Downregulation of GEP100 Improved the Growth Inhibition Effect of Erlotinib Through Modulating Mesenchymal Epithelial Transition Process in Pancreatic Cancer.

OBJECTIVE: The epidermal growth factor receptor is overexpressed in the majority of pancreatic cancer. Epidermal growth factor receptor tyrosine kinase inhibitor erlotinib was approved to treat patients combining with gemcitabine. However, the sensitivity is low. Here, we try to reveal the regulatory role of guanine nucleotide exchange protein 100 (GEP100) in erlotinib sensitivity. METHODS: We investigated the correlation between GEP100 expression and sensitivity to erlotinib in different pancreatic cancer cell lines, followed by examination of the effect of GEP100 on erlotinib sensitivity by establishing the stable knocked-down cell line. The expression level of epithelial mesenchymal transition-related protein was examined by Western blot, and the regulatory mechanism was investigated by short hairpin RNA. Xenograft experiment was also performed in nude mice. RESULTS: We identified a significant correlation between sensitivity to erlotinib and expression of GEP100. GEP100 downregulation increased its sensitivity to erlotinib. E-cadherin short hairpin RNA treatment inhibited this sensitivity. Immunohistochemical staining showed a mutual exclusive expression pattern of GEP100 and E-cadherin in human pancreatic cancer tissues. Xenograft showed that downregulation of GEP100 enhanced the growth inhibition of erlotinib in nude mice. CONCLUSIONS: Our results suggested that GEP100 and E-cadherin have the predictive value for responsiveness to erlotinib in pancreatic cancer.

Multiple Lymphomatous Polyposis of the Intestine with Ileocecal Intussusception Due to Mantle Cell Lymphoma: A Case Report of a 34-Year-Old Man.

BACKGROUND Multiple lymphomatous polyposis of the gastrointestinal tract can be associated with the B-cell lymphoma variant, mantle cell lymphoma, with most cases having been described in patients who are more than 50 years-of-age. A rare case of multiple lymphomatous polyposis due to mantle cell lymphoma is reported in a 34-year-old man. CASE REPORT A 34-year-old man presented with paroxysmal abdominal pain followed by spontaneous remission, which had been previously diagnosed as gastritis. An episode of ileocecal intussusception occurred, which was confirmed on imaging studies. The diagnosis of multiple lymphomatous polyposis due to mantle cell lymphoma was confirmed following ileocecal resection and histopathology. The patient refused to receive chemotherapy following surgery. Currently, at two-year follow-up, no further abnormality has been found. A review of the literature has shown the importance of endoscopic evaluation in the diagnosis of lymphomatous polyposis. CONCLUSIONS Multiple lymphomatous polyposis due to mantle cell lymphoma has rarely been described in young patients under the age of 50 years. Gastrointestinal endoscopic examination is important for the early diagnosis of multiple lymphomatous polyposis.

Down-regulation of GEP100 causes increase in E-cadherin levels and inhibits pancreatic cancer cell invasion.

AIMS: Invasion and metastasis are major reasons for pancreatic cancer death and identifying signaling molecules that are specifically used in tumor invasion is of great significance. The purpose of this study was to elucidate the role of GEP100 in pancreatic cancer cell invasion and metastasis and the corresponding molecular mechanism. METHODS: Stable cell lines with GEP100 knocked-down were established by transfecting GEP100 shRNA vector into PaTu8988 cells and selected by puromycin. qRT-PCR and Western blot were performed to detect gene expression. Matrigel-invasion assay was used to detect cancer cell invasion in vitro. Liver metastasis in vivo was determined by splenic injection of indicated cell lines followed by spleen resection. Immunofluorescence study was used to detect the intracellular localization of E-cadherin. RESULTS: We found that the expression level of GEP100 protein was closely related to the invasive ability of a panel of 6 different human pancreatic cancer cell lines. Down-regulation of GEP100 in PaTu8988 cells significantly decreased invasive activity by Matrigel invasion assay, without affecting migration, invasion and viability. The inhibited invasive activity was rescued by over-expression of GEP100 cDNA. In vivo study showed that liver metastasis was significantly decreased in the PaTu8988 cells with GEP100 stably knocked-down. In addition, an epithelial-like morphological change, mimicking a mesenchymal to epithelial transition (MET) was induced by GEP100 down-regulation. The expression of E-cadherin protein was increased 2-3 folds accompanied by its redistribution to the cell-cell contacts, while no obvious changes were observed for E-cadherin mRNA. Unexpectedly, the mRNA of Slug was increased by GEP100 knock-down. CONCLUSION: These findings provided important evidence that GEP100 plays a significant role in pancreatic cancer invasion through regulating the expression of E-cadherin and the process of MET, indicating the possibility of it becoming a potential therapeutic target against pancreatic cancer.

[Effect of metoclopramide on capsule endoscopy examination: a randomized study].

OBJECTIVE: To investigate the effect of metoclopramide on capsule endoscopy (CE) examination. METHODS: Total 116 patients referred for CE were randomized into two groups with 58 patients in each group. In treatment group patients received 10 mg metoclopramide intramuscular injection after swallowing the capsule and in control group no metoclopramide was administered. The gastric transit time, small bowel transit time, complete endoscopy rate were observed in both groups. RESULTS: The CE examination was completed in 51 patients of treatment group (87.9%) and 48 of control group (84.2%). Mean gastric transit time was (32.45 ± 29.63) min in treatment group and (45.81 ± 40.01)min in control group, there was significant difference between two groups (P<0.05). Mean small bowel transit time was (252.69 ± 113.29) min in treatment group and (258.75 ± 83.83) min in control group, there was no significant difference between two groups (P>0.05). CONCLUSION: Metoclopramide may reduces gastric transit time, but not effect small bowel transit time,which suggests that it might increase the likelihood of complete small-bowel examination in patients undergoing capsule endoscopy.

K-Ras resides on the Arf6-mediated CIE system and its active type interacted with Arf6T27N.

Ras is known as an oncogene transferring signals from the plasma membrane. Recent studies have demonstrated that plasma membrane was not the unique platform for Ras signaling. Ras could also be endocytosed and transported to different endomembrane compartments, evoking different signal pathways there. It is of great significance to exploit the unique intracellular trafficking features of different Ras isoforms to develop new anti-Ras drugs. ADP-ribosylation factor 6 (Arf6) was known to mediate one of the clathrin-independent endocytosis (CIE) pathways. The role of Arf6 in K-Ras dynamic remains largely unknown. In this study, we showed that K-RasG12V co-localized with Arf6 at the plasma membrane, and entered the tubular endosomes or protrusions induced by cytochalasin D or aluminum fluoride in the same way as H-RasG12V does. A subcellular fractionation experiment demonstrated that Arf6 siRNA treatment reduced the plasma membrane presence of both endogenous Ras isoforms and inhibited the phosphorylation of Erk triggered by EGF. When co-expressed with Arf6Q67L, both isoforms were sequestered into the large phosphatidylinositol 4,5-biphosphate [PI(4,5)P2]-enriched vacuoles. However, when co-expressed with Arf6T27N, K-RasG12V co-localized with Arf6T27N at the tubular endosomes significantly than H-RasG12V. Immunoprecipitation and GST fusion protein pull-down studies found out for the first time that K-RasG12V interacted with Arf6T27N. Swapping mutation study showed that the above difference was due to different C-termini. Our study indicated that Arf6 was involved in the dynamic regulation of both Ras isoforms.

ADP-ribosylation factor like 7 (ARL7) interacts with alpha-tubulin and modulates intracellular vesicular transport.

ADP-ribosylation factor (ARF) like 7 (ARL7, also named ARL4C) is a member of ARL family and recent studies showed that it is involved in the AI-dependent cholesterol secretion process. Yet its biological function remains largely unknown. Using a MALDI-TOF/MS analysis, we identified alpha-tubulin interacted with ARL7. The interaction was confirmed by GST pull-down assay and co-immunoprecipitation in renal carcinoma cell 786-O in which we found the endogenous ARL7 is expressed. This is the second ARL member found interacting with tubulin after ARL8. In addition, ARL7Q72L, a GTP-binding form, promoted the transferrin transport from early endosome to recycling endosome significantly. The above data suggested that ARL7 might modulate the intracellular vesicular transport via interaction with microtubules.

Selective inhibition of cyclooxygenase-2 suppresses the growth of pancreatic cancer cells in vitro and in vivo.

Cyclooxygenase-2 (COX-2), a prostaglandin synthetase, is involved in development of certain tumors. We therefore analyzed COX-2 expression in pancreatic cancer tissues (53 samples) and Panc-1 human pancreatic cancer cells by immunohistochemistry, RT-PCR and western-blotting analyses. Also, immunohistochemistry of proliferating cell nuclear antigen (PCNA) was performed. We found expression of COX-2 was dramatically upregulated in 36 of 53 cases (67.9%) and the expression of COX-2 was associated with the diameter (> 3 cm) of the tumors (p < 0.05), but not with the age, gender, tumor location, differentiation, lymph-node metastases and TNM stage. The positivity rate of PCNA expression in the pancreatic cancer cells of the COX-2 positive group (32.88 +/- 13.26%) was significantly higher than that in the COX-2 negative group (24.56 +/- 11.51%) (p < 0.05). Then we investigated the effect of selective inhibitors of COX-2 (NS398 and celecoxib) on proliferation of Panc-1 cells by 3-(4,5 dimethyl-2-thiazolyl)-2.5-diphenyl-2H-tetrazolium bromide (MTT) assay. Either NS398 or celecoxib suppressed proliferation of Panc-1 cells dose-dependently in vitro. Furthermore, Panc-1 cells were implanted into nude mice, and celecoxib was administrated orally with feed. The volume of the tumor xenografted into nude mice was decreased by 51.6% in the celecoxib group (p < 0.01). In conclusion, the increased expression of COX-2 may be responsible for rapid proliferation of pancreatic cancer, and specific inhibition of COX-2 suppresses proliferation of Panc-1 cells in vitro and in nude mice. The selective inhibitor of COX-2 may be an effectual agent for pancreatic cancer chemoprevention.

Effect of emodin on pancreatic fibrosis in rats.

AIM: To establish the rats model of chronic fibrosing pancreatitis and to prove the anti-fibrotic effect of emodin in chronic pancreatitis with fibrosis. METHODS: Fifty rats were randomly divided into five groups, 10 rats in each group. Trinitrobenzene sulfonic acid (TNBS) was infused into the pancreatic duct to induce chronic pancreatitis in rats (except for normal group). Emodin-treated rats were fed with different doses of emodin (20, 40 and 80 mg/kg body weight) for 28 d, while normal group and control group received 0.9% sodium chloride solution. Serum levels of hyaluronic acid (HA) and laminin (LN) were determined by radioimmunoassay. Histopathological alterations were studied by optical microscopy. Expression of collagen was also examined while transforming growth factor-beta-1 (TGF-beta(1)) was localized by immunochemistry. RESULTS: In emodin-treated rats, the serum levels of HA and LN were decreased significantly (HA, 62.2 +/- 19.3 microg/L vs 112.7 +/- 26.5 microg/L, P < 0.05; LN 44.3 +/- 10.4 microg/L vs 86.2 +/- 16.5 microg/L, P < 0.05); the degree of fibrosis was ameliorated observably; the expression of collagen in pancreatic tissue was reduced especially in high-dose emodin-treated group (36% +/- 5% vs 42% +/- 6%, P < 0.05); with the increased doses of emodin, the expression of TGF-beta(1) was declined, compared with those in control group. CONCLUSION: Emodin has an anti-fibrotic effect on pancreatic fibrosis in rats. Because of its anti-fibrotic effect, it could be a potential herb for the treatment of chronic pancreatitis.

[Effect of emodin on pancreatic fibrosis: experiment with rats].

OBJECTIVE: To study the effect of emodin on pancreatic fibrosis and potential mechanism thereof. METHODS: Fifty SD rats were randomly divided into 5 equal groups: normal control group, model control group, low-dose emodin-treated group, mediate-dose emodin-treated group, and high-dose emodin-treated group. The rats of the latter 4 groups underwent infusion of trinitrobenzene sulfonic acid (TNBS) into the pancreatic duct so as to establish models of pancreatic fibrosis. The emodin-treated rats were fed with different doses of emodin (20, 40, and 80 mg/kg body weight), while the normal and model control groups received 0.9% sodium chloride solution instead. Twenty-eight days later the rats were killed, blood samples were collected, and their pancreases were taken out. The serum levels of hyaluronic acid (HA) and laminin (LN) were determined by radioimmunoassay. The histopathological alterations were studied by optical microscopy. The expression of collagen was examined by Van Gieson staining. Western blotting was used to detect the protein expression of transforming growth factor-beta(1) (TGF-beta(1)). RESULTS: (1) The serum level of HA of the low-dose, mediate-dose, and high-dose emodin-treated groups were 87 microg/L +/- 22 microg/L, 78 microg/L +/- 25 microg/L, and 62 microg/L +/- 19 microg/L respectively, all significantly lower than that of the model control group (113 microg/L +/- 27 microg/L, P < 0.05 or < 0.01). The serum levels of laminin in the low-dose, mediate-dose, and high-dose emodin-treated groups were 67 microg/L +/- 14 microg/L, 57 microg/L +/- 12 microg/L, and 44 microg/L +/- 10 microg/L respectively, all significantly lower than that of the model control group (86 microg/L +/- 17 microg/L, P < 0.05 or P < 0.01); (2) The degrees of fibrosis of the emodin-treated groups were obviously ameliorated in comparison with the model control group, the higher the dose of emodin the more improved the pathological changes, especially in the high-dose emodin-treated group (P < 0.05). (3) The percentages of collagen positive cells of the low-dose, mediate-dose, and high-dose emodin-treated groups were 39% +/- 7%, 38% +/- 4%, and 36% +/- 5% respectively, all lower than that of the model control group (42% +/- 6%), with a significant difference between the high-dose emodin-treated group and the model control group (P < 0.05). (4) The protein content of TGF-beta(1) of the low-dose, mediate-dose, and high-dose emodin-treated groups were 44.3% +/- 2.1%, 39.2% +/- 1.8%, and 28.8% +/- 1.6% respectively, all significantly lower than that of the model control group (60.7% +/- 1.7%, all P < 0.05), and the protein content of TGF-beta(1) of the high-dose emodin-treated group was significantly lower than those of the other 2 emodin-treated groups (both P < 0.05). CONCLUSION: Emoidn has an anti-fibrosis effect on pancreatic fibrosis, which maybe related to the content of TGF-beta(1) protein.

[Effect of selective cyclooxygenase-2 inhibitor celebrex on expression of vascular endothelial growth factor (VEGF) in pancreatic carcinoma].

BACKGROUND & OBJECTIVE: The previous study has identified that cyclooxygenase-2 (COX-2) may have a close relation with tumor genesis, particularly with digestive tract tumors, and its inhibitor can exert the chemoprevention role on carcinogenesis. This study was designed to investigate the effect of celebrex, a selective cyclooxygenase-2 inhibitor, on the expression of vascular endothelial growth factor (VEGF) in pancreatic carcinoma of xenografted nude mice induced by pancreatic carcinoma PC-3 cell lines. METHODS: The effect of celebrex on tumor growth was observed.The expression of VEGF in the tumors was determined by reverse transcription polymerase chain reaction (RT-PCR), Western blot analysis, and enzyme-linked immunosorbent assay (ELISA). RESULTS: Average tumor volume and tumor weight from control mice were 0.438+/-0.052 cm(3) and 0.552+/-0.064 g as compared with 0.215+/-0.038 cm(3) and 0.244+/-0.042 g from treated mice (inhibition rate:51.6%,P< 0.05). VEGF expression was significantly down-regulated in the celebrex-treated tumors. ELISA revealed that the expression levels of VEGF were 1.11+/-0.11(microg/g) in control mice and the 0.66+/-0.11(microg/g) in the treated mice. The inhibition rate of VEGF was 40.6% (P< 0.05). CONCLUSION: COX-2 may play an important role in the angiogenesis of pancreatic carcinoma. The selective COX-2 inhibitor, celebrex, can result in the inhibition of angiogenesis and tumor growth.

[Inhibition of angiogenesis in pancreatic carcinoma by cyclooxygenase-2 antisense oligodeoxynucleotides].

OBJECTIVE: To investigate the effect of cyclooxygenase-2 antisense oligodeoxynucleotides (COX-2 AS-ODNs) on the angiogenesis in pancreatic carcinoma and to evaluate the intermediary effect of prostaglandin 2 in this process. METHODS: Specific targeting COX-2 AS-ODNs were designed and synthesized, and transfected into the PC3 human pancreatic carcinoma cells cultured in vitro. Fluorescence microscopy was used to observe the PC3 cells 0.12. 24, 40, and 72 hours after the transfection. the second cultured PC3 cells were divided into 5 groups: control group, Lipo group (transfected with Lipofectin only), C1 group (transfected with 1 micro g COX-2 AS-ODN + Lipo/well), C2 group (transfected with 2 micro g COX-2 AS-ODN + Lipo/well), and C3 group (transfected with 3 micro g COX-2 AS-ODN + Lipo/well). RT-PCR was used to observe the expression of COX-2 mRNA in the PC3 cells. The third batch of PC3 cells were transfected with 3 micro g COX-2 AS-ODN + Lipo/well, and the expression of COX-2 mRNA was observed 0, 12, 24, 48, and 72 hours later by RT-PCR. 3 micro g COX-2 AS-ODN + Lipo/bottle and 9 micro g COX-2 AS-ODN + Lipo/bottle were added into the cultured PC3 cells and Western blotting was used to observe the expression of COX-2 protein 24 hours later. 24 chicken eggs were inoculated with PC3 cells into the chorio-allantoic membrane and then divided equally into 5 groups; control group, Lipo group, COX-2 AS-ODN + Lipo group, and COX-2 AS-ODN + Lipo + PGE2 group. Leica microscopy was used to observe the angiogenesis in the transplanted carcinoma. RESULTS: RT-PCR showed that the downregulation of expression of COX-2 mRNA in the PC3 cells with the increase of the COX-2 AS-ODN concentration, peaking at the concentration of 0.2 micro mol/L. The effect of COX-2 AS-ODN was strongest by the 12th hour after transfection and then began to decrease and basically disappeared 48 hours after. Western blotting showed that COX-2 AS-ODN, especially that of the concentration of the expression of 9 micro g/bottle, inhibited the expression of COX-2 AS-ODN. The angiogenesis of the transplanted carcinoma in the eggs was significantly inhibited in the Lipo + COX-2 AS-ODN group, the density of newly generated vessels in the Lipo + COX-2 AS-ODN + PGE2 group was between those of the other 2 groups. CONCLUSION: COX-2 AS-ODN significantly inhibits the angiogenesis in the pancreatic carcinoma. Endogenous COX-2 AS-ODN may play an important role in such a process and PGE2 may play an intermediate role therein.