[Experimental study of MAT1 gene silencing mediated by siRNA in pancreatic cancer].

OBJECTIVE: To investigate the inhibitory effect of gene silencing mediated by MAT1-siRNA constructed in vitro transcription for pancreatic cancer in vivo and in vitro. METHODS: 21-nt double strand siRNA targeting MAT1 gene was constructed and labeled with Cy3 fluorescent labeling reagent. Human pancreatic cancer cells of the line BxPC3 were cultured and divided into 4 groups: MAT1-siRNA transfected group, negative siRNA control group, lipid control group, and blank control group. The rate of cell duplication was determined by counting the cells for three consecutive days. Cell cycle profiles were determined by flow cytometry. Semi-quantitative analysis of the level of MAT1-mRNA expression was performed using the RT-PCR technique. The level of MAT1 protein expression was analyzed by Western-blotting. 18 nude mice were injected subcutaneously with BxPC3 cells to establish mouse tumor models, and then divided randomly into 3 equal groups: MAT1-siRNA group undergoing injection of MAT1-siRNA directly into the tumors 2 times a week for 4 weeks, blank control group, and negative MAT1-siRNA group. 4 weeks later the mice were killed to observe the weight and size of tumor and to calculate the tumor inhibition rate. RESULTS: Two of the 4 designed MAT1-siRNAs significantly suppressed the growth of the BxPC3 cells. 72 h after transfection the cell duplication was inhibited by 34.9% in the MAT1-siRNA transfection group. The cell cycle profile showed 83.9% of the MAT1-siRNA transfected cells were in the G0/G1 phase, a rate significantly higher than that in the blank control group (59.86%, P < 0.01). 48 h later the content of MAT1-mRNA of the MAT1-siRNA transfected cells was significantly reduced by 80.12%, and 72 h after the transfection the content of MAT1 protein was reduced by 50.12%, a rate significantly higher than those of the 2 control groups (both P < 0.01). The weight and volume of the transplant tumors in the MAT1-siRNA injected nude mice were significantly reduced compare with the negative siRNA injected control nude mice and blank control nude mice (both P < 0.05). The inhibition rate was 42.53%. CONCLUSION: MAT1 gene silencing mediated by siRNA significantly suppresses the growth of pancreatic cancer cells in vitro, and significantly achieves an anti-tumor effect on the subcutaneously transplanted pancreatic tumor in vivo.

[Effect of NK4 gene transfection on tumor growth of human pancreatic cancer xenograft in nude mice].

BACKGROUND & OBJECTIVE: NK4 is not only an antagonist of hepatocyte growth factor but also an angiogenesis inhibitor. Studies have confirmed that NK4 can inhibit tumor growth and metastasis, but its effect on pancreatic cancer remains unknown. This study was designed to observe the effect of NK4 gene on human pancreatic cancer in nude mice and the possible mechanisms. METHODS: The nude mouse model of pancreatic cancer was established with human pancreatic cancer cell line SW1990. The eukaryotic expression vector of NK4 gene was constructed and transfected into the tumors. The mice weight, tumor size and volume were measured before and after transfection. The apoptotic cells, microvessel density (MVD), and the expression of proliferating cell nuclear antigen (PCNA) in the tumors were observed using immunohistochemistry and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) technique. RESULTS: Four weeks after NK4 gene transfection, the tumor volume and weight was significantly smaller in NK4-transfected group than in PBS control group and empty vector group [(1.39+/-0.33) cm(3) vs. (2.06+/-0.55) cm(3) and (1.90+/-0.36) cm(3), P<0.01; (1.30+/-0.81) g vs. (3.45+/-1.88) g and (3.14+/-1.51) g, P<0.01]; the inhibition rate was 62.29%. The tumor cell apoptotic index was significantly higher in NK4-transfected group than in the rest 2 groups (9.34+/-0.91 vs. 4.13+/-0.79 and 3.94+/-1.03, P<0.001); the MVD was significantly lower in NK4-transfected group than in the rest 2 groups (12.24+/-4.63 vs. 20.13+/-7.00 and 19.70+/-6.15, P<0.05); the expression of PCNA in NK4-transfected group was not different from those of the rest 2 groups (53.88+/-4.30 vs. 56.24+/-4.03 and 54.33+/-5.41,P>0.05). CONCLUSION: NK4 gene transfection may inhibit the growth of human pancreatic cancer in mouse model through suppressing angiogenesis and enhancing the apoptosis of pancreatic cancer cells.

[The effect of recombinant adenovirus encoding antisense MAT1 on cell cycle of human pancreatic cancer cells BxPC-3].

OBJECTIVE: To investigate the regulatory effect of the human MAT1 gene on the cell cycle G(1)/S transition of human pancreatic cancer BxPC-3 cells. METHODS: To construct the replication deficient recombinant adenovirus of antisense MAT1 gene using homologous recombination by AdEasy system. Cell growth assay was carried out by counting alive cells after trypan blue exclusion. The protein expressions of MAT1, cyclin D1, cyclin E, and Rb were detected by western blotting. The cell cycle status was determined by flow cytometry. RESULTS: The recombinant adenovirus encoding antisense MAT1 fragment Ad-MAT1AS was obtained with the titer of expression was 5 x 10(10) pfu/ml. The expression of MAT1 of BxPC-3 was significantly reduced after Ad-MAT1AS infection. In this case BxPC-3 cell cycle was arrested in G(1) phase. The estimated proportion of G(0)/G(1) phase cells in the control for blank and vector cultures ranged from 40% to 44%. In contrast, 79% of the Ad-MAT1AS cells were in G(0)/G(1) phase. Cyclin E and pRb gene expression changes were observed in the infected cells. CONCLUSION: The results suggest that MAT1 gene may play an important role in the regulation of cell cycle G(1)-->S transition of BxPC-3 cells.

Antiangiogenic therapy for human pancreatic carcinoma xenografts in nude mice.

AIM: To investigate the anti-tumor effects of antiangiogenic therapy (a combination of TNP-470, an antiangiogenic compound, with gemcitabine, an antimetabolite) on human pancreatic carcinoma xenografts and its mechanism. METHODS: A surgical orthotopic implantation (SOI) model was established by suturing small pieces of SW1990 pancreatic carcinoma into the tail of pancreas in nude male mice. Mice then received either single therapy (n = 24) or combined therapy (n = 32). Mice receiving single therapy were randomly divided into control group, G100 group receiving 100 mg/kg gemcitabine IP on d 0, 3, 6 and 9 after transplantation, and T30 group receiving 30 mg/kg TNP-470 s.c on alternate days for 8 wk. Mice receiving combined therapy were randomly divided into control group, T15 group, G50 group and combination group (TNP-470 30 mg/kg and gemcitabine 50 mg/kg). Animals were killed 8 wk after transplantation. Transplanted tumors, liver, lymph node and peritoneum were removed. Weight of transplanted tumors, the T/C rate (the rate of mean treated tumor weight to mean control tumor weight), change of body weight, metastasis rate, and 9-wk survival rate were investigated. Tumor samples were taken from the control group, T30 group, G100 group and combination group. PCNA index (PI) and microvessel density (MVD) were investigated by immunohistochemical staining for PCNA and factor VIII, respectively. RESULTS: There was a significant inhibitory effect on primary tumor growth of pancreatic carcinoma in G100 group, compared to T30 group, whereas tumor metastasis was significantly inhibited in T30 group compared to G100 group. There was no significant improvement in survival rate in these two groups. No significant inhibitory effect on tumor growth and metastasis in T15 group and G50 group. However, significant anti-tumor and anti-metastatic effects were observed in the combination group with a significant improvement in survival rate. The inhibitory effect on tumor growth in combination group enhanced 2 times in comparison with G50 group and 5 times in comparison with T15 group. Moreover, 25% of the animals bearing tumors were cured by the combination therapy. The levels of MVD and PI were 14.50+/-5.93 and 0.41+/-0.02, 12.38+/-1.60 and 0.30+/-0.07, 7.13+/-2.99 and 0.37+/-0.03, and 5.21+/-1.23 and 0.23+/-0.02 respectively in the control group, G100 group, T30 group and combination group. A significant inhibitory effect on PI level and MVD level was observed in G100 group and T30 group respectively whereas both MVD and PI levels were significantly inhibited in the combination group (P<0.05). CONCLUSION: Antiangiogenic therapy shows significant anti-tumor and anti-metastatic effects, and is helpful to reduce the dosage of cytotoxic drugs and the side effects. These effects are related to the antiangiogenic effect of TNP-470 and cytotoxic effect of gemcitabine.

[Effect of transferred NK4 gene on biological characteristics of human pancreatic cancer cell line SW1990].

BACKGROUND & OBJECTIVE: Hepatocyte growth factor (HGF) plays an important role in the regulation of migration, invasion,and angiogenesis of cancer via the activation of its receptor, c-Met. NK4 is not only an antagonist of HGF but also an angiogenesis inhibitor. The blockade of HGF/c-Met signal pathway and tumor angiogenesis may be a new strategy for cancer treatment. This study was designed to construct eukaryotic expressing vector of NK4 gene, transfer it into human pancreatic cancer cell line SW1990, and observe the effect of transfected NK4 gene on the biological behaviors of SW1990 cells,and its expression in SW1990 cells. METHODS: The recombinant of pcDNA3/hNK4 plasmid was digested by restrictive enzyme,NK4 gene was cloned into a high effective eukaryotic expressing vector pRC/CMV2, and the recombinant of pRC/CMV2-hNK4 plasmid was transiently introduced into SW1990 cells by lipofectamine. Reverse transcriptase-polymerase chain reaction (RT-PCR),and Western blot were used to detect the expression of NK4 at mRNA, and protein levels,respectively. Migration, and invasion capabilities of the transfected cells were evaluated by Transwall chamber, and Matrigel invasion chamber, respectively. RESULTS: Expressions of NK4 gene after lipofectamine mediated transfection were observed in SW1990 cells, expected fragment of 453 bp has been amplified by RT-PCR, and Western blot analysis showed positive expression of NK4 protein (50 KDa). NK4 gene had no inhibitory effect on the growth of SW1990 cells (2.2x10(5) vs 2.5x10(5), P >0.05), while it had significantly suppressive effect on the migration and invasion of SW1990 cells driven by HGF or fibroblasts (P< 0.01). CONCLUSION: NK4 gene transfection may inhibit spreading and invasion of pancreatic cancer cells, which would play an important role in the anti-metastasis therapy for pancreatic cancer.

Expression of c-erbB-2 oncogene protein, epidermal growth factor receptor, and TGF-beta1 in human pancreatic ductal adenocarcinoma.

OBJECTIVE: To detect the relations of c-erbB-2 oncogene protein, epidermal growth factor receptor (EGFR) and transforming growth factor-beta1 (TGF-beta1) to the progression or metastasis of pancreatic carcinoma. METHODS: Using streptavidinbiotin complex (SABC) method, c-erbB-2 oncongene protein, we examined immunohistochemically EGFR and TGF-beta1 expressions in wax-tissue sections from 10 individuals with normal pancreas (NP), 13 patients with chronic pancreatitis (CP) and 36 patients with pancreatic ductal adenocarcinoma (PC). RESULTS: The positive expression rates of c-cerbB-2 oncogene protein, EGFR and TGF-beta1 in the NP, CP and PC groups were 0, 0, 10%; 7.7%, 7.7%, 7.7%; and 41.7%, 50.0%, 44.4%, respectively. The positive expression rates of the three specific proteins increased more significantly in the PC group than in the NP and CP groups (P<0.05). The individual expression of c-erbB-2, EGFR and TGF-beta1 was not related to the age and sex of the patients as well as the site, size and histopathological grade of tumors (P>0.05), but to the clinical stage of tumors (P<0.01). The coexpression rate of the three proteins was 27.8% (10/36). This coexpression in the PC group was correlated with the histopathological grades and clinical stages of tumors (P<0.01). CONCLUSION: Detection of c-erbB-2 oncogene protein, EGFR, and TGF-beta1 expressions in pancreatic tissue is helpful to judge the malignancy, progression, and metastasis of PC.

Experimental study of gene therapy with angiostatin gene in pancreatic cancer.

OBJECTIVE: To study the effects of angiostatin(AS) gene mediated by liposome on human pancreatic cancer cell line SW1990. METHODS: Angiostatin gene was cloned into the eukaryotic expression vector pRC/CMV. The recombinant of pRC/CMV-AS was introduced into the pancreatic cancer cell line, SW1990. The mechanism of anti-tumor was studied and tested. RESULTS: The eukaryotic expression vector pRC/CMV-AS was identified by the restriction digest. pRC/CMV-AS was stably integrated into the target cells and expressed by Western blot and drug-sensitivity tests, and inhibited the vascular endothelial cells proliferation in vitro. In addition, the effects of the angiostatin vector on reducing the volume of tumors implanted in nude mouse models were also noted. CONCLUSION: This study demonstrated that the recombinant pRC/CMV-AS mediated by liposome may play a potential role in the treatment of pancreatic cancer in the future.