OBJECTIVESTo study the change of ability to transform from 5'-deoxy-fluorouracil monophosphate (5'-DFUR) to fluorouracil (5-FU) in human colon cancer cell lines SW480 and LOVO which transfected with thymidine phosphorylase (TP) gene. And to discuss the anti-cancer activity of 5'-DFUR to SW480 and LOVO cells.

METHODSTP cDNA were transfected into human colorectal cancer cell lines SW480 and LOVO with the lentiviral vector, pLenti6.3_MCS_IRES2-EGFP. The transfection efficiency was analyzed by flow cytometer, the mRNA expression of TP was detected by RT-PCR, and the TP protein expression was detected by Western blot, and the volumes of 5-FU converted from 5'-DFUR both in 2 cells and medium were detected by high performance liquid chromatography (HPLC). The 50% inhibitory concentration (IC50) of 5'-DFUR on these 2 colon cancer cell lines both wild type and TP-transfected cells were evaluated by MTT assay.

RESULTSThe colorectal cancer cell lines SW480 and LOVO transfected with human TP cDNA were monitored 5 generations, and the transfections efficiency rate wea about 95%. Compared with wild type cell SW480 and LOVO, the RQ values of mRNA expression of SW480-TP and LOVO-TP were (695 ± 171) folds (t = -7.00, P = 0.002) and (282 ± 87) folds (t = -5.61, P = 0.030), respectively. Also TP protein expression in SW480-TP and LOVO-TP were higher than their parent cells shown by Western blot. The volume of 5-FU converted from 5'-DFUR in the medium cultured SW480-TP and LOVO-TP were increased compared with their parent cells, respectively (t = 19.406-66.921, P < 0.01), whereas few of 5-FU was detected both in wild, and TP-transfected cells. After transfected with TP cDNA, the IC50 of 5'-DFUR on SW480-TP and LOVO-TP were (587 ± 17) µmol/L and (1088 ± 89) µmol/L respectively, and there were significantly less than their parent cells (t = -32.59 and -8.52, P < 0.01).

CONCLUSIONSThe stabilized transfections of SW480 and LOVO with higher TP expression could be built with lentiviral vector. Transfected TP cDNA into SW480 and LOVO, could improve the expression both of TP mRNA and TP protein, increase the volume of 5-FU converted from 5'-DFUR in medium, and result in an enhancement of anticancer effect on these 2 cells.

Cell Line, Tumor ; Colonic Neoplasms ; pathology ; Floxuridine ; metabolism ; Fluorouracil ; metabolism ; Humans ; Thymidine Phosphorylase ; genetics ; Transcription, Genetic ; Transfection

OBJECTIVE: To investigate the expressions of secreted frizzled-related protein 4 (SFRP4) in stage Ⅱ DNA mismatch repair-deficient (dMMR) and mismatch repair- proficient (pMMR) colorectal cancers and explore their clinical significance. METHODS: We collected fresh stage Ⅱ colon cancer tissues with different MMR status detected by immunohistochemistry (IHC). The differentially expressed mRNAs between dMMR and pMMR tumors were identified by Affymetrix Human oeLncRNA gene chip, and the expression of SFRP4 in these cancer tissues and in colorectal cancer cell lines were detected using Western blotting and real- time quantitative PCR. The apoptosis rates of HCT116 cells with and without siRNA- mediated transient SFRP4 knockdown were determined using flow cytometry. We further investigated the expression pattern of Ki-67 and its correlation with SFRP4 expression. RESULTS: Compared with pMMR colon cancer tissues or cells, both dMMR colon cancer tissues (=0.014) and cells (=0.0079) showed significantly increased expression of SFRP4, which was in negative correlation with Ki-67 (=0.041). In HCT116 cells, transient SFRP4 knockdown resulted in decreased cell apoptosis, including both early apoptosis (=0.003) and late apoptosis (=0.024). CONCLUSIONS Up-regulation of SFRP4 in dMMR stage Ⅱ colon cancer promotes apoptosis and inhibits proliferation of the cancer cells, and may improve the prognosis of dMMR colon cancer.
Apoptosis ; Cell Proliferation ; Colon ; metabolism ; pathology ; Colonic Neoplasms ; genetics ; metabolism ; pathology ; Colorectal Neoplasms ; genetics ; metabolism ; pathology ; DNA Mismatch Repair ; Gene Knockdown Techniques ; HCT116 Cells ; Humans ; Ki-67 Antigen ; metabolism ; Prognosis ; Proto-Oncogene Proteins ; genetics ; metabolism ; Up-Regulation

OBJECTIVETo investigate the relationship between SNC19 protein and cancer metastasis.

METHODSExpression of SNC19 protein in cancer cell lines and tissues was examined by Western blot analysis using anti-SNC19 monoclonal antibody. In addition, Psectag2A-SNC19(ORF) eukaryotic expression vector was constructed and transfected into BCAP37 cells. After the target protein was expressed and purified, processing forms of SNC19 protein were further identified using anti-His mAb and each form was assayed for its gelatinase activity.

RESULTSDifferent expression and post-translational processing of the SNC19 proteins in the cancer cell lines and intestinal tissues were detected.BCAP37 cells transfected full-length SNC19 (ORF) generated two different sized proteins in cell lysates, 120 and 75 kD; 75 kD was detected to have proteolytic activity by gelatin zymography.

CONCLUSIONSNC19 protein presents different expression and post-translational processing in the cancer cells and tissues, of which 75 kD was identified to have gelatinase activity.

Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Colonic Neoplasms ; genetics ; metabolism ; pathology ; DNA, Complementary ; chemistry ; Female ; Gelatinases ; metabolism ; Humans ; Neoplasm Metastasis ; Neoplasm Proteins ; chemistry ; genetics ; metabolism ; Protein Processing, Post-Translational ; Serine Endopeptidases ; chemistry ; genetics ; metabolism ; Transfection

OBJECTIVETo establish a colon cancer cell line with stable expression of carcinoembryonic antigen(CEA).

METHODSRecombinant lentivirus conjugated with CEACAM5 cDNA were used to transfect wild CT26 cells. Antibiotics were given for 2 weeks to select CEA positive cells. A single transfected clone was obtained using limiting dilution. The 7th and 14th passages of cells cultured in vitro were detected for CEACAM5 mRNA by RT-PCR, and protein by western blot. The location of CEACAM5 expression was examined using fluorescent microscope and immunocytochemistry. The 14th passage cells were injected subcutaneously into mice to create BALB/c model and CEACAM5 protein was detected by in vivo fluorescence image analysis system and immunohistochemistry.

RESULTSCEACAM5 mRNA and protein were found in the 7th and 14th passages of CT26CEA cells, which were proved to locate in the cytoplasm by fluorescence microscope and immunohistochemistry. Abundant CEACAM5 protein was found in subcutaneous tumors by in vivo fluorescence image analysis system and immunohistochemistry.

CONCLUSIONColon cancer cell line CT26 with stable expression of CEA in vitro and in mice can be used as a suitable tool to facilitate research on the impact and mechanism of CEA on colon cancer under normal immune environment.

Animals ; Carcinoembryonic Antigen ; genetics ; metabolism ; Cell Line, Tumor ; Colonic Neoplasms ; genetics ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; RNA, Messenger ; genetics ; Transfection

OBJECTIVETo investigate the anti-tumor effect of adenovirus-mediated Bcl-XL shRNA on colon cancer cells in vitro.

METHODSA recombinant Bcl-xl adenovirus was constructed, amplified, and purified. The effect on mRNA expression of Bcl-XL was assessed by RT-PCR, and the effect on apoptosis-induction of colon cancer(Lovo cell line) in vitro was assessed by MTT assay and cell clonogenic assay.

RESULTSRT-PCR showed that Ad/Bcl-XL shRNA significantly down-regulated the mRNA expression of Bcl-XL in Lovo cells. Ad/Bcl-XL shRNA suppressed the proliferation of Lovo cells in a dose-dependent as well as a time-dependent manner compared with Ad/GFP (P<0.05). Treatment with Ad/Bcl-XL shRNA dramatically suppressed the colony formation of Lovo cells in a dose-dependent manner (P<0.05). Ad/Bcl-XL shRNA showed no effect on normal human fibroblast.

CONCLUSIONAd/Bcl-XL shRNA exhibits cytotoxic effect on Lovo cells and may have the potential value in the treatment of colon cancer.

Adenoviridae ; genetics ; Cell Line, Tumor ; Cell Proliferation ; Colonic Neoplasms ; metabolism ; pathology ; Humans ; RNA, Small Interfering ; genetics ; bcl-X Protein ; genetics ; metabolism

OBJECTIVETo investigate the effect of fragile histidine triad (FHIT) gene transfection on human colorectal cancer cell line SW480 through up-regulation of caspase-8 expression.

METHODSThe eukaryotic expression plasmid containing FHIT, pRc/CMV2-FHIT was prepared and purified, and then identified by restrictive enzyme digestion. pRc/CMV2-FHIT was transfected into SW480 cells, and positive cell clones (SW480-FHIT, study group) were selected and amplified. Empty plasmid-transfected SW480 cells(SW480-pRc/CMV2, negative control) and normal SW480 cells (bland control) were used as control. Methyl thiazolyl tetrazolium (MTT) assay was used to test the changes in the proliferation of SW480 cells. Cell-cycle kinetics and apoptosis were analyzed by flow cytometry (FCM). The changes of pro-caspase-8, caspase-8 mRNA and caspase-8 relative activity were analyzed by Western blot, semi-quantitative RT-PCR and colorimetric assay with pan labeled substrate, respectively.

RESULTSAt 96 hours after transfection, cell inhibition rates of the study group and the negative control group were 71.7% and 16.9%. G0/G1 ratio was (63.2±3.5)% and (50.6±2.1)%, optical density of caspase-8 mRNA band 107 and 41, and relative activity of caspase-8 0.43 and 0.25, respectively. All the differences above were statistically significant (P<0.05). When FHIT inhibitor was added, the relative activity of caspase-8 decreased to 0.22, comparable to that in the control group.

CONCLUSIONSFHIT gene transfection can significantly inhibit the proliferation and induce G0/G1 arrest in human colon cancer cell line SW480. The mechanism is related to the up-regulation of caspase-8 expression.

Acid Anhydride Hydrolases ; genetics ; Apoptosis ; Caspase 8 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Colonic Neoplasms ; genetics ; pathology ; Humans ; Neoplasm Proteins ; genetics ; RNA, Messenger ; genetics ; Transfection

OBJECTIVETo investigate the expression differences of minichromosome maintenance 2 (MCM2) mRNA and protein among colon adenocarcinoma, colon adenoma and normal mucosa, and among different clinicopathological types of adenomas.

METHODSFifty specimens, including 33 colonic adenomas, 12 colonic adenocarcinomas and 5 normal colonic mucosa were selected. Each specimen was divided into two parts, one for immunohistochemistry and the other for real-time RT-PCR. Expression differences of MCM2 mRNA among the colonic adenocarcinoma, adenoma and normal colonic mucosa were evaluated by REST-XL software.

RESULTSThe expression of MCM2 was observed in the basal third to half of the colonic crypts in normal mucosa, while throughout the epithelium in the colonic adenocarcinomas and adenomas. However, the expression of MCM2 mRNA in the adenocarcinomas was significantly higher than that in the adenomas(P=0.001). The MCM2 mRNA expression was elevated in the adenoma with villous type, in the conditions of high-grade dysplasia, larger size, sessile morphology and in patients of older ages, but the difference was not significant by REST-XL (P>0.05).

CONCLUSIONThe difference of MCM2 expression between the adenoma and the adenocarcinoma indicates its potential value in the early diagnosis of colonic cancer.

Adenocarcinoma ; metabolism ; pathology ; Adenoma ; metabolism ; pathology ; Adult ; Aged ; Biomarkers, Tumor ; metabolism ; Cell Cycle Proteins ; genetics ; metabolism ; Colonic Neoplasms ; metabolism ; pathology ; Female ; Humans ; Male ; Middle Aged ; Minichromosome Maintenance Complex Component 2 ; Nuclear Proteins ; genetics ; metabolism ; RNA, Messenger ; Young Adult

OBJECTIVETo investigate the expressions of X-linked inhibitor of apoptosis protein (XIAP)-associated factor-1 (XAFI) and heat-shock transcription factor 1 (HSF1) and their relationship in human gastrointestinal cancers.

METHODSImmunoblotting was used to analyze the expressions of HSF1 and XAF1 in gastric and colon cancer tissues and in gastrointestinal cancer cells. The gastrointestinal cancer cells were tranfected with a eukaryotic expression vector containing HSF1 gene fragment or subjected to RNA interference to induce up- or down-regulation of HSF1 expression, and the consequence changes in XAF1 expression in the cells was measured. XAF1 expression was also assayed in the cells after stress stimulation for HSF1 expression.

RESULTSThe expression of HSF1 was higher in gastrointestinal cancer tissues than in normal tissues. The expression of XAF1 and HSF1 was inversely correlated in the cancer cell lines, and stress stimuli of the cells up-regulated the expression of HSF1 but down-regulated XAF1 expression.

CONCLUSIONHSF1 expression is increased in gastrointestinal cancer tissues to result in suppressed expression of XAF1, which may be one of the reasons for the low expression of XAF1 in association with the defect of the apoptosis mechanisms in the cancer cells

Cell Line, Tumor ; Colonic Neoplasms ; genetics ; metabolism ; pathology ; DNA-Binding Proteins ; biosynthesis ; genetics ; Heat Shock Transcription Factors ; Humans ; Immunoblotting ; Intracellular Signaling Peptides and Proteins ; Neoplasm Proteins ; biosynthesis ; genetics ; RNA Interference ; Stomach Neoplasms ; genetics ; metabolism ; pathology ; Transcription Factors ; biosynthesis ; genetics ; Transfection

BACKGROUNDHypoxia promotes tumor angiogenesis and hypoxia-inducible factor-1 alpha (HIF-1α) plays a pivotal role in this process. Recently identified pro-angiogenic factor, semaphorin4D (Sema4D) also promotes angiogenesis and enhances invasive proliferation in some tumors. Furthermore, tumor-associated macrophages (TAMs) can increase the expression of HIF-1α and Sema4D in cancer cells and thus influence tumor growth and progression. The purpose of this study was to evaluate the effect of TAMs on the expression of Sema4D and HIF-1α and the impact of biologic behavior in colon cancer cells.

METHODSImmunohistochemistry was used to analyze HIF-1α and Sema4D expression in 86 curatively resected colon cancer samples and 52 normal colon tissues samples. The relationship between their expression and clinicopathological factors was analyzed. Furthermore, macrophage-tumor cell interactions, such as metastasis, angiogenesis, were also studied using in vitro co-culture systems. Statistical analysis was performed using SPSS 17.0 software (SPSS Inc., USA). Differences between two groups were analyzed with Student's t test.

RESULTSHIF-1α (58%) and Sema4D (60%) were expressed at a significantly higher level in tumors than in normal tissues (P < 0.01, for both). Furthermore, HIF-1α and Sema4D expression was significantly correlated with lymphatic metastasis, specific histological types and TNM stages (P < 0.05), but not with age and tumor size (P > 0.05). Sema4D expression was correlated with that of HIF-1α (r = 0.567, P < 0.01). TAMs markedly induced HIF-1α and Sema4D expression in colon cancer cells and subsequently increased their migration and invasion.

CONCLUSIONSHIF-1α and Sema4D expression are closely related to lymphatic metastasis, specific histological types and TNM stages in colon cancer. Furthermore, TAMs promote migration and invasion of colon cancer cells and endothelial tube formation, possibly through up-regulation of HIF-1α and Sema4D.

Adult ; Aged ; Antigens, CD ; genetics ; metabolism ; Cell Line, Tumor ; Colonic Neoplasms ; genetics ; metabolism ; Female ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Immunohistochemistry ; Macrophages ; immunology ; metabolism ; Male ; Middle Aged ; Neoplasm Metastasis ; genetics ; pathology ; Semaphorins ; genetics ; metabolism

OBJECTIVETo investigate the effect of microRNA143 on cell proliferation and K-ras expression in colorectal carcinoma.

METHODSNorthern blot was used to examine the expression of miR-143 in colorectal carcinoma and adjacent normal tissues. A miR-143 expression vector was constructed and transfected into a human colon adenocarcinoma cell line SW480. Cell proliferation was evaluated by MTT assay. RT-PCR and Western blot were used to examine the expression of K-ras oncogene in transfected cells.

RESULTSThe level of mature miR-143 was lower in tumors compared with adjacent normal tissues in 81% of colorectal carcinoma specimens. In transfected cells, the increased accumulation of miR-143 inhibited the cell proliferation, and resulted in approximately 40.3% decrease of K-ras protein levels, but had no effect on level of K-ras mRNA.

CONCLUSIONThe increased accumulation of miR-143 inhibits the proliferation of transfected cells, and results in down-regulation of K-ras protein in colorectal carcinoma.

Adenocarcinoma ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Colonic Neoplasms ; genetics ; metabolism ; pathology ; Down-Regulation ; Genes, ras ; Genetic Vectors ; Humans ; MicroRNAs ; genetics ; metabolism ; Plasmids ; RNA, Messenger ; metabolism ; Transfection ; ras Proteins ; metabolism