Marco Ramadani

Cyclooxygenase-2 is overexpressed in chronic pancreatitis.

Introduction Cyclooxygenase enzymes catalyze a critical step in the conversion of arachidonic acid to prostaglandins, which are important mediators of acute and chronic inflammation. The constitutively expressed cyclooxygenase-1 (COX-1) appears to regulate many normal physiologic functions in several cell types, whereas the inducible cyclooxygenase-2 (COX-2) enzyme mediates the inflammatory response. Aims and Methodology We investigated the expression of COX-2 in tissues of 35 patients with chronic pancreatitis, 6 patients with pancreatic cancer, and 5 control patients by immunohistochemical analysis and correlations to clinicopathologic features. Results We found an overexpression of COX-2 in the atrophic acinar cells (80% of patients), hyperplastic ductal cells (86% of patients), and islets cells (97% of patients) but not in normal pancreatic tissues. The COX-2 overexpression in the tissue of patients with chronic pancreatitis was significantly correlated with the frequency of acute attacks of pancreatitis. Tissue from patients who had more than five acute attacks of pancreatitis (n = 10) exhibited COX-2 immunoreactivity of a significantly higher score in atrophic acinar cells (p = 0.004). No correlation could be found with other examined clinical features such as duration of the disease, diabetes, alcohol consumption, smoking, or pain. Conclusion Our results support the hypothesis that COX-2 may be involved in inflammatory responses in chronic pancreatitis and in the progression of this chronic inflammatory disease.

Epidermal growth factor induces cyclin D1 in human pancreatic carcinoma: evidence for a cyclin D1-dependent cell cycle progression.

Introduction We recently showed that cyclin D1 is overexpressed in human pancreatic carcinoma cells, and that this overexpression correlates significantly with a poor prognosis. Aims To assess the interrelations of epidermal growth factor (EGF), EGF receptor (EGFR), and cyclin D1 in human pancreatic carcinoma. Methodology and Results In pancreatic carcinoma cell lines (BxPC-3, AsPC-1), cell cycle analysis revealed an increase in cells in the S/G1 phase between 18 and 30 hours after stimulation with 50 ng/mL EGF. Cyclin D1 mRNA increased after 2 hours, corresponding to an increase in cyclin D1 protein, with the maximum level between 7.5 and 10 hours after stimulation, as demonstrated by Western blot analysis. We performed immunohistochemical analysis on 61 adenocarcinoma tissues for the expression of EGF, EGFR, and cyclin D1 and demonstrated an overexpression in the tumor cells in 51%, 54%, and 62.3%, respectively, whereas normal human pancreas stained negative for all of the three factors. Interestingly, EGF and EGFR expression correlated significantly with the cyclin D1 expression in human pancreatic tumor cells (p < 0.001 and p < 0.01, respectively). Conclusion These results demonstrate that cyclin D1 overexpression in the tumor cells of pancreatic carcinoma tissue is at least partly dependent on the mitogenic effects of EGF signaling through the EGFR.

Systemic immune dysfunction in pancreatic cancer patients

Background and aimsWe investigated the immune status in 32 pancreatic cancer patients (PC) in comparison with healthy controls (HC).Materials and methodsUsing flow cytometry, peripheral blood lymphocytes (PBL) were characterized by the expression of surface markers for T helper cells (CD4), T suppressor cells (CD8), B cells (CD19) and NK cells (CD56). The blastogenic response of PBL was analyzed after stimulation with concavalin A (ConA), phytohemagglutinin (PHA), pokeweed mitogen (PWM) and anti-CD3 antibodies. The serum levels of TNF-α, IL-1β, IL-2, IL-10, IL-12, IL-18, IL-1RA, sIL-2R and TGF-β were determined by ELISA.ResultsNo differences in the distribution of peripheral immunocytes in PC were found, whereas the blastogenic response of peripheral blood lymphocytes (PBL) after stimulation with PHA or anti-CD3 antibodies was significantly decreased in PC. In PC, we found reduced serum levels of IL-2 and significantly elevated levels of TNF-α, TGF-β1, IL-10, IL-2R, IL-1β and IL-1RA.ConclusionThese data provide evidence for a systemic immune dysfunction in pancreatic cancer patients characterized by a shift towards a T helper cell type 2 cytokine profile, a significant elevation of substances related to T cell suppression and a reduced blastogenic response to PHA and anti-CD3 antibodies of PBL.

Down-Regulation of BRCA1 in Chronic Pancreatitis and Sporadic Pancreatic Adenocarcinoma

Purpose: BRCA1 and BRCA2 are considered to be breast cancer susceptibility genes that may also contribute to pancreatic cancer development because family studies revealed mutation carriers to have an increased risk of developing pancreatic cancer. However, as demonstrated for breast and ovarian cancer, inactivation of BRCA in sporadic diseases is based on alteration in gene expression or functional alteration. Experimental Design: To study a potential correlation of BRCA1 and BRCA2 to chronic pancreatitis and development of sporadic pancreatic adenocarcinoma, we have analyzed the expression of these genes by quantitative PCR and performed immunohistochemical analyses in normal pancreatic tissues, chronic pancreatitis, and pancreatic cancer specimens. Results: BRCA1 expression was down-regulated in chronic alcoholic pancreatitis, in particular on the RNA level. Furthermore, our data indicate suppressed BRCA1 expression in pancreatic cancer on both the RNA and protein levels. Quantitative analysis of BRCA1 protein expression demonstrated regular staining in 50% of tumor specimens tested and reduced staining in 50% of tumor specimens tested. Correlation with the clinical outcome revealed a significantly better 1-year overall survival for patients with BRCA1-regular as compared with BRCA1-reduced or BRCA1-absent tumors. In contrast, no substantial differences in BRCA2 expression were found in chronic pancreatitis and pancreatic cancer samples. Conclusions: Our data demonstrate alteration of BRCA1 expression in chronic pancreatitis and sporadic pancreatic adenocarcinoma. We, for the first time, provide evidence for a role of BRCA1 in pancreatic carcinogenesis of noninherited tumors and for clinical outcome.

Distributional and functional alterations of immunocompetent peripheral blood lymphocytes in patients with chronic pancreatitis.

ObjectiveTo investigate whether the chronic inflammatory process in patients with chronic pancreatitis affects their immune function. Summary Background DataChronic pancreatitis is a chronic inflammatory disease of the exocrine pancreas. In approximately 30% of patients, an inflammatory mass of the pancreatic head is found, representing an indication for surgery. MethodsThis study comprised 28 patients with chronic pancreatitis. Sixteen patients were also reevaluated 1 year after resection of the pancreatic head for chronic pancreatitis. ResultsCompared with an age- and gender-matched control group, the number of CD3+ cells was significantly increased in patients with chronic pancreatitis, with an increase of both CD3+CD4+ and CD3+CD8+ cells. The number of natural killer cells or B lymphocytes did not differ between the patients and the control group. After stimulation with phytohemagglutinin or anti-CD3 antibodies, the blastogenic response was significantly attenuated in the patients with chronic pancreatitis. One year after resection of the pancreatic head for chronic pancreatitis, the distribution and the blastogenic response to phytohemagglutinin and anti-CD3 antibodies had returned to normal compared with preoperative values. ConclusionThe chronic inflammatory process in chronic pancreatitis markedly affects the distribution and function of peripheral immunocompetent blood cells, and elimination of the chronic inflammatory focus by pancreatic head resection restores the suppressed immune function in these patients.

4-Hydroxy-1-(3-pyridyl)-1-butanone, an Indicator for 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone–Induced DNA Damage, Is Not Detected in Human Pancreatic Tissue

Tobacco smoking is the only known etiologic agent that causes pancreatic cancer. The tobacco-specific nitrosamine 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone (NNK) is a potent carcinogen in laboratory rodents that, independent of the route of administration, induces primarily lung adenocarcinoma

Inhibition of caspase-1 induces cell death in pancreatic carcinoma cells and potentially modulates expression levels of bcl-2 family proteins.

Caspase‐1 (interleukin‐1β‐converting enzyme) is reported to play an important role in the regulation of apoptosis. We investigated the inhibition of caspase‐1 by the cell permeable caspase‐1 inhibitor Ac‐AAVALLPAVLLALLAP‐YVAD.CHO in pancreatic carcinoma cells. Inhibition of caspase‐1 induced a non‐apoptotic/‘necrotic‐like’ cell death in AsPC‐1, BxPC‐3, MiaPaCa‐2 and Panc‐1 cells. Expression levels of bcl‐2 and bax were up‐regulated in caspase‐1 inhibitor‐treated cells while that of bcl‐xL remained unaltered. Our observations support our previous findings that caspase‐1 is potentially involved in anti‐apoptotic processes in pancreatic carcinoma.

Overexpression of Caspase-1 (Interleukin-1β Converting Enzyme) in Chronic Pancreatitis and Its Participation in Apoptosis and Proliferation

Caspase-1, formerly designated interleukin-1&bgr; converting enzyme, was the first described member of a group of cysteine proteases called caspases. It is suggested that caspases play an important role in apoptosis, but recent observations could show that caspase-1 might also be involved in cellular proliferation. We investigated the expression of caspase-1 in 38 chronic pancreatitis tissues, six pancreatitis tissues from patients with pancreatic carcinoma and nine normal pancreatic tissues by immunohistochemistry. Western blot analysis was used to confirm the immunohistochemical findings. We found a clear expression of caspase-1 in chronic pancreatitis, but not in normal pancreatic tissues. Interestingly, we found expression of caspase-1 in three distinct morphologic compartments: (i) in atrophic acinar cells (31 of 35; 89%), (ii) proliferating cells of ductal origin (33 of 38; 87%), and (iii) in acinar cells redifferentiating to form tubular structures (26 of 31; 83%). These immunohistochemical findings were confirmed by Western blot analysis, which showed an expression of caspase-1 in 85% of the tissues. No correlation was found between any of the examined clinicopathologic features and the caspase-1 expression in chronic pancreatitis. In conclusion, the expression of caspase-1 is a frequent event in chronic pancreatitis and its distribution pattern may reflect two functions of this protease: on one hand its participation in the apoptotic pathway in atrophic acinar cells and, on the other hand, its role in proliferation and differentiation in proliferating duct cells.

CD44v6 cell surface expression is a common feature of macrophages and macrophage-like cells – implication for a natural macrophage extravasation mechanism mimicked by tumor cells

Soluble CD44standard (sCD44s) and CD44v6 (sCD44v6) cannot only be detected in sera of patients with pancreatic carcinoma but also of healthy blood donors. To investigate whether sCD44s and sCD44v6 are derived from white blood cells, we stimulated whole blood with phytohemagglutinin and interleukin‐2, which induced expression of CD44v6 only on monocytes. For further investigations, we used the promyelocytic leukemia cell line Hl‐60. Only Hl‐60 cells differentiating along the macrophage pathway showed increased expression of CD44s and CD44v6. Furthermore, only macrophages showed increased secretion of sCD44s and sCD44v6. Our data suggest that CD44s and CD44v6 are common adhesion molecules on macrophages and macrophage‐like cells.

Overexpression of caspase-1 in pancreatic disorders: implications for a function besides apoptosis.

The caspases are known to play a crucial role in the triggering and execution of apoptosis in a variety of cell types. We assessed the expression of caspase-1 in 42 pancreatic cancer tissue samples, 38 chronic pancreatitis specimens, and nine normal pancreatic tissues by immunohistochemistry and Western blot analysis. We found a clear overexpression of caspase-1 in both disorders, but differences in the expression patterns in distinct morphologic compartments. Pancreatic cancer tissue showed a clear cytoplasmatic overexpression of caspase-1 in tumor cells in 71% of the tumors, whereas normal pancreatic tissue showed only occasional immunoreactivity. In chronic pancreatitis an overexpression of caspase-1 was found in atrophic acinar cells (89%), hyperplastic ducts (87%), and dedifferentiating acinar cells (84%). Although in atrophic cells a clear nuclear expression was found, hyperplastic ducts and dedifferentiating acinar cells showed clear cytoplasmic expression. Western blot analysis revealed a marked expression of the 45 kDa precursor of caspase-1 in pancreatic cancer and chronic pancreatitis (80% and 86%, respectively). Clear bands at 30 kDa, suggested to represent the pl0-p20 heterodimer of active caspase-1, were found in 60% of the cancer tissue and 14% of the pancreatitis tissue specimens. Since we found a highly significant correlation between cytoplasm overexpression of caspase-1 in pancreatic cancer and overexpression of the known prognostic factors cyclin D1, epidermal growth factor, and epidermal growth factor receptor, it is plausible that caspase-1 has a yet unknown function in proliferative processes in addition to its well-known role in the apoptotic pathway.