Fan Daiming

EpCAM is overexpressed in gastric cancer and its downregulation suppresses proliferation of gastric cancer

PurposeTo identify the role of epithelial cellular adhesion molecule (EpCAM) in gastric cancer growth and explore the potential value of EpCAM monoclonal antibody as new therapeutic strategy for gastric cancer.MethodsThe expression of EpCAM was determined by immunohistochemistry staining in gastric cancer tissues, RT-PCR and Western blot in cell lines. EpCAM expression in cell lines was downregulated by small interfering RNA. Then the effects of EpCAM on gastric cancer cell growth in vivo and in vitro were determined by MTT, FCM analysis, clone formation assay and tumor formation assay. Additionally, western blot was used to detect the effect of EpCAM on cell cycle-relevant factor cyclin D1.ResultsEpCAM was found to be overexpressed in gastric cancer tissues and cell lines. Downregulation of EpCAM resulted in a decrease of cell proliferation and cell cycle arrest in AGS and SGC7901 cells, which had high endogenous EpCAM expression. EpCAM downregulation also suppressed tumor formation in nude mice. Moreover, EpCAM repression in gastric cancer cells could downregulate cyclin D1.ConclusionsEpCAM was a potential oncogene and contributed to the growth of gastric cancer. Our data first provided compelling evidence of potential value of EpCAM in the therapy of gastric cancer in clinic.

Expression and significance of TWIST basic helix-loop-helix protein over-expression in gastric cancer

Aims: TWIST protein has been implicated in neoplastic transformation and development of some cancers. In this study, we aimed to investigate the expression of TWIST in gastric cancer and its clinical significance. Methods: A total of 76 cases of archival gastric cancer tissues were immunohistochemically evaluated for TWIST expression, and its expression was correlated with clinicopathological parameters. Semi‐quantitative reverse transcriptase polymerase chain reaction (RT‐PCR) was used to detect the mRNA of TWIST in four gastric cancer cell lines and a normal immortalised gastric epithelial cell line (GES‐1). The expression of TWIST protein in these cell lines and 14 pairs of fresh gastric carcinoma and adjacent normal tissue samples was detected by Western blotting. Results: TWIST expression increased in diffuse‐type gastric carcinoma compared with intestinal‐type gastric carcinoma (26/42, 61.9% versus 9/34, 26.5%, p<0.05). TWIST expression was significantly increased in 35 (46.1%) of the 76 cancers and correlated with lymph node metastasis (node positive rate 60.4%; node negative rate 21.4%; p<0.05). The expression of TWIST protein was higher in 9/14 (64.3%) fresh cancer tissues compared with adjacent normal tissues. The expression of mRNA and protein of TWIST in gastric cancer cell lines was up‐regulated compared with that in GES‐1. Conclusions: TWIST was highly expressed in gastric cancer. Its up‐regulation was associated with the neoplastic transformation and subsequent development of gastric cancer. Therefore, TWIST maybe a useful prognostic marker and target for gastric cancer therapy.

The inhibitory effect of p75 neurotrophin receptor on growth of human hepatocellular carcinoma cells

p75 neurotrophin receptor (p75NTR), a member of the TNF receptor superfamily, is a focus for study at present. Up to now, its role and functions in hepatocellular carcinoma were not fully elucidated. In this study, we investigated the expression of p75NTR in hepatocellular carcinoma and the impact of its alteration on tumor growth. We found that the expression of p75NTR was decreased significantly in 158 cases of hepatocellular carcinoma tissues as compared with their adjacent noncancerous counterparts, and its expression was also significantly decreased in various human hepatocellular carcinoma cell lines. Down-regulating p75NTR by specific siRNA promoted the growth of normal liver cell lines, whereas up-regulating p75NTR inhibited the growth of hepatocellular carcinoma cell lines in vitro and caused dramatic attenuation of tumor growth in vivo by induction of cell cycle arrest. Furthermore, we found that up-regulating p75NTR could down-regulate the expression of cyclin A, cyclin D1, cyclin E, cdk2, p-Rb and PCNA, but up-regulate the expression of Rb. Conversely, the results were inverse when p75NTR was down-regulated by specific siRNA. Therefore, we provided the evidence that p75NTR was a potential tumor suppressor and might be used as a therapeutic target for hepatocellular carcinoma.

Mitochondrial DNA from colorectal cancer cells promotes the malignant phenotype of NIH3T3 cells

We investigated the potential role of mitochondrial DNA (mtDNA) in colorectal carcinogenesis by constructing a eukaryotic expression vector of the mitochondrial D‐loop gene from colorectal cancer cell SW480 and transfected NIH3T3 cells. The NIH3T3/SW480 cells exhibited a significantly increased growth rate and colony formation rate, and also had a decreased apoptotic rate. Polyploidy and aberrant chromosomes were detected in the NIH3T3/SW480 cells by chromosome karyotype analysis. Our results suggested that mtDNA from colorectal cancer cells promotes the malignant phenotype of NIH3T3 cells. Further study of the biological functions of NIH3T3/SW480 cells might be helpful in understanding the role of mtDNA in colorectal carcinogenesis.

Screening and Identification of Recombinant Anti-Idiotype Antibodies against Gastric Cancer and Colon Cancer Monoclonal Antibodies by a Phage-Displayed Single-Chain Variable Fragment Library

Several monoclonal antibodies (McAbs) have been developed that show high sensitivity and specificity to gastric cancer and colorectal cancer. However, few of the antigens recognized by these antibodies have been identified. The authors now report the selection of anti-idiotype (anti-id) antibodies of MGb1 McAb against gastric cancer and MC5 McAb against colorectal cancer using phage-displayed single-chain variable fragment (ScFv) libraries. After purification, the anti-id antibodies were approximately 30 kd and could be recognized by MGb1/MC5 McAb. Anti-id antibodies significantly blocked the binding of MGb1 and MC5 to gastric cancer/colorectal cancer cells, respectively, suggesting that the antibodies were specific to MGb1 and MC5. Antibodies against gastric and colorectal cancer could be detected in mice at 6 weeks after immunization with the anti-id antibodies. At week 8, antibody titers reached 1:400. The anti-id antibodies may be useful as novel reagents for developing vaccines against gastric cancer and colorectal cancer.

Establishment of immuno-PCR technique for the detection of tumor associated antigen MG7-Ag on the gastric cancer cell line

The gastric cancer associated antigen MG7-Ag was detected by means of a newly established method, termed Immuno-PCR. A McAb-recombinant DNA chimeric molecule was made which possesses bispecific binding affinity for antigen that had been immobilized on microtiter wells and the segment of the attached DNA was amplified by PCR. The antigen of gastric cancer cell line KATO III was monitored by this method. Analysis of PCR products by agarose gel electrophoresis after staining with ethidium bromide allowed as few as 20 cells to be detected readily and reproducibly. Immuno-PCR showed a 104 enhancement in detection sensitivity compared with ELISA assay. When the same numbers of cells (2×106/ml) were immobilized and then the serial diluted chimeric molecule was added, 3.8 × 10−14 moles and 3.0 × 10−11 moles were needed to give positive results with the Immuno-PCR and ELISA assay, respectively. Therefore, Immuno-PCR could give an enomous amplification capability with good specificity, and has a sensitivity much higher than any existing techniques for antigen detection.

Cytotoxicity of indirect immunotoxin mediated by anti-gastric cancer monoclonal antibodies on tumor cells

In the present study, an indirect assay was employed to investigate 5 anti-gastric cancer monoclonal antibodies for their cytotoxic potential as ricin A chain-containing immunotoxins. The tumor cells were treated with dilutions of tested antibody followed by ricin A chain coupled to goat anti-mouse immunoglobulin. The cytotoxic effect was determined with tetrazolium colorimetric assay. The results showed that among the 5 antibodies chosen. MGb2 and MG7 could be well used for preparation of effective A chain immunotoxins.

Selective killing of gastric cancer cells by monoclonal antibody conjugated with a chain of ricin

A specific cytotoxic agent against gastric cancer was constructed by covalently coupling the ricin A chain to monoclonal artibody, MGb2, MGb2 was modified by SPDP to introduce the 3-(2-pyridylthio) propionyl radical and then treated with a reduced A chain to give a disulfide linked conjugate, that retained the original binding specificity of the antibody moiety. The conjugate obtained retained the activity of the antibody and the biological activity of the A chain well.