OBJECTIVETo investigate the differences about RNA interference (RNAi) technique which focuses on single or multiple sites to suppress colon cancer LoVo cell line's epidermal growth factor receptor (EGFR) mRNA and protein expression, induce cell apoptosis and enhance 5-fluorouracil (5-FU) sensitivity.

METHODSThe human colon cancer LoVo cells were transfected by liposome with pU6-EGFR-shRNA-1 and pU6-EGFR-shRNA-2 expressive vectors which were established by p Genesil-1 plasmid and EGFR short hairpin RNA (shRNA) synthesized in vitro, then were selected for 4 weeks by using G418. Five groups were selected for the study: Group 1: the normal cultured LoVo cells; Group 2: the negative control plasmid HK; Group 3: pU6-EGFR-shRNA-1 plasmid vector; Group 4: pU6-EGFR-shRNA-2 plasmid vector; Group 5: pU6-EGFR-shRNA-1 and pU6-EGFR-shRNA-2, half for each. The mRNA and protein expression were assessed using Real Time PCR and Western blot, the cell apoptosis was determined via flow cytometry, and the suppressive rate and IC(50) to LoVo cells by 5-FU of different concentrations and time points were carried out by using Cell Counting Kit-8 (CCK-8).

RESULTSExpression plasmids encoding shRNA were successfully established and transfected into the LoVo cells. In group 3, 4 and 5, the mRNA expression was decreased by (80.2 +/- 3.4)%, (81.3 +/- 2.8)% and (90.6 +/- 2.8)%, respectively, and protein expression was decreased by (74.1 +/- 4.0)%, (73.4 +/- 2.3)% and (90.4 +/- 3.3)%, respectively; meanwhile, cell apoptosis increased by (10.4 +/- 0.5)%, (10.1 +/- 0.4)% and (14.2 +/- 0.5)%, respectively. The IC(50) of 5-FU and cell suppressive rate analysis demonstrated that there were significant differences among group 5, groups 3 and 4, and groups 1 and 2, but there were no significant difference between group 1 and group 2, as well as group 3 and group 4.

CONCLUSIONSBoth pU6-EGFR-shRNA-1 and pU6-EGFR-shRNA-2 were capable of suppressing EGFR expression of LoVo cells, and therefore promoting apoptosis and increasing the cell toxicity of 5-FU. The targeting double combined sites RNAi technique was significantly better than single site interference. The new therapeutic modalities in the treatment of human colon cancer are suggested by this study.

Apoptosis ; genetics ; Cell Line, Tumor ; Colonic Neoplasms ; genetics ; metabolism ; pathology ; therapy ; Combined Modality Therapy ; Fluorouracil ; pharmacology ; Genetic Therapy ; Humans ; RNA ; genetics ; RNA Interference ; Receptor, Epidermal Growth Factor ; biosynthesis ; genetics ; Transfection

OBJECTIVETo explore the feasibility of IGF2 imprinting system in target gene therapy for tumors.

METHODSThe mouse H19 enhancer, DMD and promoter H19 were amplified by PCR from mouse genomic DNA and then cloned into the plasmid pDC312. The EGFP and DT-A fragments were amplified by PCR and cloned into the recombinant plasmid, and then the shuttle plasmid were transfected into HEK293 cells together with the adenoviral vector Ad5, namely, Ad-EGFP and Ad-DT-A. Adenovirus hexon gene expression was applied to confirm the presence of adenovirus infections. The effect of the IGF2 imprinting system was tested by fluorescence microscopy. RT-PCR and Western blotting after transfection of the recombinant adenoviral vectors into cancer cells were used to show loss of IGF2 imprinting (LOI) and maintenance of IGF2 imprinting (MOI), respectively. The anti-tumor effect was assessed by MTT and flow cytometry after the HCT-8 (LOI). Human breast cancer cell line MCF-7 (MOI) and human normal gastric epithelial GES-1 (MOI) cell line were transfected with Ad-DT-A in vitro. The anti-tumor effect was detected by injecting the Ad-DT-A in nude mice carrying HCT-8 tumors.

RESULTSThe expression of EGFP protein, DT-A mRNA and DT-A protein were seen to be positive only in the HCT-8 tumor cell line. Infection with Ad-DT-A resulted in obviously growth inhibition in HCT-8 cells (75.4 ± 6.4)% compared with that in the control group, and increased the percentage of apoptosis in the HCT-8 cells (20.8 ± 5.9)%. The anti-tumor effect was further confirmed by injecting the recombinant adenoviruses in HCT-8 tumor-bearing nude mice, and the results showed that the Ad-DT-A inhibited the tumor growth, with on inhibition rate of 36.4%.

CONCLUSIONSThe recombinant adenoviruses carrying IGF2 imprinting system and DT-A gene have been successfully constructed, while Ad-DT-A can effectively kill the tumor cells showing loss of IGF2 imprinting. It might play an important role in future target gene therapy against malignant tumors based on loss of IGF2 imprinting.

Adenoviridae ; genetics ; Animals ; Apoptosis ; Breast Neoplasms ; genetics ; pathology ; Colonic Neoplasms ; genetics ; pathology ; therapy ; Diphtheria Toxin ; biosynthesis ; genetics ; Female ; Genetic Therapy ; methods ; Genetic Vectors ; Genomic Imprinting ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; Insulin-Like Growth Factor II ; genetics ; metabolism ; MCF-7 Cells ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Peptide Fragments ; biosynthesis ; genetics ; Plasmids ; RNA, Messenger ; metabolism ; Random Allocation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection

OBJECTIVETo investigate anti-tumor effect of the recombined adenovirus encoding NK4 gene regulated by human telomerase reverse transcriptase (HTERT) promoter (Ad HTERTp-NK4) on human colon cancer.

METHODSColon cell line HCT116 was infected with Ad HTERTp-NK4. NK4 expression was determined by RT-PCR and Western blot. The cell-growth inhibition rate and the invasive capacity of cells were evaluated by MTT method and reconstituted basement membrane invasion assay. The model of subcutaneous tumor was generated by injection of HCT116 cells into the dorsum of nude mice. Ad HTERTp-NK4 was injected around the tumor tissues, and tumor growth was observed.

RESULTSNK4 gene was highly expressed in infected HCT116 cells. The cell growth inhibition rate and the invasive inhibition rate in Ad HTERTp-NK4 cells were 47.14% and 40.63% respectively, which were significantly higher than those in the control cells (2.75% and 2.31%, P<0.05). Tumor growth was significantly inhibited in mice injected with Ad HTERTp-NK4, and the tumor growth inhibition rate was 47.3%, which was significantly higher than that in the controls (4.6%, P<0.05).

CONCLUSIONAd HTERTp-NK4 can inhibit tumor growth and decrease the invasive capacity of tumor cells, which makes it an ideal candidate for new gene therapy for colon carcinoma.

Adenoviridae ; genetics ; Animals ; Cell Line, Tumor ; Cell Proliferation ; Colonic Neoplasms ; genetics ; metabolism ; pathology ; therapy ; Genetic Therapy ; Humans ; Mice ; Mice, Nude ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Promoter Regions, Genetic ; Telomerase ; genetics ; Transfection ; Xenograft Model Antitumor Assays

OBJECTIVETo study the selective killing effect of adenovirus (Ad)-mediated double suicide gene system driven by the KDR promoter (KDR-CDglyTK) on human colon adneocarcinoma SW480 cells.

METHODSKDR-expressing SW480 cells and LS174T cells that did not express KDR were infected by KDR-CDglyTK, and the infection efficiency and the expression of CDglyTK in the cells were detected by RT-PCR. The infected cells were treated with the prodrugs 5-FC and GCV at different concentrations, and the cell-killing effects and bystander effects were evaluated by MTT method. DNA content and the cell cycle changes in SW480 cells were detected by flow cytometry.

RESULTSThe expression of green fluorescent protein (GFP) was observed in 95% of the infected SW480 and LS174T cells with a multiplicity of infection (MOI) of 100. RT- PCR demonstrated that the product of CD/TK gene existed in SW480 cells infected by Ad- KDR- CD/TK, but not in infected LS174 cells. The infected SW480 cells exhibited high sensitivity to the prodrugs, but the infected LS174T cells did not (P<0.01). Bystander effects of the double suicide gene system were observed in the coculture of the infected and non-infected SW480 cells. At the MOI of 100, treatment of the infected cells with the prodrugs resulted in increased cell percentage in G(0)-G(1) phase and decreased percentage in S phase and the prodrug-treated cells showed an apoptotic peak in flow cytometry.

CONCLUSIONCDglyTK fusion gene system driven by the KDR promoter selectively kills and induces the apoptosis of the KDR-CDglyTK SW480 cells.

Adenocarcinoma ; genetics ; pathology ; Adenoviridae ; genetics ; metabolism ; Apoptosis ; genetics ; Cell Line, Tumor ; Colonic Neoplasms ; genetics ; pathology ; Cytosine Deaminase ; biosynthesis ; genetics ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; Promoter Regions, Genetic ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Thymidine Kinase ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; metabolism

BACKGROUND AND OBJECTIVEColon cancer is one of the most common malignant tumors, and its pathogenesis is not fully understood. Transcriptional silencing by DNA methylation is believed to be an important mechanism of carcinogenesis. E-cadherin can suppress tumor cell invasion and metastasis, and is considered as an invasion/metastasis suppressor gene. Inactivation of E-cadherin gene often occurs in colon carcinoma. This study was to investigate the correlation between E-cadherin gene expression and the methylation status of E-cadherin 5' CpG islands in human colon carcinoma cell line HT-29, and to explore the mechanism of carcinogenesis of colon cancer.

METHODSImmunocytochemical dicho-step method and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect the expression of E-cadherin protein and mRNA in HT-29 cells after 5-Aza-CdR treatment; methylation specific PCR was used to analyze the methylation status at promoter of E-cadherin gene.

RESULTSThe expression of E-cadherin gene could be restored by 5-Aza-CdR treatment, immunocytochemical staining showed the positive expression ratio of E-cadherin increased from (21+/-7)% (1 micromol/L) to (39+/-13)% (5 micromol/L); E-cadherin genes were methylated and not expressed in HT-29 cells in the colon carcinoma.

CONCLUSIONSE-cadherin methylation plays an important role in the carcinogenesis of colon carcinoma cells and can re-express after the treatment with 5-Aza-CdR.

Antimetabolites, Antineoplastic ; pharmacology ; Azacitidine ; analogs & derivatives ; pharmacology ; Cadherins ; genetics ; metabolism ; Cell Proliferation ; drug effects ; Colonic Neoplasms ; drug therapy ; DNA Methylation ; Gene Expression Regulation, Neoplastic ; HT29 Cells ; metabolism ; pathology ; Humans ; RNA, Messenger ; metabolism

Induction of tumor vasculature occlusion by targeting a thrombogen to newly formed blood vessels in tumor tissues represents an intriguing approach to the eradication of primary solid tumors. In the current study, we construct and express a fusion protein containing vascular endothelial growth factor (VEGF) and tissue factor (TF) to explore whether this fusion protein has the capability of inhibiting tumor growth in a colon carcinoma model. The murine cDNA of VEGF A and TF were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), and then cloned into prokaryotic expression plasmid pQE30 with a linker. The expression product recombinant VEGF-TF (rVEGF-TF) was purified and proved to have comparable enzyme activity to a commercial TF and the capability of specific binding to tumor vessels. Significant decrease of tumor growth was found in the mice administered with rVEGF-TF on Day 6 after initiated rVEGF-TF treatment (P<0.05), and the tumor masses in 2 of 10 mice were almost disappeared on Day 14 after the first treatment. In addition, valid thrombogenesis and tumor necrosis were observed in the tumor tissues injected with rVEGF-TF. Our results demonstrate that occlusion of tumor vasculature with rVEGF-TF is potentially an effective approach for cancer therapy.
Animals ; Cell Line, Tumor ; Cloning, Molecular ; Colonic Neoplasms ; blood supply ; drug therapy ; pathology ; Disease Models, Animal ; Disease Progression ; Gene Expression ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Plasmids ; genetics ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; metabolism ; therapeutic use ; Thromboplastin ; genetics ; isolation & purification ; metabolism ; therapeutic use ; Thrombosis ; drug therapy ; pathology ; Vascular Endothelial Growth Factor A ; genetics ; isolation & purification ; metabolism ; therapeutic use

E2F-1 and p53 are sequence specific transcription factors that are intimately involved in the regulation of the cell cycle. In addition to their role in cell cycle control, both E2F-1 and p53 have been identified as tumor suppressors and mediators of apoptosis. We have shown previously that adenoviral-mediated E2F-1 overexpression induces efficient apoptosis in colon adenocarcinoma cells. Previous reports have suggested that E2F-1 and p53 cooperate to mediate apoptosis and therefore, in this study, we examined the efficacy of combination gene therapy using adenovirus vectors expressing E2F-1 and p53 in human colon adenocarcinoma cell lines, HT-29 and SW620 (both mutant p53). Cells were treated by mock infection or infection with adenoviral vectors expressing b-galactosidase (LacZ), E2F-1, p53 or a combination of E2F-1 and p53. IC25 concentrations of each virus were estimated and used for each treatment in order to detect any synergistic or cooperative effects on tumor cell death in the combination therapy. By 5 days post infection, E2F-1-overexpressing cells exhibited growth inhibition and approximately 40-50% cell death in both cell lines. Co-expression of p53 with E2F-1 abrogated E2F-1-mediated growth inhibition and cell death. Cell cycle analysis revealed that overexpression of E2F-1 resulted in an accumulation of cells in G2/M phase, while overexpression of p53 resulted in a G1 phase accumulation. However, co-expression of E2F-1 and p53 counteracted each other as fewer cells accumulated in G1 and G2/M when compared to either p53 or E2F-1 alone. Furthermore, co-expression of p53 with E2F-1 resulted in decreased levels of E2F-1 protein expression. Mechanistically, upregulation of the CDK inhibitory protein, p21(WAF1/CIP1), was demonstrated in HT-29 cells following overexpression of either E2F-1, p53 or the combination E2F-1/p53 therapy. However, in SW620 cells, only the cells infected with Ad-p53 alone or in combination resulted in upregulation of p21(WAF1/CIP1). These results suggest that p53 and p21(WAF1/CIP1) may cooperate to inhibit the expression and activity of E2F-1. In conclusion, combination adenoviral vector-mediated E2F-1 and p53 gene transfer was not therapeutically advantageous in this in vitro model of human colon adenocarcinoma.
Adenocarcinoma/*metabolism/pathology ; Adenoviridae/genetics ; Apoptosis/*physiology ; Cell Cycle ; Cell Division ; Colonic Neoplasms/*metabolism/pathology ; Comparative Study ; Cyclins ; Gene Expression ; Gene Therapy ; Gene Transfer Techniques ; *Genes, p53 ; Genetic Vectors ; HT29 Cells ; Human ; Protein p53/genetics/*metabolism ; Recombinant Proteins/metabolism ; Transcription Factors/genetics/metabolism/physiology ; Tumor Cells, Cultured ; Up-Regulation

E2F-1 and p53 are sequence specific transcription factors that are intimately involved in the regulation of the cell cycle. In addition to their role in cell cycle control, both E2F-1 and p53 have been identified as tumor suppressors and mediators of apoptosis. We have shown previously that adenoviral-mediated E2F-1 overexpression induces efficient apoptosis in colon adenocarcinoma cells. Previous reports have suggested that E2F-1 and p53 cooperate to mediate apoptosis and therefore, in this study, we examined the efficacy of combination gene therapy using adenovirus vectors expressing E2F-1 and p53 in human colon adenocarcinoma cell lines, HT-29 and SW620 (both mutant p53). Cells were treated by mock infection or infection with adenoviral vectors expressing b-galactosidase (LacZ), E2F-1, p53 or a combination of E2F-1 and p53. IC25 concentrations of each virus were estimated and used for each treatment in order to detect any synergistic or cooperative effects on tumor cell death in the combination therapy. By 5 days post infection, E2F-1-overexpressing cells exhibited growth inhibition and approximately 40-50% cell death in both cell lines. Co-expression of p53 with E2F-1 abrogated E2F-1-mediated growth inhibition and cell death. Cell cycle analysis revealed that overexpression of E2F-1 resulted in an accumulation of cells in G2/M phase, while overexpression of p53 resulted in a G1 phase accumulation. However, co-expression of E2F-1 and p53 counteracted each other as fewer cells accumulated in G1 and G2/M when compared to either p53 or E2F-1 alone. Furthermore, co-expression of p53 with E2F-1 resulted in decreased levels of E2F-1 protein expression. Mechanistically, upregulation of the CDK inhibitory protein, p21(WAF1/CIP1), was demonstrated in HT-29 cells following overexpression of either E2F-1, p53 or the combination E2F-1/p53 therapy. However, in SW620 cells, only the cells infected with Ad-p53 alone or in combination resulted in upregulation of p21(WAF1/CIP1). These results suggest that p53 and p21(WAF1/CIP1) may cooperate to inhibit the expression and activity of E2F-1. In conclusion, combination adenoviral vector-mediated E2F-1 and p53 gene transfer was not therapeutically advantageous in this in vitro model of human colon adenocarcinoma.
Adenocarcinoma/*metabolism/pathology ; Adenoviridae/genetics ; Apoptosis/*physiology ; Cell Cycle ; Cell Division ; Colonic Neoplasms/*metabolism/pathology ; Comparative Study ; Cyclins ; Gene Expression ; Gene Therapy ; Gene Transfer Techniques ; *Genes, p53 ; Genetic Vectors ; HT29 Cells ; Human ; Protein p53/genetics/*metabolism ; Recombinant Proteins/metabolism ; Transcription Factors/genetics/metabolism/physiology ; Tumor Cells, Cultured ; Up-Regulation

KITENIN (KAI1 C-terminal interacting tetraspanin) promotes invasion and metastasis in mouse colon cancer models. In the present study, we evaluated the effects of KITENIN knockdown by intravenous administration of short hairpin RNAs (shRNAs) in an orthotopic mouse colon cancer model, simulating a primary or adjuvant treatment setting. We established orthotopic models for colon cancer using BALB/c mice and firefly luciferase-expressing CT-26 (CT26/Fluc) cells. Tumor progression and response to therapy were monitored by bioluminescence imaging (BLI). In the primary therapy model, treatment with KITENIN shRNA substantially delayed tumor growth (P = 0.028) and reduced the incidence of hepatic metastasis (P = 0.046). In the adjuvant therapy model, KITENIN shRNA significantly reduced the extent of tumor recurrence (P = 0.044). Mice treated with KITENIN shRNA showed a better survival tendency than the control mice (P = 0.074). Our results suggest that shRNA targeting KITENIN has the potential to be an effective tool for the treatment of colon cancer in both adjuvant and metastatic setting.
Animals ; Carrier Proteins/*genetics/metabolism ; Cell Line, Tumor ; Colonic Neoplasms/genetics/mortality/pathology/*therapy ; Disease Progression ; Liver Neoplasms/prevention & control/*secondary ; Membrane Proteins/*genetics/metabolism ; Mice ; Mice, Inbred BALB C ; Neoplasm Metastasis/*prevention & control ; Neoplasm Recurrence, Local/genetics/*prevention & control ; RNA Interference ; RNA, Small Interfering/*therapeutic use ; Tumor Markers, Biological/genetics

Use of adenoviruses as vehicle for gene therapy requires that target cells express appropriate receptors such as coxsakievirus and adenovirus receptor (CAR). We show here that CAR-deficiency in cancer cells, that limits adenoviral gene delivery, can be overcome by using adenovirus complexed with the liposome, Ad-PEGPE [1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly-ethylene glycol)-2000]. We first confirmed that CT-26 mouse colon cancer cells are deficient in CAR by RT-PCR, and then showed that CT-26 cells infected with Ad-GFP/PEGPE exhibited highly enhanced expression of green fluorescent protein (GFP), compared with those infected with Ad-GFP. GFP expression depends on the dose of liposome and adenovirus. Luciferase expression in livers treated with Ad-luc/PEGPE was about 1,000-fold less than those infected with Ad-luc. In a liver metastasis mouse tumor model developed by intrasplenic injection of CT-26 cells, luciferase expression following i.v. injection of Ad-luc/PEGPE was significantly higher in tumors than in adjacent non-neoplastic liver. Following systemic administration of Ad-GFP/PEGPE, GFP expression increased in tumors more than in adjacent liver while the reverse was true following administration of Ad-GFP. In the latter case, GFP expression was higher in liver than in tumors. This study demonstrates that systemic delivery of PEGPE-adenovirus complex is an effective tool of adenoviral delivery as it overcomes limitation due to CAR deficiency of target cells while reducing hepatic uptake and enhancing adenoviral gene expression in tumors.
*Adenoviridae/genetics/metabolism ; Animals ; Colonic Neoplasms/*genetics/metabolism/pathology/*therapy ; Dose-Response Relationship, Drug ; Gene Therapy ; *Gene Transfer Techniques ; Genetic Vectors ; Green Fluorescent Proteins/genetics ; Liposomes/administration & dosage/chemistry/pharmacokinetics/*therapeutic use ; Liver/drug effects/metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Transgenic ; NIH 3T3 Cells ; Phosphatidylethanolamines/administration & dosage ; Polyethylene Glycols/administration & dosage ; Receptors, Cytoplasmic and Nuclear/deficiency/genetics ; Receptors, Virus/deficiency/*genetics ; Transcription Factors/deficiency/genetics ; Tumor Cells, Cultured