeks and correlated with that of TGF-β1, its elevated expression tended to be prolonged. Pancreatic fibrosis peaked at 16 weeks after the peak of TGF-β1 expression. These results suggest that TGF-β1 expression may be a trigger of the fibrotic process of chronic pancreatitis in the WBN/Kob rat.

Undifferentiated (anaplastic) carcinoma of the pancreas showing sarcomatous change and neoplastic cyst formation

SummaryConclusionPancreatic undifferentiated (anaplastic) carcinoma contained two components: sarcomatous change of spindle-cell type and a neoplastic cyst. Immunohistochemical analysis suggested that both the sarcomatous and the cystic portions were of epithelial origin. This case showed a “dual” differentiation both into the sarcomatous change and into the cystic lesion.BackgroundPancreatic sarcoma or sarcomatous change is very rate. The origin of the sarcomatous change is unknown. On the other hand, pancreatic adenocarcinoma sometimes shows necrosis and forms a cystic lesion during its growth, but a neoplastic cyst formation is very rare except for cystadenomas.MethodsWe report a case of pancreatic undifferentiated (anaplastic) carcinoma associated with sarcomatous change and neoplastic cyst formation. Clinicopathological and immunohistochemical analyses were performed.ResultsA 75-yr-old male was admitted because of low back pain and body weight loss. He died 7 d after admission presenting obstructive jaundice. Autopsy disclosed that the large mass was a poorly differentiated ductal adenocarcinoma, which mostly consisted of sarcomatous components of spindle-cell type. The cystic lesion was a neoplastic cyst with the wall composed of epithelial tumor cells. There was no necrosis or hemorrhage in the cystic cavity. Immunohistochemical analyses suggested that both the tumor and the cystic lesion were of epithelial origin. Sarcomatous changes were recognized also in the metastatic lesions in the liver and lymph nodes. The tumor is considered to be labeled undifferentiated (anaplastic) carcinoma of the pancreas.

Curcumin enhanced antiproliferative effect of mitomycin C in human breast cancer MCF-7 cells in vitro and in vivo.

Aim:To investigate the efficacy of mitomycin C (MMC) in combination with curcumin in suppressing human breast cancer in vitro and in vivo.Methods:Human breast cancer MCF-7 cells were used. Cell viability was measured using MTT assay. The cell cycle phase was detected with flow cytometric analysis. Cell cycle-associated proteins were examined using Western blot analysis. MCF-7 breast cancer xenografts were established to monitor tumor growth and cell cycle-associated protein expression.Results:Curcumin inhibited MCF-7 breast cancer cell viability in a concentration-dependent manner (IC50 value=40 μmol/L). Similarly, MMC inhibited the cell viability with an IC50 value of 5 μmol/L. Combined treatment of MMC and curcumin showed a synergistic antiproliferative effect. In the presence of curcumin (40 μmol/L), the IC50 value of MMC was reduced to 5 μmol/L. In MCF-7 xenografts, combined administration of curcumin (100 mg/kg) and MMC (1-2 mg/kg) for 4 weeks produced significantly greater inhibition on tumor growth than either treatment alone. The combined treatment resulted in significantly greater G1 arrest than MMC or curcumin alone. Moreover, the cell cycle arrest was associated with inhibition of cyclin D1, cyclin E, cyclin A, cyclin-dependent kinase 2 (CDK2) and CDK4, along with the induction of the cell cycle inhibitor p21 and p27 both in MCF-7 cells and in MCF-7 xenografts. These proteins were regulated through p38 MAPK pathway.Conclusion:The results suggest that the combination of MMC and curcumin inhibits MCF-7 cell proliferation and cell cycle progression in vitro and in vivo via the p38 MAPK pathway.

Antifibrotic effect of the herbal medicine Saiko-keishi-to (TJ-10) on chronic pancreatitis in the WBN/Kob rat.

Chronic pancreatitis is characterized by fibrosis. We reported an anti-inflammatory effect of the herbal medicine Saiko-keishi-to (TJ-10) on chronic pancreatitis. This study aimed to elucidate the antifibrotic effect of TJ-10. Four-week-old male WBN/Kob rats were fed a special pellet diet (MB-3) with or without TJ-10 (80 mg/100 g body weight) for 20 weeks. Pancreata were histopathologically examined at every 4 weeks, and the expression of fibrosis-related factors such as transforming growth factor &bgr;1 (TGF-&bgr;1), fibronectin (FN), &agr;-smooth muscle actin (&agr;-SMA), and type III collagen was analyzed. In untreated WBN/Kob rats, chronic pancreatitis developed at 12 weeks and progressed with marked fibrosis at 16 weeks, and the expression of TGF-&bgr;1 and FN peaked at 12 weeks. However, in the TJ-10–treated rats, the rate of pancreatic fibrosis and the expression of TGF-&bgr;1, FN, &agr;-SMA, and type III collagen at 12 and 16 weeks decreased significantly compared to those in the untreated rats. These results suggest that TJ-10 inhibits the pancreatic fibrosis by the suppression of TGF-&bgr;1 expression.

Expression of pancreatitis-associated protein (PAP) mRNA in gastrointestinal cancers

SummaryConclusionPancreatitis-associated protein (PAP) mRNA is expressed in some cases of gastric and colorectal cancers resulting from an ectopic expression in dedifferentiated cancer cells.BackgroundThe PAP gene is identical to the hepatoma-intestine-pancreas (HIP) gene, which is expressed in hepatoma. Expression in cancer might be another characteristic of PAP.MethodsFresh surgical specimens of 100 gastrointestinal cancers, 14 benign digestive diseases, and six normal organs were studied with nonisotopicin situ hybridization (ISH) using biotin-labeled cDNA probe.ResultsPAP mRNA was detected in 10% (6/60) of gastric cancers, 21.4% (6/28) of colorectal cancers, 20.0% (1/5) of pancreatic cancers and 0% of biliary tract (three), esophageal (one), and hepatocellular cancers (three). Reverse transcription-polymerase chain reaction (RT-PCR) detected PAP mRNA in these ISH-positive cases. PAP mRNA was not detected in noncancerous portions, benign disease tissues, or normal organs except for the small intestine. There was no relationship between PAP mRNA expression and any clinicopathological factors.

Effect of camostat mesilate on the expression of pancreatitis-associated protein (PAP), p8, and cytokines in rat spontaneous chronic pancreatitis.

An oral protease inhibitor, camostat mesilate (CM) has been used clinically for chronic pancreatitis (CP) in Japan, but it lacks enough scientific evidence of its effectiveness. The aim of this study was to analyze the effect of CM on the gene expressions of pancreatitis-associated protein (PAP), p8, and cytokines such as interleukin-6 and transforming growth factor-&bgr; 1 in spontaneous CP model (WBN/Kob rats). CM (10 mg/100 g body weight), mixed in MB-3 diet, was administered orally and gene expressions were analyzed by reverse transcription-polymerase chain reaction. In untreated WBN/Kob rats, the gene expressions of all the four factors peaked at 12 weeks, whereas they were significantly suppressed in the CM-treated rats. CM significantly increased the body weight and pancreatic wet weight, and it significantly inhibited inflammatory changes and fibrosis of the pancreas. These results suggest that CM inhibits pancreatic inflammation and fibrosis through the suppression of gene expressions of PAP, p8, and cytokines in CP.

Arginine induces apoptosis and gene expression of pancreatitis-associated protein (PAP) in rat pancreatic acinar AR4-2J cells

Arginine-induced pancreatic acinar cell injury has been reported in vivo, but the mechanism involved is unknown. In this study we investigated the effects of arginine on the cell morphology and pancreatitis-associated protein (PAP) gene expression in rat pancreatic acinar AR4-2J cells in vitro. Arginine inhibited the proliferation of AR4-2J cells in a dose-dependent manner. This decrease in proliferation was due to an increase in apoptosis, as assessed by cell morphology and DNA fragmentation. PAP messenger RNA (mRNA) was expressed at doses of 2.5 and 5.0 mg/ml of arginine, and a time-course study showed that the expression started 2 h after arginine addition and peaked at 6 h. Apoptosis was rarely seen when PAP mRNA was highly expressed, but occurred when PAP mRNA expression was decreased. These results suggest that arginine induces apoptosis and PAP gene expression in pancreatic acinar cells and that PAP might inhibit the induction of apoptosis.