[Validation of candidate immunogenic membrane antigens of human pancreatic cancer screened by proteomics].

OBJECTIVE: To validate those obtained immunogenic membrane antigens candidate of human pancreatic cancer in the performed research. METHODS: In the pre-studies, serum IgG purified from clinically collected sera of pancreatic cancer patients underwent immunoblot with human pancreatic cancer cell line SW1990 membrane protein, totally obtained 9 positive protein spots. Number 5 and 6 positive dots of immunoblot were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and peptide mass fingerprinting matching. The candidate membrane antigens were further validated in cell lines by RT-PCR and real-time PCR. RNA of human normal pancreatic tissue and pancreatic cancer tissue was extracted respectively, different gene expression level of prohibitin 2 was studied by real-time PCR. RESULTS: Number 5 and 6 positive dots were identified as prohibitin 2 and prohibitin. RT-PCR and real-time PCR all showed that gene of prohibitin 2 and prohibitin were expressed in the human pancreatic cancer cell line SW1990, AsPc and P3 respectively, especially in P3 cell with highest expression (t = 7.442, P < 0.01). In addition, gene expression level of prohibitin 2 was significant higher in human pancreatic cancer than that of normal pancreatic tissue (t = 0.893, P < 0.01). CONCLUSIONS: Prohibitin 2 and prohibitin are both differently expressed in the pancreatic cancer cell line SW1990, AsPc and P3. Prohibitin 2 is obvious highly expressed in human pancreatic cancer tissue. Prohibitin 2 and prohibitin might be the candidate immunogenic membrane antigens of human pancreatic cancer.

[Candidate immunogenic membrane antigens of human pancreatic cancer].

OBJECTIVE: To verify the obtained immunogenic membrane antigens candidate of pancreatic cancer in the performed research. METHODS: Pancreatic cancer cell line SW1990 membrane protein underwent immunoblot with serum IgG purified from clinically collected sera of 66 pancreatic cancer patients. Number 3 and number 8 positive dots of immunoblot were identified by MALDI-TOF mass spectrometry and peptide mass fingerprinting matching. The candidate membrane antigens were further validated in cell lines by RT-PCR, real-time PCR and Western blot, and their different expression level of gene and protein in pancreatic cancer cell lines were contrastly studied. RESULTS: Number 3 and number 8 positive dots were identified as: voltage-dependent anion channel (VDAC3) and catechol-o-methyltransferase (COMT). RT-PCR, real-time PCR and Western blot showed that gene and protein of VDAC3 and COMT were expressed in the pancreatic cancer cell line SW1990, AsPc and P3 respectively. CONCLUSION: VDAC3 and COMT might be the candidate immunogenic membrane antigens of human pancreatic cancer, and their gene and protein are differently expressed in the pancreatic cancer cell line SW1990, AsPc and P3.

[Research of immunogenic membrane antigens of pancreatic cancer].

OBJECTIVE: To screen and obtain the validate immunogenic membrane antigens in pancreatic cancer. METHODS: Pancreatic cancer cell line SW1990 membrane protein was extracted and separated by two-dimensional gel electrophoresis (2-DE). One of the three parallel 2-DE gels underwent Coomassie blue staining while the other two underwent immunoblot. Serum IgG was purified from clinically collected sera of 66 pancreatic cancer patients and 24 chronic pancreatitis patients and used as the primary antibody of the immunoblot. Positive dots of immunoblot were identified by MALDI-TOF mass spectrometry and PMF matching. The candidate membrane antigens were further validated respectively in cell lines and tissues by RT-PCR, real-time PCR, Western blot, and their different expression level of gene and protein between pancreatic cancer cell line and normal pancreatic tissue were compared studied. RESULTS: The immunoblot of SW1990 membrane protein with serum IgG from cancer patients showed nine positive dots which were not the same as those from immunoblot with serum IgG from chronic pancreatitis patients. One talent dot was identified with MALDI and PMF as VDAC2. RT-PCR and real-time PCR showed that the gene of VDAC2 was expressed in the pancreatic cancer cell line. Western blot showed that the expression of protein level of VDAC2 in the pancreatic cancer cell line was obviously higher than in normal pancreatic tissue. CONCLUSIONS: VDAC2 might be the candidate immunogenic membrane antigens of pancreatic cancer, and its gene is all expressed in the pancreatic cancer cell line SW1990, AsPc and P3. The protein level of VDAC2 is significantly overexpressed in pancreatic cancer cell line than in normal pancreatic tissue.

Microarray analysis of gene expression profile of multidrug resistance in pancreatic cancer.

BACKGROUND: Chemotherapy is the most frequently adopted adjuvant therapy of pancreatic ductal adenocarcinoma (PDAC), but the development of drug resistance reduces its effectiveness. Clarification of the mechanism of multidrug resistance (MDR) development in PDAC is needed to improve the therapeutic effect of chemotherapy. This study was aimed to investigate the molecular mechanism of MDR of PDAC and to identify genes associated with MDR development. METHODS: The gene expression profiles of cell line SW1990 and three drug-selected pancreatic chemoresistant sub-lines, SW1990/5-Fu, SW1990/ADM and SW1990/GEM, were obtained using an oligonucleotide microarray (Affymetrix HG U133 2.0 plus) that contained approximately 38,000 human genes. The microarray results were validated by real-time quantitative polymerase chain reaction and Western blot analysis. RESULTS: There were 165 genes and expressed sequence tags, some of which have never been linked to drug resistance, that were up- or down-regulated at least 2-fold in all resistant sub-lines when compared with SW1990. According to Gene Ontology annotation, differentially expressed genes related to MDR in pancreatic cancer belong to many functional families and with diverse biological processes. Genes related to antioxidant activity, apoptosis, the cell cycle, signal transduction and intracellular adhesion may undergo epigenetic changes preceding MDR development. A hierarchical clustering was conducted and several interesting clusters were discovered that may be primarily related to cell cycle and developmental regulation. A prediction rule was built from the expression profiles of 117 genes after support vector machine (SVM) analysis, and the prediction result was examined by cytotoxic testing. As a result, a differential gene expression pattern was constructed in multidrug resistant pancreatic cancer cells. CONCLUSIONS: The findings of this study prove that construction of a chemoresistance prediction rule, based on gene expression patterns, is practical. These data provide new insights into the molecular mechanism of pancreatic cancer MDR development and may be useful for the detection and treatment of MDR in pancreatic cancer patients.

[The relationship between the changes of proangiogenic factors serum concentrations and progression of pancreatic carcinoma patients].

OBJECTIVE: To investigate the relationship between VEGF, bFGF and IGF-1 serum concentration and progression of pancreatic carcinoma. METHODS: Fifty-six patients with pancreatic carcinoma were divided into resectable group (n = 32) and unresectable group (n = 24). Another group was normal group (n = 20). The expression and significance of these proangiogenic factors were respectively analyzed in different groups. RESULTS: For pancreatic carcinoma group, concentrations of VEGF and bFGF were significantly higher than these of normal group (P < 0.01). Serum VEGF was significantly correlated with the resection of pancreatic carcinoma (P < 0.05) while bFGF and IGF were not. According to univariate analysis, serum VEGF was correlated with tumor grade, nodal disease, vascular invasion, distant metastases and tumor stage. Serum bFGF was associated with tumor size and grade. Serum IGF-1 was correlated with vascular invasion. CONCLUSIONS: Angiogenic factors play important roles in growth, invasion and metastasis. Detection of serum proangiogenic factors may have potential value in diagnosis and prognosis of pancreatic carcinoma.

[Selection of genes related to multidrug resistance of pancreatic ductal adenocarcinoma by microarray analysis].

OBJECTIVE: To investigate the genes concerning multidrug resistance (MDR) of pancreatic ductal adenocarcinoma with microarray analysis. METHODS: Gene expression profile of pancreatic cancer cell line SW1990 and resistance subline SW1990/5-FU, SW1990/ADM, SW1990/GEM were screened in two independent replicates using oligonucleotide microarray (Affymetrix HG U133 2.0 plus) which contained 38,500 human genes. And advanced bioinformatics analysis was conducted. RESULTS: Totally, 165 genes and expressed sequence tags (ESTs), which were seldom reported to be related with drug resistance before,were statistically difference and the fold change was up- or down-regulated at least 2 folds in all 3 resistant sub-lines when compared with SW1990. According gene ontology, the genes related to oxidoreductase activity, apoptosis, cell cycle, signal transduction and cell adhesion might be some epigenetic changes for MDR development. Hierarchical clustering analysis, showed several interesting clusters, namely, TNKS2, PRDX4 and CCDC4. CONCLUSIONS: MDR of pancreatic cancer is a complicated and multifactorial process. In the present study, a widespread differential gene expression pattern was constructed in PDAC multidrug resistant cells. Advanced study will provide new targets for MDR research and cast insights into research of the molecular mechanism of MDR.

[Relationship between apoptosis induced by 2-butylamino-2-demethoxy-hypocrellin B in human pancreatic cancer cells Capan-1 and photosensitization of mitochondria].

OBJECTIVE: To explore the possible mechanism of apoptosis induced by photodynamic therapy (PDT) in human pancreatic cancer cells Capan-1 with 2-butylamino-2-demethoxy-hypocrellin B (BAHB) as photosensitizer. METHODS: The localization of BAHB in Capan-1 cells was studied, apoptosis was determined by DNA gel electrophoresis after PDT. The mitochondria membrane potential (DYm) and cytochrome C release were observed by laser scan confocal microscopy and Western blotting. RESULTS: The low concentration photosensitizer was mainly localized in mitochondria and also in lysosomes when the concentration is high. DNA ladder analysis showed characteristic of apoptosis. The mitochondria membrane potential (DYm) showed a loss of 30% around, after 6 hours by PDT under laser scan confocal microscopy, which is caused by a sudden increase in the permeability of mitochondria membrane accompanied with apoptosis. In Western blotting, cytochrome C release was observed from the mitochondria into the cytoplasm during BAHB-induced apoptosis. CONCLUSION: The research suggests that BAHB-induced apoptosis is related to photosensitization of mitochondria.

[Expression and significance of GCS gene in human pancreatic cancer cell SW1990 and its drug-resistant sublines].

OBJECTIVE: To study the expression and significance of GCS gene in human pancreatic cancer cell line SW1990 and its drug-resistant sublines. METHODS: SW1990 and its drug-resistant sublines, SW1990/FU, SW1990/ADM and SW1990/GEM were cultured in vitro. CCK-8 (Cell Counting kit-8) was used to detect the drug resistance of the sublines. Relative quantitation of GCS mRNA expression was evaluated by real-time PCR and Western blot was adopted to evaluate the expression of GCS protein. RESULTS: The drug resistance indexes of SW1990/FU, SW1990/ADM and SW1990/GEM were 339.7, 11.9 and 56.6, respectively. The GCS mRNA and protein were expressed in all SW1990 and its drug-resistant sublines. There was a higher expression of GCS mRNA in all the sublines and a significant difference of GCS protein expression was detected in SW1990/ADM and SW1990/GEM compared with SW1990. CONCLUSIONS: GCS gene is expressed in SW1990 and its drug-resistance sublines. The high expression of GCS protein in SW1990/ADM and SW1990/GEM might be one reason of resistance to ADM and GEM in the sublines.

[The method of inducing and establishing human pancreatic cancer cell sublines with radiation resistance].

OBJECTIVE: To explore the method of inducing and building pancreatic cancer cell sublines with radiation resistance. METHODS: Simulating the clinical radiotherapy, the pancreatic cell lines SW1990, Capan-1 (Cap), AsPC-1 (ASPC), P3, PANC-1 (Pan-1) and MIAPaCa-2 (MIA) were repeatedly given individual dose of X-rays with liner accelerator to induce radiation resistance, the changes of cell morphology, cell cycle and radio sensibility in the induced cell lines were compared with the parental cell lines at the end of inducing course. RESULTS: Compared with the parental cells, there were significant changes in morphology in the pancreatic cancer cell sublines after the radiation. Cell cycle analysis suggested that SW1990-R, ASPC-R, MIA-R, PAN-R and P3-R had lower G(2)/M and greater SF(2) (survival fraction after 2 Gy irradiation) compared with the parental cell lines. CONCLUSIONS: The method of radiating cells step by step and repeatedly is viable to establish radio-resistant pancreatic cancer cell lines.

[Drug resistance and activity changes of thioredoxin reductase in pancreatic cancer cells strain SW1990 induced by gemcitabine].

OBJECTIVE: To establish gemcitabine-resistant pancreatic cancer cell strain and study the role of thioredoxin reductase (TrxR) in drug-resistant process. METHODS: Gemcitabine-resistant pancreatic cancer cell strain SW1990/GZ was induced by increasing drug dosage intermittently, then the changes of its biological features and the activity of TrxR were examined. RESULTS: Stable drug-resistant SW1990/GZ cell strain was established by culturing with gemcitabine for 9 months. The morphology and growth characteristics of the cell strain changed remarkably. The cells shrunk and became rounder; its endoplasm expanded; granular substances increased; and the doubling-time was prolonged. Resistance of the cell line to gemcitabine, fluorouracil, adriamycin, and mitomycin significantly increased. The TrxR activity of the drug-resistant cells was increased markedly. CONCLUSION: SW1990/GZ has certain multidrug resistance to some chemotherapy drugs, and TrxR plays a role in the drug-resistant process.

[Effects of hydrogen peroxide on intracellular free Ca2+ content in rat liver oval cells].

OBJECTIVE: To study the effects and mechanism of hydrogen peroxide (H2O2) of low concentration on dynamic changes of intracellular free calcium contents ([Ca2+]i) in cultural rat liver oval cells (WB-F344 cells). METHODS: Using Fluo-3/Am as fluorescent indicator of [Ca2+]i and it was measured by laser scanning confocal microscope system. RESULTS: The results showed that: (1) A rapid transient spiking of [Ca2+]i occurred after the stimulation of H2O2 of low concentration (800 nmol/L). (2) The [Ca2+]i increase was abolished by pretreated with catalase (CAT) or by incubated in D-Hank's solution containing EGTA, the chelate of extracellular Ca2+. (3) The [Ca2+]i increase was not inhibited by pretreated nifedipine, Ca2+ channel blocker, but was abolished by pretreated with anthracere-9-cardoxylic acid (A9C), the Cl-channel blocker and which also blocked calcium activated non-selective cation channel (CAN). CONCLUSIONS: These results suggest that the increase of [Ca2+]i induced by H2O2 of low concentration may be due to the influx of extracellular Ca2+ through CAN.