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Chemotherapy with 5-fluorouracil (5-FU) is commonly used in combination therapy for esophageal squamous cell carcinoma (ESCC), but its efficacy is limited in certain patients. Recent studies suggest that constitutive activation of nuclear factor-κB (NF-κB) has a critical role in tumorigenesis and is associated with poor prognosis and resistance to chemoradiation therapy in many types of human cancers. In the present study, we evaluated the effect of small interfering RNA targeting NF-κB (NF-κB siRNA) combined with 5-FU on the proliferation of two cell lines of cultured ESSCs. Immunofluorescence and immunoblot analyses revealed that the NF-κB protein was localized mostly in the cytoplasm of ESCCs. When cultured ESCCs were exposed to tumor necrosis factor-α, NF-κB was transferred to the nucleus and activated. ESCCs with activated NF-κB had poor sensitivity to 5-FU. When cells were transfected with NF-κB siRNA, the levels of NF-κB protein were significantly decreased in the cytoplasm and the nucleus. Transcriptional activity of NF-κB was significantly suppressed in cells treated with 5-FU and NF-κB siRNA compared to cells treated with 5-FU alone. 5-FU consistently suppressed proliferation of ESCCs in a dose-dependent manner, and this effect was significantly enhanced when combined with NF-κB siRNA. These results suggest that combination therapy of 5-FU with NF-κB siRNA may provide a new therapeutic option for ESCC.

We examined the susceptibility of six human gastric carcinoma cell lines to infection with recombinant p53 adenovirus vector (AxCA-p53). AxCA-p53 infection at a muliplicity of infection (MOI) of 50 resulted in apoptotic cell death (MKN-1 cells), growth arrest (MKN-45, MKN-74 and KATO-III cells), or non-effectiveness (TMK-1 and OCUM-2M cells). Western blot analysis revealed increasing expression levels of p21/WAF1 protein after infection with AxCA-p53 in all the cell lines. After infection with AxCA-p53, the expression levels of bax or bcl-XL protein changed in MKN-1, but not in the other cell lines. These results suggest that the apoptotic pathway (dependence on the expressions of bcl-2 family proteins) dominates the growth arrest pathway (dependence on the expressions of p21/WAF1 protein) after infection with AxCA-p53. Thus, the bcl-2 family might play a crucial role in p53-mediated growth arrest and apoptosis in human gastric carcinoma cells.

We investigated the clinicopathological significance of dendritic cell infiltration (DCsI) in esophageal squamous cell carcinoma and in regional lymph nodes of 88 patients. The expression of mutated p53 protein and the degree of positive cancer cells of proliferating cell nuclear antigen (PCNA labeling index) in tumors were analyzed as biological markers. These factors were compared with the degree of DCsI in tumors and in lymph nodes. The number of dendritic cells (DCs) were counted and scored as per mm2 in each case. The degree of DCsI of tumors with expression of p53 (19/mm2, n=50) was significantly lower than that of DCsI in 38 tumors without expression of p53 (27/mm2, P=0.0411). However, no significant correlation was detected between the PCNA labeling index and the degree of DCsI in 88 primary tumors (P=0.1273). The degree of DCsI in 53 metastatic lymph nodes (30/mm2) was significantly lower than that of DCsI in 264 cancer-free regional lymph nodes (48/mm2, P=0. 02). Although the degree of DCsI in tumors was not an independent prognostic factor for the 78 surviving patients (P=0.2647), the 3-year survival rate of patients in stage III and IV who underwent curative operation and who had tumors with high DCsI (>9/mm2, n=16, 72%) was significantly higher than that of the 24 patients who had tumors with low DCsI (< or = 9/mm2, 21%, P=0.008). These findings indicate that DCs infiltrated in and around the esophageal cancer may play a defensive role of the hosts against the tumors. This immune defense of the hosts might be an important prognostic factor for patients with advanced esophageal cancer. However, cancer cells which express a mutated p53 protein might regulate the function or activity of DCs.

Adenovirus has attracted much attention as a vector for gene therapy. Integrin alphavbeta3 and alphavbeta5 mediate adenovirus internalization into cells. We examined adenoviral transduction efficiency and expression of integrin alphavbeta3 alphavbeta5 in 9 human gastric carcinoma cell lines. The percentage of -gal-positive cells was more than 85% 3 days after infection with recombinant adenovirus carrying the bacterial LacZ gene (AxCALacZ) at a dose of 25 MOI in 7 cell lines and the transduction efficiency was 32 and 42% in HSC-39 and MKN-28 cells, respectively. Adenoviral transduction efficiency did not correlate with the histological cell types. Flow cytometric analysis revealed relatively high expression of integrin alphavbeta5 in MKN-28 and OCUM-2M cells, followed by MKN-1, MKN-7, MKN-45, MKN-74, TMK-1 and KATO-III cells. HSC-39 cells minimally expressed integrin alphavbeta5. On the other hand, integrin alphavbeta3 was expressed only in MKN-1 cells. These results suggest that the adenovirus vector might be useful for gene transduction into human gastric carcinoma cells and the transduction efficiency partly correlates with the expression levels of integrin alphavbeta5 but not integrin alphavbeta3.

We analyzed taxol-induced apoptosis in human colon carcinoma cell lines. Cells expressing Bcl-2 (COLO320) and those that did not (DLD-1), underwent apoptosis after 24 h exposure to 1 mu M taxol. Flow cytometry showed that the numbers of cells arrested in G(2)/M decreased and that of apoptotic cells increased time-dependently. The molecular weight of Bcl-2 was increased to above 26 kDa in COLO320 and LoVo cells after a 4 h exposure to taxol. Incubating the cells with genistein, a protein tyrosine kinase inhibitor, inhibited the taxol-induced modified Bcl-2 expression and apoptosis. These results suggest that taxol induces apoptosis in cells arrested in G(2)/M phase which might be partly explained by Bcl-2 inactivation by phosphorylation in human colon carcinoma cell lines.

Many anticancer agents induce an active process that leads to cell death, known as, apoptosis, in sensitive tumor cells. The fragmentation of DNA, an indicator of apoptosis, was analyzed in two different lines of human gastric cancer cells (HSC-39 and MKN-28) that had been exposed to adriamycin and cisplatin. The fragmentation of DNA was detected in HSC-39 cells (signet ring cell gastric carcinoma) after a 1-h incubation with 0.18 mu M and after a 6-h incubation with 0.09 mu M adriamycin, as well as after a 1-h incubation with 1.67 mu M cisplatin. However, in MKN-28 cells (moderately differentiated gastric adenocarcinoma), the fragmentation of DNA was not detected after a 6-h incubation with 0.18 mu M adriamycin or a 6-h incubation with 3.33 mu M cisplatin. The results suggest that signet ring cell gastric carcinoma is more sensitive to adriamycin and to cisplatin than moderately differentiated gastric adenocarcinoma.