Correlation between HER-2/neu(erbB-2) expression level and therapeutic effect of combination treatment with HERCEPTIN and chemotherapeutic agents in gastric cancer cell lines.

INTRODUCTION: Although advanced gastric cancer has many limitations and response rate is marginal in chemotherapy. Overexpression of human epidermal growth factor receptor 2(HER-2/neu) gene and its protein are associated with increased cell division and a high rate of tumor growth and have been reported in several malignancies. Especially, approximately 30% of breast cancer patients have overexpression of HER-2/neu protein and the overexpression metastasize faster, induces resistance of the chemotherapy and down-regulate function of estrogen receptor. Recombinant humanized anti-HER2 antibody (Herceptin) inhibits proliferation of HER-2/neu overexpressing tumor cells and the use of that in combination in metastatic breast cancer have increased cytotoxicity of chemotherapeutic agents. METHODS: We evaluated the expression of HER-2/neu protein in gastric cell lines by FACS and then comparing the cytotoxicity in chemotherapeutics (doxorubicin, cisplatin, paclitaxel, 5-FU) alone and in combination with Herceptin according to the expression of HER-2/neu protein by MTT assay. RESULTS: 1. NCI-N87 (88%) gastric cancer cell line and SK-BR-3 (89%) breast cancer cell line with strong positivity of HER-2/neu expression. YBC-2 (55%) and YBC-3 (48%) gastric cancer cell line with intermediated, weak positivity respectively. Negative control U-87 MG (6%) brain cancer cell line were showed low expression of HER-2/neu. 2. Cell growth was dose-dependently inhibited in HER-2/neu positive, control cell line SK-BR-3 by Herceptin treatment but not observed in HER-2/neu negative control cell line U-87 MG. Effective growth inhibition was not observed in gastric cancer cell lines with single treatment of Herceptin, all cell lines observed the dose-dependent growth inhibition to chemotherapeutic agents (doxorubicin, cisplatin, paclitaxel and 5-FU). 3. Combination of Herceptin with doxorubicin observed synergistic effects in all cancer cell lines except YBC-3, combination of Herceptin with cisplatin observed NCI-N87 and SK-BR-3 and combination of Herceptin with paclitaxel observed synergistic effects in YBC-2. Combination of Herceptin with 5-FU observed antagonistic effects in all cancer cell lines. CONCLUSIONS: According to HER-2/neu expression level, effect of anti-cancer agents was observed differently in combination of Herceptin with chemotherapeutic agents. This suggests that HER-2/neu expression level can be applied standard of combination drug selection in combination of Herceptin With chemotherapeutic agents in gastric cancer.

Identification of differentially expressed genes in gastric cancer by high density cDNA microarray.

The identification of molecular markers for diagnosis, treatment, and prognosis is a significant issue in the management of patients with gastric cancer. We compared the expression profiles of 23 gastric cancers and 22 normal gastric tissues using cDNA microarrays. We divided the samples into two sets, 11 pairs as a training set and 12 unpaired gastric cancer and 11 unpaired normal gastric tissues as a test set. We selected significant genes in the training set and validated the significance of the genes in the test set. We obtained 238 classifier genes that showed a maximum cross-validation probability and clear hierarchical clustering pattern in the training set, and showed excellent class prediction probability in the independent test set. The classifier genes consisted of known genes related to the biological features of cancer and 28% unknown genes. We obtained genome-wide molecular signatures of gastric cancer, which provides preliminary exploration data for the pathophysiology of gastric cancer.

Application of mesenchymal stem cells as a vehicle to deliver replication-competent adenovirus for treating malignant glioma.

Although gene therapy was regarded as a promising approach for glioma treatment, its therapeutic efficacy was often disappointing because of the lack of efficient drug delivery systems. Mesenchymal stem cells(MSCs) have been reported to have a tropism for brain tumors and thus could be used as delivery vehicles for glioma therapy. Therefore, in this study, we attempted to treat glioma by using MSCs as a vehicle for delivering replication-competent adenovirus. We firstly compared the infectivity of type 3, type 5, and type 35 fiber-modified adenoviruses in MSCs. We also determined suitable adenovirus titer in vitro and then used this titer to analyze the ability of MSCs to deliver replication-competent adenovirus into glioma in vivo. Our results indicated that type 35 fiber-modified adenovirus showed higher infectivity than did naked type 3 or type 5 fiber-modified adenovirus. MSCs carrying replication-competent adenovirus significantly inhibited tumor growth in vivo compared with other control groups. In conclusion, MSCs are an effective vehicle that can successfully transport replication-competent adenovirus into glioma, making it a potential therapeutic strategy for treating malignant glioma.

Optimizing DC vaccination by combination with oncolytic adenovirus coexpressing IL-12 and GM-CSF.

Dendritic cell (DC)-based vaccination is a promising strategy for cancer immunotherapy. However, clinical trials have indicated that immunosuppressive microenvironments induced by tumors profoundly suppress antitumor immunity and inhibit vaccine efficacy, resulting in insufficient reduction of tumor burdens. To overcome these obstacles and enhance the efficiency of DC vaccination, we generated interleukin (IL)-12- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-coexpressing oncolytic adenovirus (Ad-ΔB7/IL12/GMCSF) as suitable therapeutic adjuvant to eliminate immune suppression and promote DC function. By treating tumors with Ad-ΔB7/IL12/GMCSF prior to DC vaccination, DCs elicited greater antitumor effects than in response to either treatment alone. DC migration to draining lymph nodes (DLNs) dramatically increased in mice treated with the combination therapy. This result was associated with upregulation of CC-chemokine ligand 21 (CCL21(+)) lymphatics in tumors treated with Ad-ΔB7/IL12/GMCSF. Moreover, the proportion of CD4(+)CD25(+) T-cells and vascular endothelial growth factor (VEGF) expression was decreased in mice treated with the combination therapy. Furthermore, combination therapy using immature DCs also showed effective antitumor effects when combined with Ad-ΔB7/IL12/GMCSF. The combination therapy had a remarkable therapeutic efficacy on large tumors. Taken together, oncolytic adenovirus coexpressing IL-12 and GM-CSF in combination with DC vaccination has synergistic antitumor effects and can act as a potent adjuvant for promoting and optimizing DC vaccination.

Genetic analysis of liver metastatic cell lines with different metastatic potential.

Metastasis is the major feature of malignant tumors that causes 90% of cancer deaths. Our laboratory has already established liver metastatic clones with YCC-16, isolated from the blood of a gastric cancer patient and expanded in vitro culture using a repeated orthotopic implantation method, and had reported biologic behaviour of the parental YCC-16, the orthotopic primary S1L0, and S1L1, S2L2 and S3L3 liver metastatic clones. Here, using these cell lines, we screened from chromosomal abnormalities using karyotype analysis and micro-CGH matching. There were 31 genes screened using PCA method which were functionally related to cell adhesion. Also, there were 23 genes selected which were related to the liver specific metastasis but excluded genes related to adhesion. There were 4 genes which demonstrated reduced or increased expression stepwise with passage. In conclusion, our results should contribute to exploring the mechanisms of liver metastasis by gastric cancer.

Therapeutic and tumor-specific immunity induced by combination of dendritic cells and oncolytic adenovirus expressing IL-12 and 4-1BBL.

Recently, gene-based cytokine treatment has been actively pursued as a new promising approach in treating cancer. In an effort to augment the efficiency of antitumor effect by cytokine-mediated immunotherapy, we selected both interleukin (IL)-12 and 4-1BB ligand (4-1BBL) as suitable cytokines to fully activate the type-1 immune response. Coexpression of IL-12 and 4-1BBL mediated by oncolytic adenovirus (Ad) greatly enhanced the antitumor effect. Further, synergistic enhancement in interferon (IFN)-gamma levels were seen in mice treated with oncolytic Ad expressing both IL-12 and 4-1BBL. Next, to improve the overall antitumor immune response, we coadministered IL-12- and 4-1BBL-coexpressing oncolytic Ad with dendritic cells (DCs). Combination treatment of IL-12- and 4-1BBL-coexpressing oncolytic Ad and DCs elicited greater antitumor and antimetastatic effects than either treatment alone. Moreover, enhanced type-1 antitumor immune response and higher migratory abilities of DCs in tumors were also observed in the combination arms. The nature of the enhanced antitumor immune response seems to be mediated through the enhanced cytolytic activity of cytotoxic T lymphocytes (CTLs) and IFN-gamma-releasing immune cells. Taken together, these data highlight the potential therapeutic benefit of combining IL-12- and 4-1BBL-coexpressing oncolytic Ad with DCs and warrants further evaluation in the clinic.