Expression of HIP/PAP in hepatocellular carcinoma and effect of siRNA on migration and invasion in HCC cells

OBJECTIVE@#To investigate the expression of HIP/PAP in hepatocellular carcinoma (HCC) patients and explore its role in migration and invasion of HCC.@*METHODS@#The expression of HIP/PAP in HCC tissue and corresponding adjacent noncancerous tissue was assessed by IHC, RT-PCR and Western blot. The correlation between clinicopathological features and HIP/PAP expression was analyzed. The role of HIP/PAP on invasion and migration of HCC cells was observed by RNA interference, wound healing and Transwell assay.@*RESULTS@#Both mRNA and protein expression of HIP/PAP was upregulated in HCC tissues compared to tumor-adjacent tissue and correlated with poor tumor differentiation, advanced tumor stage and vascular invasion. HIP/PAP expression was also upregulated in HCC cells, and silencing its expression by specific siRNA could inhibit the invasion and migration of HCC cells.@*CONCLUSIONS@#HIP/APA is overexpressed in HCC and contributes to the migration and invasion of HCC cells.

Expression of HIP/PAP in hepatocellular carcinoma and effect of siRNA on migration and invasion in HCC cells

Objective: To investigate the expression of HIP/PAP in hepatocellular carcinoma (HCC) patients and explore its role in migration and invasion of HCC. Methods: The expression of HIP/PAP in HCC tissue and corresponding adjacent noncancerous tissue was assessed by IHC, RT-PCR and Western blot. The correlation between clinicopathological features and HIP/PAP expression was analyzed. The role of HIP/PAP on invasion and migration of HCC cells was observed by RNA interference, wound healing and Transwell assay. Results: Both mRNA and protein expression of HIP/PAP was upregulated in HCC tissues compared to tumor-adjacent tissue and correlated with poor tumor differentiation, advanced tumor stage and vascular invasion. HIP/PAP expression was also upregulated in HCC cells, and silencing its expression by specific siRNA could inhibit the invasion and migration of HCC cells. Conclusions: HIP/APA is overexpressed in HCC and contributes to the migration and invasion of HCC cells.

Lentivirus-mediated reversion-inducing cysteine-rich protein with Kazal motifs gene transfer suppresses pancreatic cancer invasion in vitro

To investigate the biological functions of reversion-inducing cysteine-rich protein with Kazal motifs [RECK] over-expression in pancreatic cancer cell line Panc-1. This study was carried out in the Department of General Surgery, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China from January to September 2009. The design of the study was to construct a recombinant lentivirus carrying the RECK gene [LV-RECK] to be used to infect Panc-1 cells, and investigate the biological functions of RECK in pancreatic cancer in vitro. The RECK expression was measured by reAl time polymerase chain reaction [PCR] and Western blotting. Cell proliferation and apoptosis were examined by 3-[4, 5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide [MTT] assay and flow cytometry. The ability of invasion was detected using Transwell chambers. The expression and activity of matrix metalloproteinase [MMP]-2 and matrix metalloproteinase-9 [MMP-9] was measured by reAl time PCR, Western blotting, and gelatin zymography. We successfully constructed LV-RECK and proved it can over-express the RECK gene in Panc-1 cells. The RECK over-expression potently suppressed the invasion ability of Panc-1 cells in vitro without affecting cell proliferation and apoptosis. The RECK over-expression potently inhibited the secretion and activity of MMP-2, and the secretion of MMP-9 without affecting the messenger ribonucleic acid, and protein expression of MMP-2 and MMP-9. The RECK gene exerts repressive effects on the invasion ability of the pancreatic cancer cell line Panc-1, and it may be a novel therapeutic target for pancreatic cancer

GM-CSF gene-modified dendritic cell vaccine enhances antitumor immunity in vitro / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate if granulocyte-macrophage colony stimulating factor (GM-CSF) gene-modified dendritic cells (DC) enhance antitumor immunity in vitro.</p><p><b>METHODS</b>Mice were injected with chemokine ligand 3 (CCL3) via the tail vein. Fresh B220(-)CD11c(+) cells were sorted from the peripheral blood mononuclear cells (PBMCs) and cultured into DCs by cytokines.DCs were transfected with AdGM-CSF gene at different ratios of multiplicity of infection (MOI) to determine the optimal gene transfection conditions, and the expression of GM-CSF was detected after transfection. The variation of GM-CSF gene-modifiedDCs were analyzed by morphological examination, phenotype analysis, and mixed lymphocyte reaction (MLR).DCs were loaded with gastric cancer antigen obtained by freezing and thawing method. The killing effect of DCs vaccine-stimulated T lymphocytes on gastric cancer cells was assessed by MTT assay. INF-gamma production was determined with the INF-gamma ELISA kit.</p><p><b>RESULTS</b>B220(-)CD11c(+) cells increased obviously after CCL3 injection. The ELISA results showed that after GM-CSF gene modification, DCs could produce high level of GM-CSF. When DCs were transfected with AdGM-CSF gene at MOI equal to 100, the GM-CSF level in culture supernatants reached saturation [(130.00 +/- 12.61) pg/ml]. After GM-CSF gene-modification, DCs tend to be more maturated as detected by morphological observation and phenotype analysis. At the same time, the capacity of activating the proliferation of allogeneic T lymphocytes was enhanced greatly. T lymphocytes stimulated by DCs transfected with GM-CSF gene showed a specific killing effect on gastric carcinoma cells and produced high level of INF-gamma [(1245.00 +/- 13.75) pg/ml].</p><p><b>CONCLUSION</b>After GM-CSF gene modification, DCs can produce high level of GM-CSF, which tend to be more maturated, and the capacity of activating the proliferation of allogeneic T lymphocytes is enhanced greatly. GM-CSF gene modified DCs can induce specific CTL to target tumor cells in vitro.</p>

Effect of recombinant adenovirus vector mediated human interleukin-24 gene transfection on pancreatic carcinoma growth / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Pancreatic cancer is a highly malignant tumor affecting an ever increasing number of patients with a mean 5-year survival rate below 4%. Therefore, gene therapy for cancer has become a potential novel therapeutic modality. In this study we sought to determine the inhibitory effects of adenovirus-mediated human interleukin-24 (AdhIL-24) on pancreatic cancer.</p><p><b>METHODS</b>Human interleukin-24 gene was cloned into replication-defective adenovirus specific for patu8988 tumor cells by virus recombination technology. Reverse transcription-polymerase chain reaction and Western blotting analysis were used to determine the expression of human interleukin-24 mRNA in patu8988 cells in vitro. Induction of apoptosis by overexpression of human interleukin-24 in patu8988 cells was determined by flow cytometry. In vivo efficacy of adenoviral delivery of human interleukin-24 was assessed in nude mice (n = 10 for each group) bearing patu8988 pancreatic cancer cell lines by determining inhibition of tumor growth, endothelial growth factor and CD34 expression, and intratumoral microvessel density (MVD).</p><p><b>RESULTS</b>The recombinant adenovirus vector AdVGFP/IL-24 was constructed with a packaged recombinant retrovirus titer of 1.0 x 10(10) pfu/ml and successfully expressed of both mRNA and protein in patu8988 cells. The AdVGFP/IL-24 induced apoptosis of patu8988 tumor cells in vitro and significantly inhibited tumor growth in vivo (P < 0.05). The intratumoral MVD decreased significantly in the treated tumors (P < 0.05).</p><p><b>CONCLUSION</b>The recombinant adenovirus AdGFP/IL-24 can effectively express biologically active human interleukin-24, which results in inhibition of pancreatic cancer growth.</p>

Effect of endothelial PAS domain protein 1 and hypoxia inducible factor 1alpha on vascular endothelial growth factor expression in human pancreatic carcinoma / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Transcription factors hypoxia inducible factor 1alpha (HIF 1alpha) and endothelial PAS domain protein 1 (EPAS1) promote the transcription of vascular endothelial growth factor (VEGF). VEGF enhances angiogenesis and vascular permeability of tumours, which promotes tumour growth and facilitates entry of cancer cells into blood circulation and metastasizing. This study examined whether HIF 1alpha and EPAS1 stimulated angiogenesis through activation of VEGF in human pancreatic carcinoma.</p><p><b>METHODS</b>Specimens from pancreatic carcinoma and healthy parts of same pancreas were taken from 60 patients. Real time quantitative reverse transcription polymerase chain reaction estimated expression of HIF 1alpha, EPAS1, and VEGF mRNAs. Western blotting and immunohistochemical, streptavidin peroxidase method assessed expression of HIF 1alpha, EPAS1, and VEGF proteins. Microvessel density (MVD) was assessed.</p><p><b>RESULTS</b>Highly significant increases in expression of EPAS1, VEGF, and MVD were found in pancreatic carcinoma tissue but not in normal pancreatic tissue: VEGF at mRNA and protein levels (t = 17.32, P = 0.0001; t = 98.41, P = 0.0001); EPAS1 protein level (t = 22.51, P = 0.0001). Expression of HIF 1alpha was similar in pancreatic carcinoma and normal pancreatic tissues at both mRNA and protein levels. Significant correlations were observed between EPAS1 and VEGF (r = 0.736, P = 0.0041), between VEGF and MVD (r = 0.858, P = 0.0001), and between EPAS1 and MVD (r = 0.641, P = 0.0003). No significant correlations were observed between HIF 1alpha and VEGF, or between HIF 1alpha and MVD. MVD and expression of EPAS1 and VEGF were significantly related with TNM staging, so was EPASI and VEGF with size of tumour.</p><p><b>CONCLUSIONS</b>EPAS1 and VEGF, but not HIF1alpha, are overexpressed in pancreatic carcinoma. The expression of EPAS1 is correlated with that of VEGF and MVD. EPAS1 may be involved in the angiogenesis of pancreatic carcinoma by upregulating the expression of VEGF. Targeting EPAS1 may be a new method of antiangiogenic tumour therapy for pancreatic carcinoma.</p>