共查询到20条相似文献,搜索用时 62 毫秒
1.
Sadoulet MO Franceschi C Aubert M Silvy F Bernard JP Lombardo D Mas E 《Glycobiology》2007,17(6):620-630
In human pancreatic adenocarcinoma, alterations of glycosylation processes leads to the expression of tumor-associated carbohydrate antigens, representing potential targets for cancer immunotherapy. Among these pancreatic tumor-associated carbohydrate antigens, the J28 glycotope located within the O-glycosylated mucin-like C-terminal domain of the fetoacinar pancreatic protein (FAPP) and expressed at the surface of human tumoral tissues, can be a good target for anticancer therapeutic vaccines. However, the oncodevelopmental self character of the J28 glycotope associated with the low immunogenicity of tumor-associated carbohydrate antigens may be a major obstacle to effective anti-tumor vaccine therapy. In this study, we have investigated a method to increase the immunogenicity of the recombinant pancreatic oncofetal J28 glycotope by glycoengineering Galalpha1,3Galss1,4GlcNAc-R (alphaGal epitope) which may be recognized by natural anti-alphaGal antibody present in humans. For this purpose, we have developed a stable Chinese hamster ovary cell clone expressing the alphaGal epitope by transfecting the cDNA encoding the alpha1,3galactosyltransferase. These cells have been previously equipped to produce the recombinant O-glycosylated C-terminal domain of FAPP carrying the J28 glycotope. As a consequence, the C-terminal domain of FAPP produced by these cells carries the alphaGal epitope on oligosaccharide structures associated with the J28 glycotope. Furthermore, we show that this recombinant "alpha1,3galactosyl and J28 glycotope" may not only be targeted by human natural anti-alphaGal antibodies but also by the mAbJ28, suggesting that the J28 glycotope remains accessible to the immune system as vaccinating agent. This approach may be used for many identified tumor-associated carbohydrate antigens which can be glycoengineered to carry a alphaGal epitope to increase their immunogenicity and to develop therapeutic vaccines. 相似文献
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Bile salt-dependent lipase (BSDL) was detected in human SOJ-6 and rat AR4-2J pancreatic cells. Whereas AR4-2J cells actively secreted the enzyme, BSDL was retained within the Golgi compartment of SOJ-6 cells. Because Rab6 is involved in vesicle transport in the Golgi apparatus and the trans-Golgi network, we confirmed the presence of Rab6 in these cells. In rat AR4-2J cells, Rab6 as well as Rab1A/B and Rab2, partitioned between the cytosol and microsomes. In SOJ-6 cells Rab1A/B and Rab2 also partitioned between the cytosol and microsomes, but Rab6 was strictly associated with microsome membranes, suggesting a specific defect of Rab6 cycling in human SOJ-6 cells. The apparent defect of cycling in these cells is not due to the expression of a defective Rab6 since its correct sequence was confirmed. We further demonstrated that AR4-2J and SOJ-6 cells express the Rab-GDIbeta and Rab-GDIalpha isoforms, respectively. However, the sequence of Rab-GDIbeta, which may be the main form expressed by SOJ-6 cells, identified a few substitutions located in regions that are essential for Rab-GDI function. We conclude that the deficient secretion of BSDL by SOJ-6 cells could be due to the expression of defective Rab-GDIbeta. In spite of the alterations in Rab-GDIbeta, membrane proteins such as CD71 and NHE3 were correctly localized to the cell plasma membrane of SOJ-6 cells, suggesting that two functional distinct secretory pathway coexist in pancreatic cells. 相似文献
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Franceschi C Collignon A Isnardon D Benkoel L Vérine A Silvy F Bernard JP Lombardo D Beraud E Olive D Mas E 《Journal of immunology (Baltimore, Md. : 1950)》2011,186(7):4067-4077
Aberrant glycosylation or overexpression of cell-surface glycosylated tumor-associated Ags (TAA) distinguish neoplastic from normal cells. Interactions of TAA MUC1 and HER2/neu with dendritic cells (DC) preclude efficient processing, which impairs immune responses. It is thus important to define the mechanisms of interactions between DC and glycosylated TAA and their trafficking and processing for further T cell activation. In this work, we study interactions between DC and the oncofetal fucose-rich glycovariants of bile salt-dependent lipase (BSDL), expressed in pancreatic cancer tissues and referred to as pathological BSDL carrying the fucosylated J28 glycotope (pBSDL-J28) because it is characterized by the mAb J28. The expression of pBSDL-J28 was assessed by immunohistochemistry and quantified by confocal microscopy. Nontumoral pancreatic tissues and cells do not express pBSDL-J28. Using multidisciplinary approaches and functional studies, we provide the first evidence, to our knowledge, that this tumoral glycoprotein is rapidly internalized by human DC through macropinocytosis and endocytosis via mannose receptors and then transported to late endosomes for processing. Interestingly, pBSDL-J28 per se induced DC maturation with increased expression of costimulatory and CD83 molecules associated with cytokine secretion (IL-8 and IL-6). Surprisingly, DC retained their full ability to internalize Ags, making this maturation atypical. Finally, the allogeneic pBSDL-J28-treated DC stimulated lymphocyte proliferation. Besides, pulsing DC with pBSDL-J28 C-terminal glycopolypeptide and maturation with CD40L triggered CD4(+) and CD8(+) T cell proliferation. Therefore, interactions of pBSDL-J28, expressed on tumoral pancreatic tissue, with DC may lead to adequate Ag trafficking and processing and result in T cell activation. 相似文献
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Aubert-Jousset E Garmy N Sbarra V Fantini J Sadoulet MO Lombardo D 《Structure (London, England : 1993)》2004,12(8):1437-1447
Structure similarity searches using a combinatorial extension approach revealed that a protein fold structurally related to the sphingolipid binding domain (SBD) of HIV-1 gp120 (V3 loop) is present on pancreatic bile salt-dependent lipase (BSDL). A synthetic peptide derived from the predicted V3-like domain of BSDL interacted with reconstituted monolayers of sphingolipids such as GalCer and GlcCer. Using Chinese hamster ovary cells stably transfected with the cDNA encoding the rat BSDL (CHO-3B clone) or pancreatic SOJ-6 cells expressing the human BSDL as models, we showed that the enzyme cofractionates with caveolin-1. The secretion of BSDL by CHO-3B cells was inhibited by permeable drugs affecting rafts structure (D609, PDMP, and filipin). Data suggest that the functional interaction between the BSDL SBD and lipid rafts is physiologically relevant and could be essential for sensing the BSDL folding prior to secretion. A tentative model accounting for the phosphorylation-induced dissociation of BSDL from rafts is presented. 相似文献
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Jeong Youp Park Yukihiko Hiroshima Jin Young Lee Ali A. Maawy Robert M. Hoffman Michael Bouvet 《PloS one》2015,10(3)
The goal of this study was to determine whether MUC1 antibody conjugated with a fluorophore could be used to visualize pancreatic cancer. Anti-MUC1 (CT2) antibody was conjugated with 550 nm or 650 nm fluorophores. Nude mouse were used to make subcutaneous and orthotopic models of pancreatic cancer. Western blot and flow cytometric analysis confirmed the expression of MUC1 in human pancreatic cancer cell lines including BxPC-3 and Panc-1. Immunocytochemistry with fluorophore conjugated anti-MUC1 antibody demonstrated fluorescent areas on the membrane of Panc-1 cancer cells. After injecting the conjugated anti-MUC1 antibodies via the tail vein, subcutaneously transplanted Panc-1 and BxPC-3 tumors emitted strong fluorescent signals. In the subcutaneous tumor models, the fluorescent signal from the conjugated anti-MUC1 antibody was noted around the margin of the tumor and space between the cells. The conjugated anti-MUC1 antibody bound the tumor in orthotopically-transplanted Panc-1 and BxPC-3 models enabling the tumors to be imaged. This study showed that fluorophore conjugated anti-MUC1 antibodies could visualize pancreatic tumors in vitro and in vivo and may help to improve the diagnosis and treatment of pancreatic cancer. 相似文献
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X Wang J Zhao J Huang H Tang S Yu Y Chen 《Biochemical and biophysical research communications》2012,425(1):51-57
Carcinogenesis is driven by an accumulation of mutations and genetic lesions, which leads to activation of oncogenes and inactivation of tumor suppressor genes. However, the molecular mechanisms by which the expression of these genes was regulated in pancreatic cancer remains unclear. In this study, we investigated the regulatory effects of microRNA and methylation on the expression of k-ras, TP53 and PTEN genes in pancreatic cancer cells. The protein and miRNA levels were measured by Western blotting and Northern blotting, respectively. Xenograft pancreatic tumor models were established by inoculating BxPC-1, Capan-2, and Panc-1 tumor cells into athymic nu/nu mice. A disparate level of KRAS, p53, PTEN, Dnmts, and Dicer 1 proteins as well as let-7i, miR-22, miR-143, and miR-29b miRNA was observed in BxPC-1, Capan-2, and Panc-1 cells. Knockdown of Dicer 1 expression in BxPC-3 and Panc-1 cells resulted in significant increases in KRAS, p53, PTEN, and Dnmts protein levels and significant decreases in miR-22, miR-143, let-7i, and miR-29b expression. Knockdown of Dicer 1 expression in Capan-2 cells significantly increased p53 and PTEN expression, while significantly decreased miR-22 and miR-143 expression, but had no effects on PTEN, Dnmts, let-7i, and miR-29b expression. Knockdown of Dicer 1 expression significantly inhibited xenograft BxPC-3 tumor growth, but promoted xenograft Panc-1 tumor growth. In contrast, knockdown of Dicer 1 expression had no effect on xenograft Capan-2 tumor growth. Our study suggested that different pancreatic cancer cell lines exhibited obvious discrepancies in gene expression profiles, implying that different molecular mechanisms are involved in the carcinogenesis of pancreatic cancer subclasses. Our study highlighted the importance of personalized therapy. 相似文献
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Aberrant O-linked oligosaccharide biosynthesis in lymphocytes and platelets from patients with the Wiskott-Aldrich syndrome 总被引:13,自引:0,他引:13
E A Higgins K A Siminovitch D L Zhuang I Brockhausen J W Dennis 《The Journal of biological chemistry》1991,266(10):6280-6290
The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive immunodeficiency affecting B lymphocytes, T lymphocytes, and platelets. Previous studies on lymphocytes from WAS patients have revealed that leu-kosialin (CD43), a cell-surface glycoprotein bearing approximately 90 O-linked oligosaccharide chains, shows an aberrant electrophoretic mobility. To determine whether this finding reflects a different pattern of O-linked glycosylation in WAS cells, we have compared healthy individuals and WAS patients with respect to glycosyltransferase activities in T lymphocytes, platelets, and Epstein-Barr virus (EBV)-immortalized B cell lines. Stimulation of peripheral T cells from normal individuals in vitro with anti-CD3 antibodies and interleukin-2 was associated with a 3-fold increase in UDP-GlcNAc:Gal beta 3GalNAc-R (GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase (core 2 GlcNAc-T) from 0.8 to 2.2 nmol/mg/h. In contrast, peripheral T lymphocytes from WAS patients showed an inversion of this phenotype with high core 2 GlcNAc-T activity in unstimulated cells (2.3 nmol/mg/h) and a 2-3-fold decrease in activity following stimulation. Core 2 GlcNAc-T activity was also three times higher in platelets from WAS patients than in normal platelets. Glycosyltransferase activities were measured in immortalized B cell lines established from WAS and normal subjects by infection with EBV. Core 2 GlcNAc-T was less than 0.4 nmol/mg/h in WAS EBV-B cell lines compared to 2.4 nmol/mg/h in EBV-B cell lines from healthy individuals, In contrast, CMP-SA:SA alpha 2-3Gal beta 1-3GalNAc-R (where SA represents sialyl (sialic acid to GalNAc) alpha 6-sialyltransferase II activity was 2.0 nmol/mg/h in the WAS EBV-B cell and less than .01 nmol/mg/h in EBV-B cell lines derived from normal subjects. Eleven other glycosyltransferase activities were measured and found to be similar in EBV-B cell lines from WAS and normal individuals. Polylactosamine sequences were much reduced in the O-linked oligosaccharides of CD43 from WAS EBV-B cells consistent with decreased core 2 GlcNAc-T activity and expression of core 1 oligosaccharides in the cells. In conclusion, B cells, T cells, and platelets in WAS patients show abnormal expression of two developmentally regulated glycosyltransferases, consistent with the idea that the WAS immunodeficiency is due to a failure of normal lymphocyte maturation. 相似文献
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To specifically eliminate recipient anti-blood group ABO antibodies prior to ABO-incompatible organ or bone marrow transplantation, an efficient absorber of ABO antibodies has been developed in which blood group determinants may be carried at high density and by different core saccharide chains on a mucin-type protein backbone. The absorber was made by transfecting different host cells with cDNAs encoding a P-selectin glycoprotein ligand-1/mouse immunoglobulin G(2b) chimera (PSGL-1/mIgG(2b)), the H- or Se-gene encoded alpha1,2-fucosyltransferases (FUT1 or FUT2) and the blood group A gene encoded alpha1,3 N-acetylgalactosaminyltransferase (alpha1,3 GalNAcT). Western blot analysis of affinity-purified recombinant PSGL-1/mIgG(2b) revealed that different precursor chains were produced in 293T, COS-7m6, and Chinese hamster ovary (CHO)-K1 host cells coexpressing FUT1 or FUT2. FUT1 directed expression of H type 2 structures mainly, whereas FUT2 preferentially made H type 3 structures. None of the host cells expressing either FUT1 or FUT2 supported expression of H type 1 structures. Furthermore, the highest A epitope density was on PSGL-1/mIgG2(2b) made in CHO-K1 cells coexpressing FUT2 and the alpha1,3 GalNAcT. This PSGL-1/mIgG(2b) was used for absorption of anti-blood group A antibodies in human blood group O serum. At least 80 times less A trisaccharides on PSGL-1/mIgG(2b) in comparison to A trisaccharides covalently linked to macroporous glass beads were needed for the same level of antibody absorption. In conclusion, PSGL-1/mIgG(2b), if substituted with A epitopes, was shown to be an efficient absorber of anti-blood group A antibodies and a suitable model protein for studies on protein glycosylation. 相似文献
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Le Petit-Thévenin J Bruneau N Nganga A Lombardo D Vérine A 《Journal of lipid research》2001,42(8):1220-1230
In this study we demonstrated that two polyunsaturated fatty acids, arachidonic acid (AA, n-6) and docosahexaenoic acid (DHA, n-3), modulate the secretion of bile salt-dependent lipase (BSDL) by pancreatic AR4-2J cells. The effects of AA and DHA were also compared with that of the monounsaturated fatty acid, oleic acid (OA). Our results showed that the chronic treatment of cells with AA or DHA, that did not affect the biosynthesis rate of BSDL, similarly decreased the amount of secreted BSDL and perturbed the intracellular partitioning of the enzyme, whereas OA had no effect. Particularly, AA and DHA induced the retention of the enzyme in microsomes and lowered its content in the cell cytosol. We have further shown that AA treatment decreased the ubiquitination of the protein, and consequently diminished its export toward the cytosol, a result that might explain the retention of BSDL in microsomes and correlated with membrane phospholipids alteration. The retained protein was further degraded by a nonproteasomal pathway that likely involves ATP-dependent endoplasmic reticulum proteases. These findings concerning the regulation of the pancreatic BSDL secretion by two polyunsaturated acids, AA and DHA, might be of physiological importance in the plasmatic and cellular cholesterol homeostasis. 相似文献
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Le Petit-Thevenin J Verine A Nganga A Nobili O Lombardo D Bruneau N 《Biochimica et biophysica acta》2001,1530(2-3):184-198
Bile salt-dependent lipase (BSDL, EC 3.1.1.13) is a lipolytic enzyme normally secreted by the pancreatic acinar cell. Co- and post-translational modifications, such as N- and O-linked glycosylation, regulate the secretion of this enzyme; therefore it was of first importance to determine the behaviour of BSDL under conditions that impaired its secretion. Using AR4-2J pancreatic cells as model, we showed, particularly when BSDL secretion is impaired, that proteasome inhibitors increased the amount of intracellular BSDL, suggesting that the proteasome is involved in the degradation of this protein. This was strengthened by the detection of ubiquitinated BSDL and of degradation product. Our results suggested that both ubiquitination and degradation of the enzyme occurred at the level of the cytosolic side of microsome membranes. ATP hydrolysis appears essential in ubiquitinated BSDL association with membranes and degradation. Furthermore, under normal secretory conditions, we have shown that a fraction of ubiquitinated BSDL is neither O-glycosylated nor N-glycosylated, suggesting that the N-glycosylation-deficient proteasome substrate does not reach the Golgi and could be degraded by the ER-associated degradation machinery. However, another fraction of ubiquitinated BSDL that is deficient in O-glycosylation, carries out endoglycosidase H-insensitive N-linked glycans, meaning that a second system, that detects abnormal BSDL molecules, could also operate at the level of the Golgi compartment. Consequently, it appears that impairment of BSDL secretion consecutive to secretion inhibition or to a deficient glycosylation leads to the proteasome-ubiquitin-dependent degradation of the protein. Therefore, this pathway is part of the quality control involved in BSDL secretion. 相似文献
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At least 6 N-acetylglucosaminyltransferases (GlcNAc-T I, II, III, IV, V and VI) are involved in initiating the synthesis of the various branches found in complex asparagine-linked oligosaccharides (N-glycans), as indicated below: GlcNAc beta 1-6 GlcNAc-T V GlcNAc beta 1-4 GlcNAc-T VI GlcNAc beta 1-2Man alpha 1-6 GlcNAc-T II GlcNAc beta 1-4Man beta 1-4-R GlcNAc T III GlcNAc beta 1-4Man alpha 1-3 GlcNAc-T IV GlcNAc beta 1-2 GlcNAc-T I where R is GlcNAc beta 1-4(+/- Fuc alpha 1-6)GlcNAcAsn-X. HPLC was used to study the substrate specificities of these GlcNAc-T and the sequential pathways involved in the biosynthesis of highly branched N-glycans in hen oviduct (I. Brockhausen, J.P. Carver and H. Schachter (1988) Biochem. Cell Biol. 66, 1134-1151). The following sequential rules have been established: GlcNAc-T I must act before GlcNAc-T II, III and IV; GlcNAc-T II, IV and V cannot act after GlcNAc-T III, i.e., on bisected substrates; GlcNAc-T VI can act on both bisected and non-bisected substrates; both Glc-NAc-T I and II must act before GlcNAc-T V and VI; GlcNAc-T V cannot act after GlcNAc-T VI. GlcNAc-T V is the only enzyme among the 6 transferases cited above which can be essayed in the absence of Mn2+. In studies on the possible functional role of N-glycan branching, we have measured GlcNAc-T III in pre-neoplastic rat liver nodules (S. Narasimhan, H. Schachter and S. Rajalakshmi (1988) J. Biol. Chem. 263, 1273-1281). The nodules were initiated by administration of a single dose of carcinogen 1,2-dimethyl-hydrazine.2 HCl 18 h after partial hepatectomy and promoted by feeding a diet supplemented with 1% orotic acid for 32-40 weeks. The nodules had significant GlcNAc-T III activity (1.2-2.2 nmol/h/mg), whereas the surrounding liver, regenerating liver 24 h after partial hepatectomy and control liver from normal rats had negligible activity (0.02-0.03 nmol/h/mg). These results suggest that GlcNAc-T III is induced at the pre-neoplastic stage in liver carcinogenesis and are consistent with the reported presence of bisecting GlcNAc residues in N-glycans from rat and human hepatoma gamma-glutamyl transpeptidase and their absence in enzyme from normal liver of rats and humans (A. Kobata and K. Yamashita (1984) Pure Appl. Chem. 56, 821-832). 相似文献
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Sialyl Lewis A (SLe(a)), Lewis A (Le(a)), and Lewis B (Le(b)) have been studied in many different biological contexts, for example in microbial adhesion and cancer. Their biosynthesis is complex and involves beta1,3-galactosyltransferases (beta3Gal-Ts) and a combined action of alpha2- and/or alpha4-fucosyltransferases (Fuc-Ts). Further, O-glycans with different core structures have been identified, and the ability of beta3Gal-Ts and Fuc-Ts to use these as substrates has not been resolved. Therefore, to examine the in vivo specificity of enzymes involved in SLe(a), Le(a), and Le(b) synthesis, we have transiently transfected CHO-K1 cells with relevant human glycosyltransferases and, on secreted reporter proteins, detected the resulting Lewis antigens on N- and O-linked glycans using western blotting and Le-specific antibodies. beta3Gal-T1, -T2, and -T5 could synthesize type 1 chains on N-linked glycans, but only beta3Gal-T5 worked on O-linked glycans. The latter enzyme could use both core 2 and core 3 precursor structures. Furthermore, the specificity of FUT5 and FUT3 in Le(a) and Le(b) synthesis was different, with FUT5 fucosylating H type 1 only on core 2, but FUT3 fucosylating H type 1 much more efficient on core 3 than on core 2. Finally, FUT1 and FUT2 were both found to direct alpha2-fucosylation on type 1 chains on both N- and O-linked structures. This knowledge enables us to engineer recombinant glycoproteins with glycan- and core chain-specific Lewis antigen substitution. Such tools will be important for investigations on the fine carbohydrate specificity of Le(b)-binding lectins, such as Helicobacter pylori adhesins and DC-SIGN, and may also prove useful as therapeutics. 相似文献
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In previous studies, we have shown that the bile salt-dependent lipase (BSDL) associates with the Grp94 molecular chaperone, an association that appears to play essential roles in the folding of BSDL. More recently, combined biochemical and immunocytochemical investigations were carried out to show that the transport of BSDL occurs via an association with the Grp94 all along the pancreatic secretory route (ER-Golgi-granules). The Grp94-BSDL complex is secreted with the pancreatic juice into the acinar lumen and reaches the duodenal lumen, where it is internalized by enterocytes. The dissociation of the complex could take place within the endosomal compartment because BSDL continues further on its way to the basolateral membrane of the enterocyte. To localize the affinity binding sites of pancreatic BSDL in pancreatic and duodenal tissues, we have used an affinity-gold ultrastructural technique. BSDL coupled to gold particles appears to interact with specific sites in tissue sections. This was confirmed by another indirect morphological approach using biotin-labeled BSDL and streptavidin-gold complexes on tissue sections. We have shown that BSDL associates with sites in the pancreatic secretory pathway compartments and in the microvilli, the endosomal compartment, and the basolateral membrane of enterocytes. By biochemical approaches, biotin-labeled BSDL displayed affinities with proteins of 180-190 kD in both pancreatic and duodenal tissues. We have also shown that the Grp94-BSDL complexes, which are insensitive to denaturing conditions, are present in pancreatic homogenate but not in duodenal lysate. Thus, BSDL is able to bind protein complexes formed by either BSDL-Grp94 or Grp94 dimers. (J Histochem Cytochem 48:267-276, 2000) 相似文献
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Herein, we found that salidroside suppressed hypoxia-inducible factor 1 alpha (HIF-1α) and lysyl oxidase-like protein 2 (LOXL2) within human pancreatic cancer BxPC-3 cells cultured both under normoxia and hypoxia condition. To investigate the effect of salidroside on tumorigenesis of BxPC-3 cells and whether HIF-1α and LXCL2 were involved in this process, cells transfected with or without LOXL2 overexpression vector, were treated with 50 μg/mL of salidroside or 50 μM of KC7F2 (a HIF-1α inhibitor) under hypoxia. Cell viability and invasion were assessed using CCK-8 and Transwell chamber assay, respectively. Expression of E-cadherin and matrix metalloproteinase 2/9 (MMP 2/9) was determined, by Western blot analysis, to assess cell mobility at molecular levels. We confirmed that hypoxia increased LOXL2 and induced tumorigenesis of BxPC-3 cells, as evidenced by promoted cell proliferation and invasion, enhanced MMP2/9 while reduced E-cadherin. Interestingly, hypoxia-induced carcinogenesis was significantly retarded by both salidroside and KC7F2, however, enhanced with LOXL2 overexpression. Besides, salidroside and KC7F2 reduced LOXL2, and reversed the tumorigenesis of BxPC-3 cells induced by LOXL2 overexpression. Given the inhibitory effect of salidroside on HIF-1α expression, our data suggested that: (1) LOXL2 was the mechanism, whereby salidroside and KC7F2 showed inhibitory effect on cancer progression of BxPC-3 cells; (2) salidroside exerted its anticancer effect, most likely, by a HIF-1α/LOXL2 pathway. In conclusion, salidroside was a novel therapeutic drug in pancreatic cancer, and downregulation of HIF-1α and LXCL2 was the underlying mechanism. 相似文献
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目的:化疗是晚期胰腺癌的主要治疗手段,但临床效果有限。为提高胰腺癌化疗效果,本研究将化疗药物奥沙利铂(OXA)联合肿瘤全细胞抗原负载的树突状细胞(DC)体外作用于胰腺癌BxPC-3细胞系,观察对其增殖的影响。方法:自健康人外周血中分离培养DC和T细胞。反复冻融BxPC.3细胞,使其裂解并提取全细胞抗原后致敏DC,再以DC激活T细胞。ELISA检测T细胞培养上清中IL-2、IL-4、IL-10和IFN-g的含量。将T细胞与不同浓度的OXA联合作用于BxPC-3细胞,MTT法检测杀伤率。结果:负载BxPC.3全细胞抗原的DC能显著激活T细胞使其成为效应性T细胞,并使其分泌IL-2和IFN—Y。不同浓度的OXA与效应性T细胞联合作用于BxPC.3细胞可明显抑制其增殖,且杀伤效果与OXA的浓度呈正相关。结论:负载BxPC-3全细胞抗原的DC可诱导出抗肿瘤的效应性T细胞,联合OXA能显著提高对BxPC-3细胞的杀伤作用。生物治疗联合化疗抑制胰腺癌细胞增殖的作用明显,具有较好的临床应用前景。 相似文献
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In previous studies, we have shown that the bile-salt-dependent-lipase (BSDL), secreted by pancreatic acinar cells and secreted into the duodenal lumen, can be transcytosed through intestinal cells up to the lamina propria. In this study, we used an in vitro system to provide insights into the apical to basolateral transport of BSDL, across the intestinal barrier. The Int407 human epithelial cell line, grown under conditions that optimize polarity, was used as a tight epithelium model. We attempted to delineate uptake mechanisms and the transcytotic pathway followed by this pancreatic enzyme within the intestinal Int407 cells, which do not produce BSDL. When added to the apical reservoir of Transwell-grown Int407 cells, BSDL was shown to first interact with the apical membrane. Further, BSDL forms clusters that are internalized via clathrin-coated pits. Following endocytosis, BSDL is directed to a nocodazole- and colchicin-sensitive multivesicular compartment. Interestingly, this protein transits through the Golgi apparatus, where it was found to colocalize with the KDEL retrieval-receptor. Finally, enzymatically active intact BSDL was released at the basolateral membrane level. This is the first demonstration for an apical-to-basolateral transcytotic pathway of a secreted pancreatic digestive enzyme through polarized intestinal cells. 相似文献