NSAID对胰腺癌中COX-2的表达及其生长活力的影响

目的 :检测胰腺癌中COX 2表达 ,探讨COX 2抑制剂非甾体类消炎药 (NSAID)的抑癌机制。方法 :胰腺癌组织和细胞株中的COX 2检测分别采用免疫组化和细胞免疫化学分析 ,并使用MTT法及流式细胞仪检测细胞株生长活力和凋亡。结果 :胰腺癌组织中COX 2的表达增强 ,阳性率 73.3% (P <0 .0 5 )。SW 1 990和Capan 2细胞株均有COX 2表达 ,前者高表达 ,后者低表达。两种NSAID(NS398及ASA)均可抑制两种细胞株的生长 ,诱导细胞凋亡率显著升高 ,并与细胞株COX 2表达强度有关 ;对SW 1 990细胞株的作用强于Capan 2 ,NS398的抑制作用又强于ASA。结论 :COX 2在胰腺癌组织和细胞株中表达增强 ,NSAID抗胰腺癌机制可能是通过抑制COX 2活性 ,诱导胰腺癌细胞凋亡     

Antiproliferative effects of interferon alpha on hepatic progenitor cells in vitro and in vivo

Hepatic progenitor cells (called oval cells in rodents) proliferate during chronic liver injury. They have been suggested as targets of malignant transformation in chronic liver diseases, including chronic hepatitis C. Interferon alpha therapy reduces the risk of hepatocellular carcinoma (HCC) in chronic hepatitis C regardless of viral clearance. The aim of this study was to determine whether interferon alpha could reduce the risk of HCC by modifying preneoplastic events in the hepatic progenitor cell population. Pre- and post-treatment liver biopsies were evaluated for changes in t he hepaticprogenitor cell population in 16 patients with non-responding chronic hepatitis C Interferon alpha-based treatment significantly reduced the numbers of c-kit-positive hepatic progenitor cells by 50%. To determine the mechanism of cell number reduction, the effects of interferon alpha on murinehepatic progenitor cells were studied in vitro. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) proliferation assay and proliferating cell nuclear antigen staining showed that interferon alpha had a dose-dependent, anti-proliferative effect Interferon alpha stimulated hepatocytic and biliary differentiation of the oval cell lines reflected by increased expression of albumin and cytokeratin19 accompanied by decreased expression of alphafetoprotein and Thy-1. To validatethese results in vivo, mice were placed on the choline-deficient, ethionine-supplemented diet to induce liver injury and oval cell proliferation and treated with pegylated interferon alpha 2b for 2 weeks. This resulted in a significant four-fold reduction in the number of oval cells (P < .05). In conclusion, interferon alpha-based treatment reduced the number of hepatic progenitor cells in chronic liver injury by modulating apoptosis, proliferation, and differentiation. Supplementay material for this article can     

Local secretion of complement C3 in the exocrine pancreas: Ductal epithelial cells as a possible biosynthetic site

BACKGROUND & AIMS: The complement system participates in the local immune system in various tissues. In this study, we investigated the local secretion of complement C3 into the pancreatic fluid and attempted to determine a possible biosynthetic site. METHODS: C3 protein in human pancreatic fluid was analyzed by, immunoblotting. The C3 messenger RNA (mRNA) expression in several pancreatic carcinoma cell lines was analyzed by the polymerase chain reaction and/or Northern blotting. The secretion of C3 by these pancreatic carcinoma cells was assessed by metabolic labeling and immunoprecipitation experiments. RESULTS: In five samples of human pancreatic fluid, C3 was detected as a molecule composed of alpha and beta chains. C3 mRNA expression was observed in the ductal cell carcinoma lines (PANC-1 and MIA PaCa-2) but not in the acinar cell line (HPC-YO and AR-42J). C3 production in these cells was enhanced by interleukin 1 beta and tumor necrosis factor alpha at both the protein and the mRNA levels. CONCLUSIONS: (1) Complement C3 is secreted into the exocrine fluids of the pancreas. (2) Ductal epithelial cells are possible biosynthetic sites for C3. (3) The proinflammatory cytokines, interleukin 1 beta and tumor necrosis factor alpha are effective stimulators of local C3 production in the pancreas. (Gastroenterology 1996 Jun;110(6):1919-25)     

Decreased expression of transforming growth factor alpha during differentiation of human pancreatic cancer cells.

The relationship between cell differentiation and transforming growth factor alpha (TGF-alpha) expression in human pancreatic cancer cells was analyzed in Capan 1 cells. These cells differentiate either spontaneously or after butyrate treatment. During differentiation (spontaneous or butyrate induced), TGF-alpha messenger RNA (mRNA) levels decreased, whereas the TGF-beta 1 mRNA levels remained unchanged. TGF-alpha was present in cells as proTGF-alpha, which decreased after butyrate treatment. Secretion of TGF-alpha was not found. Under the two conditions of differentiation, the membrane-bound protein kinase C activity was also reduced. Conversely, long-term phorbol ester treatment increased both membrane-bound protein kinase C activity (260%) and TGF-alpha mRNA level (500%), a not significant increase of TGF-beta 1 mRNA was observed. However, phorbol 12-myristate-13-acetate did not induce TGF-alpha synthesis or secretion. These data suggest that expression of TGF-alpha can be reduced in cancer cells; they also suggest the existence of a relationship between TGF-alpha expression and cell differentiation. In addition, the protein kinase C-induced TGF-alpha mRNA level was not followed by the increase of TGF-alpha biosynthesis, suggesting a translational control. Finally, the expression of TGF-alpha and -beta 1 messengers appears to be differently regulated.     

Molecular cloning of the human kidney differentiation antigen gp160: human aminopeptidase A.

gp160 is a cell surface differentiation-related glycoprotein of 160 kDa expressed by epithelial cells of the glomerulus and proximal tubule cells of the human nephron but only by a subset of renal cell carcinomas (RCCs). We have reported that gp160 expression correlates with the resistance of cultured RCCs to the antiproliferative effects of alpha interferon, while lack of expression correlates with sensitivity to alpha interferon. In this study, we have purified gp160 protein, obtained partial sequences of random peptides, and isolated a full-length cDNA. The gp160 cDNA possesses 78% homology to the murine BP-1/6C3 antigen, a B-lymphocyte differentiation protein that exhibits aminopeptidase A (APA; EC 3.4.11.7) activity. Enzymatic assays on human RCC cell lines indicated a 100% concordance between APA activity and gp160 expression. APA activity of gp160-expressing RCC cells was increased or decreased by a panel of APA activators or inhibitors, respectively. Furthermore, anti-gp160 monoclonal antibodies immunoprecipitate APA activity from RCC cell lysates and selectively deplete APA activity from RCC cell extracts. These data indicate that the gp160 human kidney/RCC glycoprotein is human APA.     

Regulation of epidermal growth factor receptor in human colon cancer cell lines by interferon alpha

BACKGROUND AND AIM: The biology of growth factor receptor expression has implications for receptor specific cancer therapy. In this study, we examined: (a) regulation of epidermal growth factor receptor (EGFR) expression in a panel of 10 human colon cancer cell lines using interferon alpha (IFN-alpha); (b) ability of IFN-alpha to inhibit cell proliferation; and (c) sensitivity of IFN-alpha pretreated cells to EGF. METHODS: Cell proliferation was measured both by crystal violet colorimetric and clonogenic assays. Cell surface, intracellular, and/or total cell protein expression of EGFR was assessed by indirect immunofluorescence flow cytometry and/or fluorescein isothiocyanate (FITC)-EGF binding and internalisation flow cytometric assay. RESULTS: IFN-alpha treatment upregulated expression of cell surface EGFR in seven of 10 colon cancer cell lines within 16 hours, reaching a peak within 48-96 hours; this was accompanied by transient elevation of intracellular EGFR and marked growth inhibition. IFN-alpha treated cancer cells were still sensitive to EGF proliferative stimulation. CONCLUSIONS: Our results indicate that cytostatic concentrations of IFN-alpha can enhance cell surface and intracellular EGFR expression in a proportion of human colon cancer cells. The antiproliferative action of IFN-alpha could not block the signal transduction of the EGF-EGFR pathway. This may have clinical implications for improving treatment based on targeting of EGFR.     

Interferon increases HLA synthesis in melanoma cells: interferon-resistant and -sensitive cell lines.

We report that human leukocyte interferon preparations increase the expression of beta 2-microglobulin by 100-200% on the surface of normal fibroblast and melanoma cell lines sensitive to interferon. This increase in expression can be correlated with an increase in HLA synthesis as measured by incorporation of [35S]methionine in these antigens. This enhanced HLA synthesis, which is 5- to 17-fold, is time dependent and dose related. Synchronized cells in the G0/G1 phase of the cell cycle appear to be more sensitive to this interferon action. Neither an increase in surface expression nor in HLA synthesis is observed in a melanoma cell line resistant to the antiviral and antigrowth effects of interferon. Furthermore, there appears to be a stronger correlation between this increased HLA synthesis and the antiviral function than between it and the antiproliferative action of interferon.     

Growth inhibitory effects of interferon-alpha subtypes vary according to human liver cancer cell lines

BACKGROUND: Interferon (IFN)-alpha preparations used in the treatment of viral and neoplastic disease consist of single or multiple IFN-alpha subtypes that may possess different biological activity, but there are no data on liver cancer cells. METHODS: Antiproliferative effects and the mechanisms of growth inhibition of five IFN-alpha subtypes (alpha1, alpha2, alpha5, alpha8 and alpha10) were examined in vitro using 13 human liver cancer cell lines. RESULTS: The antiproliferative effect of each IFN-alpha subtype was different in each cell line. The 50% growth inhibitory concentration (IC50) on an antiviral unit basis showed that alpha5 presented the most potent antiproliferative effects in 11 of the 13 cell lines, and alpha8 in two cell lines. On average, the antiproliferative effects were strong in descending order from alpha5, alpha8, alpha10, alpha2 to alpha1. On weight basis, the most potent antiproliferative effect was shown by alpha8 in nine of the 13 cell lines, alpha5 in four cell lines, and the potency of the effects on average in descending order was alpha8, alpha5, alpha10, alpha2 and alpha1. No significant difference was observed between natural and recombinant alpha2. The mechanism of growth inhibition of each subtype in HAK-1B and KMCH-1 cell lines were apoptosis and S-phase arrest, and their induction levels were related to a certain degree to the antiproliferative effects. CONCLUSIONS: Our findings show that the antiproliferative effect of each IFN-alpha subtype varies according to the cell line, but that the cells are relatively or absolutely responsive to alpha5 and alpha8 subtypes.     

Effect of interferon alpha, gamma, and tumor necrosis factor alpha on the HLA-A, B, C expression of cell lines derived from human liver

Our study was undertaken to determine whether human recombinant interferon alpha(rIFN alpha), gamma(rIFN gamma), and tumor necrosis factor alpha(rTNF alpha) exert an effect on the HLA-A, B, C expression of human liver cell lines. The HLA-A, B, C expression was assayed by immunoperoxidase staining and enzyme-linked immunosorbent assay. rIFN alpha and gamma enhanced the HLA-A, B, C expression of the three cell lines tested, Chang cells, SK-Hep-1, and PLC/PRF/5. The activity of rIFN gamma proved more than 8000 times more potent than that of rIFN alpha in Chang cells, 30 times in SK-Hep-1, and 20 times in PLC/PRF/5, respectively. rTNF alpha also enhanced the HLA-A, B, C expression of the three cell lines. The enhancement of HLA-A, B, C expression by rIFN alpha and gamma reached a peak on day 3, and that by rTNF alpha on day 5. These findings suggest that IFN alpha, IFN gamma, and TNF alpha may play similar roles in enhancement of HLA-A, B, C expression of hepatocytes in hepatitis and hepatoma cells.     

Alteration of integrins by interleukin-1alpha in human pancreatic cancer cells

INTRODUCTION: Adhesion of tumor cells to extracellular matrix (ECM) proteins plays an important role in tumor invasion and metastasis. AIMS: To investigate the expression of integrins in human pancreatic cancer cell lines and its alteration by interleukin (IL)-1alpha to examine the mechanism of adhesion of metastatic human pancreatic cancer cells to ECM proteins. METHODOLOGY: The expression of integrin subunits and their alteration by IL-1alpha were examined by flow-cytometric analysis and cellular enzyme-linked immunosorbent assay in three metastatic human pancreatic cancer cell lines (AsPC-1, BxPC-3, and SW1990) and two nonmetastatic cancer cell lines (PaCa-2 and PANC-1). In addition, assays of cancer cell adhesion to ECM proteins were performed to investigate if increased integrin expression actually affected the adhesive interaction between cancer cells and the putative integrin ECM ligands. RESULTS: The alpha(6) subunit expressed in metastatic cancer cells was enhanced by IL-1alpha. Metastatic cancer cells also showed preferential adherence to laminin compared with nonmetastatic cancer cells, and this was enhanced by IL-1alpha. CONCLUSION: In pancreatic cancer, the enhancement of alpha(6)beta(1) integrin by IL-1alpha through IL-1 receptor type I, as well as the expression of alpha(6)beta(1) integrin, plays an important role in metastasis formation.     

Tumor Cell-Induced Platelet Aggregation in Vitro by Human Pancreatic Cancer Cell Lines

Background: Tumor cell-induced platelet aggregation (TCIPA) is considered to be a critical step in hematogenous metastasis. Methods: TCIPA was studied in vitro in six human pancreatic carcinoma cell lines (PC 3, PC 44, AsPC1, BxPC3, Capan2, Pane1). Results: Whereas all cell lines induced aggregation of washed platelets in the presence of minimal amounts of platelet-poor plasma, five cell lines also induced aggregation of platelets in platelet-rich plasma. The thrombin-antagonist hirudin inhibited TCIPA in all cell lines, indicating that TCIPA is thrombin-dependent. Since pretreatment of tumor cells with phospholipase A₂ or C inhibited TCIPA, the thrombin-generating activity might be confined to the tumor cell surface. Further support for a prothrombinase activity was provided by the observation that all cell lines were able to induce the aggregation of washed platelets after addition of purified coagulation factors II and V. Conclusions: Pancreatic carcinoma cells are able to induce platelet aggregation via activation of thrombin. This might support metastasis in pancreatic cancer and possibly explain the incidence of thrombosis in this tumor.     

Correlation between interferon (IFN) alpha resistance and deletion of the IFN alpha/beta genes in acute leukemia cell lines suggests selection against the IFN system.

Homozygous and hemizygous deletions of the interferon A (IFNA) and IFNB genes have been frequently observed in acute leukemia cell lines, primary acute leukemia cases, and gliomas. Because IFNs have an antiproliferative effect, selection against the IFN alpha/beta system could play a role or accompany the development of the malignant phenotype. Although the deletion of the IFNA/B genes could interrupt an autocrine loop that controls cell proliferation, cells would still respond to exogenous IFN alpha/beta and, thus, lesions at the receptor or signal transduction level should also be present to render cells resistant to exogenous IFN alpha/beta. To test if selection against the IFN system was operating in acute leukemias, the sensitivity to the antiproliferative effect of IFN alpha 2 was studied in acute leukemia cell lines with and without alterations of the IFNA/B genes. We found that 10 of 11 acute leukemia cell lines with alterations of the IFNA/B genes were resistant to the antiproliferative effect of IFN alpha 2, whereas only two of eight cell lines with normal IFNA/B genes were IFN-resistant. We then examined the possibility that an alteration of the receptor expression could account for the lack of response to IFN alpha 2. No significant alteration in the expression or structure of the IFN alpha receptor was observed. We also studied the downmodulation of the alpha subunit of the IFN alpha receptor upon IFN alpha 2 binding. One cell line with deletion of the IFNA/B genes showed impaired downmodulation of the IFN alpha receptor. The data presented here suggest that selection against the IFN alpha/beta system could play a role or accompany the development of the malignant phenotype.     

Beta1 integrins play an essential role in adhesion and invasion of pancreatic carcinoma cells

To investigate the role of beta1 integrins in pancreatic carcinoma invasion, we analyzed the relationship between the activity of beta1 integrins and the invasive ability of human pancreatic carcinoma cell lines. AsPC1, BxPC3, PANC1, SU8686, KP1NL, KP2, and H48N cells had high expression of beta1 and alpha6 subunits, and various levels of alpha2, alpha3, and alpha5 expression as determined by flow cytometry. Cell adhesion assay revealed that alpha2beta1, alpha5beta1, and alpha6beta1 integrins were the predominant adhesion receptors for collagen, fibronectin, and laminin, respectively. Beta1 integrins on different cell types showed a wide range of constitutive activity. Anti-beta1 monoclonal antibody (MAB) TS2/16 rapidly activated beta1 integrins, and thus TS2/16 requirement in cell adhesion represented the levels of constitutive activity of beta1 integrins. Notably, as the result of in vitro chemoinvasion assay, the levels of constitutive activity of beta1 integrins correlated with the invasive ability of pancreatic carcinoma cells. The inhibitory anti-beta1 MAB 13 completely blocked the invasion of these cell lines. Alternatively, the stimulatory anti-beta1 MAB TS2/16 strongly inhibited the invasion. These results show an essential role of beta1 integrins in invasion of pancreatic carcinoma cells and also suggest subtle regulatory mechanisms of cell invasion.     

Interferon alpha receptors are important for antiproliferative effect of interferon-α against human hepatocellular carcinoma cells

Aim: Interferon (IFN)-alpha is a promising drug for the prevention and treatment of hepatocellular carcinoma (HCC). We reported that responders to IFN-alpha/5-fluorouracil combination therapy expressed higher IFN alpha receptor (IFNAR)2 in tumor. Herein we studied involvement of IFNARs in response to IFN-alpha in HCC cells. Methods: IFN-alpha sensitivity and expression of IFNARs were studied in six HCC cell lines (HuH7, PLC/PRF/5, HLE, HLF, HepG2, Hep3B) using growth-inhibitory and RT-PCR, Western blot assays. Short interfering RNAs (SiRNAs) against IFNAR1 and 2 were used to analyze the role of the IFNARs in IFN-alpha's effect and signal transduction. Results: The expressions of IFNAR1 and 2c mRNAs were higher in PLC/PRF/5 cells than those in other cell lines, and PLC/PRF/5 cells expressed abundant IFNAR2c on their cell membrane. When we examined the sensitivity of the HCC cell lines to the growth-inhibitory effect of IFN-alpha, PLC/PRF/5 exhibited a significant response, while the other cells were much more resistant. Knockdown of either IFNAR1 or 2 using siRNAs suppressed the IFN-alpha's signal transduction (2.5-fold), and decreased the growth-inhibitory effect (down by 69.9% and 67.3%). Conclusion: The results suggest that the expression of IFNAR1 and IFNAR2c independently are important for the antiproliferative effect of IFN-alpha in HCC cells.     

Human tumor necrosis factor produced by human B-cell lines: synergistic cytotoxic interaction with human interferon.

Human cell lines of hematopoietic origin were tested for production of tumor necrosis factor (TNF). B-cell lines transformed by Epstein-Barr virus release a factor (referred to as hTNF) that is cytotoxic for mouse L cells sensitive to mouse TNF but not for L cells resistant to mouse TNF. Exposure to 4 beta-phorbol 12 beta-myristate 13 alpha-acetate augmented production of hTNF. hTNF activity was not found in supernatants of cell lines of T-cell, monocytic, or promyelocytic origin. Partially purified hTNF has a molecular weight of approximately 70,000, has no interferon activity, is acid labile, is destroyed by heating at 70 degrees C for 1 hr, induces cross-resistance to mouse TNF in vitro, and causes hemorrhagic necrosis of Meth A mouse sarcoma in the standard in vivo mouse TNF assay. Tests with a panel of 23 human cancer cell lines showed that hTNF is cytotoxic for 7 cell lines, cytostatic for 5, and has no effect on 11. Comparative studies with human alpha, beta, and gamma interferons indicated that sensitivity to hTNF and interferon can be distinguished. Combined treatment with hTNF and alpha or gamma interferon resulted in a synergistic cytotoxic effect.     

Modification of blood group a expression in human pancreatic tumor cell lines by inhibitors of N -glycan processing

Summary Pancreatic adenocarcinomas induced in Syrian hamsters byN-nitrosobis(2-oxopropyl)amine (BOP) treatment express blood group A (BGA) antigen, which was previously shown by this lab to be expressed on multiantennary asparagine (Asn)-linked glycans attached to membrane glycoproteins. To determine if a similar expression pattern was found in humans, three human pancreatic ductal adenocarcinoma cell lines (CD18, CD11, and Capan 1) from individuals of blood type A were analyzed and shown to express BGA antigen on membrane glycoproteins similar in molecular mass to those found in hamster tumor cells. The BGA antigen was located on Asn-linked oligosaccharides in all three human cell lines, as indicated by loss of activity after peptide:N-glycosidase F (PNGase F) treatment. Also, as shown previously in hamster pancreatic tumor cells, BGA expression at the surface of the human cell lines was blocked by growth of the cells in media containing deoxymannojirimycin (dMM), an inhibitor of mannosidase I. These results demonstrate that the BGA antigen is on Asn-linked glycans in human pancreatic adenocarcinoma cells and that these glycoproteins are processed similarly to the BGA glycoproteins in hamster pancreatic adenocarcinoma.