Pancreatic Stellate Cells Increase the Invasion of Human Pancreatic Cancer Cells through the Stromal Cell-Derived Factor-1/CXCR4 Axis

Aim: Both pancreatic stellate cells (PSCs) and the stromal cell-derived factor-1(SDF-1)/CXCR4 receptor ligand system have important roles in pancreatic cancer progression. This study set out to detect if PSCs express SDF-1 and promote the invasion of pancreatic cancer through the SDF-1/CXCR4 receptor ligand axis. Methods: RT-PCR was performed to detect the expression of SDF-1 and CXCR4 in PSCs, pancreatic cancer lines and cancer tissue samples. ELISA was used to investigate the concentration of SDF-1 in PSC supernatants. An MTT assay was applied to detect the proliferation of pancreatic cancer cells. A transwell chamber migration assay was employed to detect the migration of AsPC-1 cells. An in vitro invasion assay was used to detect the invasion of AsPC-1 cells. Results: CXCR4 expression was detected in PSCs; AsPC-1, SW1990 and BxPC-3 cancer cells; and cancer tissues. SDF-1 was detected in PSCs and cancer tissues, but not in AsPC-1, SW1990 and BxPC-3 cells. SDF-1α protein was found in PSC supernatants. PSC-conditioned media can promote the proliferation, migration and invasion of pancreatic cancer cells. SDF-1 neutralizing antibody or AMD3100 can significantly inhibit these promotive effects. Conclusion: PSCs can secrete SDF-1 and increase the invasion of pancreatic cancer cells through the SDF-1/CXCR4 axis.     

Specific Amino Acid Profile in Culture Media Conditioned by Human Pancreatic Cancer Cell Lines

Background: In malignant diseases, circulating amino acid profiles correlate with organ sites of malignancy. Direct effects of malignant cells on the extracellular amino acid profile are still uncertain. Methods: Free amino acids were measured in serum-free culture media (RPMI1640) conditioned by two human pancreatic cancer cell lines (Panc-1 and HPAF), a hamster pancreatic cancer cell line (PC-1), a human epidermoid carcinoma cell line (A431), and a human fibroblastic cell line (Ag-1523). Nonconditioned RPMI-1640 medium was used as control. Results: Amino acid profiles were changed in all the conditioned media, caused by a decrease or increase in the original amino acids and by the appearance of amino acids that were not present in non-conditioned medium. Media conditioned by two human pancreatic cancer cell lines showed similar amino acid profiles, which were characterized by a decrease in glutamine, cysteine and serine, increase in glycine, proline and glutamic acid and appearance of ornithine and alanine. Conclusion: Culture media show changed amino acid profiles following incubation with cell lines of pancreatic or non-pancreatic origins. Different human pancreatic cancer cell lines cause similar changes in amino acid profiles of media.     

BRG1 expression is increased in human glioma and controls glioma cell proliferation, migration and invasion in vitro

Purpose

The purposes of our study were to elucidate the role of BRG1 in the development of human glioma and to determine the effect of BRG1 on glioma cell growth, migration and invasion.

Methods

Using tissue microarray and immunohistochemistry, we evaluated BRG1 staining in 190 glioma tissues, 8 normal brain tissues and 8 tumor adjacent normal brain tissues. We studied glioma cell proliferative ability with reduced BRG1 expression by siRNA using CCK-8 cell proliferation assay and cell cycle analysis. We studied the role of BRG1 in glioma cell migration and invasion by cell migration assay and matrigel invasion assay. We performed western blot to detect cyclin D1, cyclin B1 and MMP-2 protein expression. We also detected MMP-2 enzyme activity by gelatin zymography.

Results

Our results showed that BRG1 expression was increased in benign tumor and malignant tumor compared with tumor adjacent normal brain tissue (P?Conclusions Our data indicated that BRG1 expression is significantly increased in human glioma and it may be involved in the process of glioma cell proliferation, migration and invasion.     

Influence of Cell Cycle Checkpoints and p53 Function on the Toxicity of Temozolomide in Human Pancreatic Cancer Cells

Background: Though an increased efficacy of carmustine and temozolomide (TMZ) has been demonstrated by inactivation of O⁶-methylguanine-DNAmethyltransferase(MGMT) with O⁶-benzyl-guanine (BG) in human pancreatic tumors refractive to alkylating agents, the regulatory mechanisms have not been explored. Methods: The effects of TMZ and BG on apoptosis, cell growth, the mitotic index, cell cycle distribution, and protein expression were studied byTUNEL, cell counting, flow cytometry, and Western blot analysis, respectively. Results: The wt-p53 human pancreatic tumor cell line Capan-2 and p53-efficient mouse embryonic fibroblasts (MEFs) were more responsive to treatment with TMZ + BG than mutant p53 Capan-1 and p53-null MEFs. S phase delay with a subsequent G2/M arrest was observed in Capans in response to BG + TMZ. The G1-to-S transition delay in Capan-2 was associated with p53-dependent apoptosis and was distinctly different from the presumed mismatch repair (MMR) killing operative during the G2/M arrest. The effect of p53on BG +TMZ toxicity was supported by a marked change in apoptosis when p53 function was restored/inactivated. There was an early induction of MMR proteins in p53-efficient lines. Conclusion: p53 provokes a classic proapoptotic response by delaying G1-to-S progression, but it may also facilitate cell killing by enhancing MMR-related cell cycle arrest and cell death.     

HOXA10 Promotes Cell Invasion and MMP-3 Expression Via TGFβ2-Mediated Activation of the p38 MAPK Pathway in Pancreatic Cancer Cells

Background

HOXA10 is closely related to tumor progression in many human cancers. However, the role of HOXA10 in pancreatic cancer remains unclear. The aim of this study was to determine the involvement of HOXA10 in pancreatic cancer cell invasion and migration.

Methods

The effect of HOXA10 on the invasion and migration of pancreatic cancer cells was assessed by invasion and migration assays. The protein of transforming growth factor beta-2 (TGFβ2) was neutralized by TGFβ2 blocking antibody. The activation of p38 was inhibited by SB239063.

Results

HOXA10 could promote the invasion and migration of pancreatic cancer cells. Knockdown of HOXA10 decreased the expressions of TGFβ2 and matrix metallopeptidase-3 (MMP-3) and suppressed the activation of p38. Conversely, overexpression of HOXA10 increased the levels of TGFβ2 and MMP-3. Further experiments identified that TGFβ2 contributed to the HOXA10-promoted invasion and migration and regulated MMP-3 expression and p38 activation. Additionally, inhibition of p38 suppressed cell invasion and MMP-3 expression in pancreatic cancer cells.

Conclusions

HOXA10 promotes cell invasion and MMP-3 expression of pancreatic cancer cells via TGFβ2-p38 MAPK pathway. Thus, HOXA10 could be a useful target for the treatment of pancreatic cancer.     

Extracellular matrix metalloproteinase inducer regulates metalloproteinases in human uterine endometrium

CONTEXT: Endometrial remodeling occurs during each menstrual cycle in women and also during the establishment of endometriosis. Both processes involve the production of metalloproteinases (MMPs) by uterine endometrial cells. OBJECTIVE: The objective of this study was to determine whether tissue remodeling and endometrial invasion involve activation of MMPs by extracellular matrix metalloproteinase inducer (EMMPRIN). MAIN OUTCOME MEASURES: EMMPRIN expression was examined by immunohistochemistry and real-time PCR in ectopic and eutopic endometria. For functional assays, human uterine fibroblasts were treated in the absence or presence of IL-1beta (10 ng/ml) or purified native EMMPRIN (0.5 or 1 microg/ml) for 24 h. Cellular RNA and conditioned medium were assayed by real-time PCR or immunoblotting. RESULTS: EMMPRIN protein localized to epithelial and fibroblast cells of eutopic and ectopic endometria. The pattern of localization was regulated by ovarian hormones. EMMPRIN mRNA levels varied throughout the menstrual cycle in parallel with the cyclic changes in estradiol. EMMPRIN treatment (0.5 microg/ml) of human uterine fibroblast cells stimulated MMP-1 (5.23-fold) and MMP-2 (8.55-fold), but not MMP-3, mRNA levels over levels in control cells (P < 0.05). EMMPRIN treatment (1 microg/ml) stimulated endogenous EMMPRIN (1.6-fold) mRNA levels (P > 0.05). IL-1beta stimulated MMP-1 (5.6-fold), MMP-2 (2.8-fold), and MMP-3 (75-fold) gene expression, but not EMMPRIN, over levels in control cells (P < 0.05). Both EMMPRIN and IL-1beta treatments stimulated MMP-1, -2, and -3, but not EMMPRIN protein secretion, with 0.5 microg/ml producing the greatest response. CONCLUSIONS: The ability of EMMPRIN to stimulate MMP secretion by endometrial fibroblasts indicates its potential role in uterine remodeling and the pathogenesis of endometriosis.     

A New Invasion Score for Determining the Resectability of Pancreatic Carcinomas with Contrast-Enhanced Multidetector Computed Tomography

Objective: It was the aim of this study to evaluate a new infiltration score to determine the resectability of pancreatic carcinomas in preoperative planning. Materials and Methods: Eighty patients with suspected pancreatic tumor were examined prospectively using 16-row spiral CT. The scans were evaluated for the presence of pancreatic carcinoma, peripancreatic tumor extension and vascular invasion using a standardized questionnaire. Invasion of the surgically relevant vessels was evaluated using a new invasion score. The operative and histological findings and the clinical follow-up served as the gold standard. Results: Forty patients had a pancreatic carcinoma, 5 had metastasis of a different primary tumor, and in 35 patients, there was no malignant pancreatic disease. The sensitivity for tumor detection was 100%, with a specificity of 88% for differentiating between malignant and benign pancreatic tumors. Invasion of the surrounding vessels was evaluated correctly using the invasion score, with a sensitivity of 89% and a specificity of 99%. In evaluation of resectability, a sensitivity of 94% and a specificity of 89% were achieved. Conclusion: Using 16-row spiral CT, the invasion score is a valid tool for correctly assessing invasion in relevant vessels in cases of pancreatic carcinoma and for determining resectability.     

Resection for pancreatic cancer in the new millennium

Background and Aim ofthe Study: Complications of pancreatic resections are dangerous and costly. A literature review was therefore done to investigate the evidence for improving the results by regionalizing this demanding surgery.Results: Studies from four countries (USA, UK, the Netherlands and Finland) with advanced health care systems have shown a significant inverse correlation between case volume for pancreatic cancer resection and post-operative mortality. Further analysis reveals lower complications, reduced hospital stay, reduced hospital costs and improved survival of patients treated in high-volume hospitals. The relationship volume and outcome is with institutional volume rather than single surgeon caseload. The evidence therefore strongly supports the regionalization of pancreatic cancer surgery into large specialized multi-disciplinary units. In the UK, the National Health Service Executive has instructed Regional Health Authorities to concentrate pancreatic cancer surgery into designated Regional Centres ideally with catchment populations of 2–4 million. There is now considerable pressure to adopt a simi-lar policy in all countries with advanced health care systems.Conclusion: There is today enough evidence to advocate the regionalization of pancreatic cancer resections.     

Enhanced ENA-78 and IL-8 Expression in Patients with Malignant Pancreatic Diseases

Background/Aim: Pancreatic cancer is characterized by perineural invasion, early lymph node and liver metastases, and an extremely dismal prognosis. In the present study we aimed at investigating the expression profile of pro-inflammatory and angiogenic CXC chemokines as potential factors contributing to the aggressive biology of this gastrointestinal malignancy. Methods: Protein expression profiles of the CXC chemokines growth-related oncogene alpha (GRO-α/ CXCL1), epithelial cell-derived neutrophil-activating peptide-78 (ENA-78/CXCL5), granulocyte chemoattractant protein-2 (GCP-2/CXCL6), neutrophil-activating protein-2 (NAP-2/CXCL7), and interleukin-8 (IL-8/CXCL8) were assessed by enzyme-linked immunosorbent assay in pancreatic carcinoma, cancer of the papilla of Vater, pancreatic cystadenoma, and chronic pancreatitis specimens. Results: IL-8 and ENA-78 protein expression was most pronounced in pancreatic carcinoma specimens, showing an 11-fold and 17-fold over-expression in comparison with non-affected neighbouring tissues, a 66-fold and 24-fold upregulation compared to pancreatic cystadenoma, and a 6-fold and 9-fold overex-pression with respect to chronic pancreatitis, respectively (p <0.05 between all groups). In addition, a close correlation between IL-8 and ENA-78 protein expression and advanced pancreatic carcinomas in relation to the T category was evident (p < 0.05). Conclusion: Our results demonstrate that ELR+ CXC chemokines are differentially expressed in malignant and non-malignant human pancreatic specimens, suggesting a potential contribution of these chemokines to the pathogenesis of pancreatic carcinoma.     

Pancreatitis-Associated Protein Inhibits Human Pancreatic Stellate Cell MMP-1 and -2, TIMP-1 and -2 Secretion and RECK Expression

Background/Aims: Pancreatic stellate cells (PSCs) play a key role in fibrogenesis associated with acute and chronic pancreatitis. Pancreatitis-associated protein (PAP), an acute-phase protein, is dramatically upregulated during acute and chronic pancreatitis. Assuming a protective role of PAP, we investigated its effects on human PSCs. Methods: PSCs were obtained by outgrowth from fibrotic human pancreastissue. PAP was expressed in the yeast Pichia pastoris. PAP was added at 10 ng/ml to cultured PSCs. Cell proliferation was determined by bromodeoxyuridine incorporation. PSC migration was assessed by a wound healing assay. Collagen types I and III, fibronectin, matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs) and reversion-inducing cysteinerich protein with Kazal motifs (RECK) were demonstrated on protein and mRNA level. Results: PAP had no significant effect on PSC proliferation and migration. Cell-associated fibrillar collagen types I and III and fibronectin increased after addition of PAP to PSCs. PAP diminished the expression of MMP-1 and -2 and TIMP-1 and -2 and their concentrations in PSC supernatants. RECK was detected on the surface of PSCs and its expression was reduced after PAP application. Conclusions: Our data offer new insights into the biological functions of PAP, which may play an important role in wound healing response and cell-matrix interactions.     

Multidetector CT Evaluation of the Course of Nonresectable Pancreatic Carcinomas with Neoadjuvant Therapy

Objective: A prospective study to determine the value of multidetector CT (MD-CT) in assessing the course of nonresectable pancreatic carcinoma during therapy. Material and Methods: 26 patients with nonresectable pancreatic carcinoma underwent MD-CT before and after therapy. The examinations were evaluated with regard to tumor size and vascular invasion using an invasion score (IS) by 2 radiologists independently (κ analysis). Diagnosis was confirmed surgically, by biopsy or clinical course. Results: Sensitivity for the assessment of irresectability was 100%. Following therapy, 54% of all the tumors were smaller (14/26), 42% had increased in volume (11/26), and one tumor remained stable (1/26). The IS (veins) during follow-up changed in 26 patients (portal vein: 5 higher (mean score 10.4/16.2), 4 lower (mean score 17.5/11.5); superior mesenteric vein: 12 higher (11/14.4), 5 lower (16.2/14.6); p = 0.026). The IS (arteries) changed in 13 patients (celiac trunk: 3 higher (3.3/10); hepatic artery: 4 higher (5.7/10.2), 3 lower (11.6/10.3); superior mesenteric artery: 2 higher (4.5/9.5), 1 lower (12/11)). The k values were calculated between 0.56 and 0.87. Conclusion: MD-CT is suitable for evaluating tumor spread during therapy for nonresectable pancreatic carcinoma. The IS is useful for assessing the degree of change in vessel invasion.     

Nafamostat mesilate can prevent adhesion, invasion and peritoneal dissemination of pancreatic cancer thorough nuclear factor kappa-B inhibition

Background

Constitutive activation of nuclear factor kappa-B (NF-??B) contributes to the aggressive behavior of pancreatic cancer. Over-expression of downstream target genes of NF-??B such as intercellular adhesion molecule-1 (ICAM-1), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) leads to the promotion of cell adhesion, angiogenesis, invasion and metastasis. We previously reported that nafamostat mesilate, a synthetic serine protease inhibitor, blocks NF-??B activation in pancreatic cancer. We hypothesized that nafamostat mesilate may inhibit cell adhesion, angiogenesis, invasion and metastases in peritoneal dissemination of pancreatic cancer.

Methods

In vitro, we assessed inhibition of NF-??B, phosphorylated I??B??, ICAM-1, VEGF and MMP-9 activity by nafamostat mesilate using human pancreatic cancer cell lines (AsPC-1, BxPC-3 and PANC-1). Changes in adhesion and invasion abilities of cancer cells were then evaluated by nafamostat mesilate treatment. In vivo, the efficacy of nafamostat mesilate treatment was assessed using peritoneal dissemination of pancreatic cancer in mice.

Results

In vitro, nafamostat mesilate inhibited activities of NF-??B, phosphorylated I??B??, ICAM-1, VEGF and MMP-9. Moreover, nafamostat mesilate not only inhibited cell adhesion and invasion but also increased the sensitivity of anoikis. In vivo, tumor growth using AsPC-1 cells of the treatment group was significantly slower, and survival rate was significantly better, than those in control group (p?Conclusion Nafamostat mesilate reduced peritoneal metastasis and prolonged survival of pancreatic cancer-bearing mice.     

Power Doppler ultrasonography for the assessment of vascular invasion by pancreatic cancer

Aim: To investigate the diagnostic accuracy of power Doppler ultrasonography (US) in assessing the vascular invasion by pancreatic cancer. Methods: A prospective study of 40 consecutive patients with pancreatic cancer (head 35, body 5) was performed. All patients underwent surgery. The relationships between tumor and each vessel were classified into four types according to the closest circumferential contact of the tumor with the vessel. A type 0 indicated no contact; type 1 indicated less than one third contact; type 2 indicated one third to 99% contact, and type 3 indicated encasement. Vascular invasion was diagnosed in types 2 and 3. The diagnostic accuracy was evaluated in the portal vein and in the splanchnic arteries (celiac artery, common hepatic artery, and superior mesenteric artery). The power Doppler US findings were confirmed by the operative findings. The results of power Doppler US were compared with those of CT scan and angiography. Results: Portal vein invasion was confirmed in resected specimens in 23 cases and by operative findings in 5 cases. For the diagnosis of portal vein invasion, sensitivity, specificity, and overall accuracy of power Doppler US were, respectively, 79.3, 90.9, and 82.5%. The respective values were 79.3, 100, and 85% for CT and 72.4, 81.8, and 75% for angiography. For the diagnosis of arterial invasion, sensitivity, specificity, and overall accuracy of power Doppler US were 80, 92, and 90%, respectively. The corresponding values were 47, 88, and 73% for CT and 47, 100, and 80% for angiography. Conclusion: Power Doppler US proved to be useful for the diagnosis of vascular invasion by pancreatic cancer.     

Vigilin and enzyme expression in isolated pancreatic acini after mellitin and gamma-interferon treatment

Background/Aims: Pancreatitis goes along with changes in exocrine enzyme synthesis and secretion in pancreatic acini. The multi-KH domain protein vigilin is supposed to play an important role in t-RNA trafficking especially in cells with high protein synthesis rates and may reflect the degree of stimulation of translational machinery during pathological processes. In relation to these phenomena we explored in this connection the impact of two different inflammation mediators in a system of isolated rat pancreatic acini. Methods: Acini were prepared from male Sprague-Dawley rats by collagenase digestion and incubated with mellitin or gamma interferon. Secretion and cytosolic cell content of pancreatic trypsin and amylase as well as the expression of vigilin were determined. Results: The phospholipase A₂ activator mellitin caused morphological alterations and increased release of trypsin and amylase, while vigilin expression and the intracellular content of these enzymes decreased. Gamma-interferon, a cytokine which is involved at different steps in inflammation processes, selectively inhibits the release of trypsin(ogen) while not affecting amylase secretion and vigilin expression. Conclusion: Mellitin as well as gamma interferon causes alterations in pancreatic enzyme secretion. Additionally, mellitin seems to influence the expressed gene pattern of pancreatic acini while interferon-γ has no effect on protein synthesis but enzyme secretion.