MKN45 Gastric Carcinoma Cells Research Articles

Growth-inhibiting and apoptosis-inducing effects of Tanshinone II A on human gastric carcinoma cells

To explore the effects of Tanshinone II A on the proliferation, apoptosis and gene expression of p53 and bcl-2 in human gastric carcinoma MKN-45 cells. Cell count and MTT assay were used to study the proliferation-inhibiting effect of Tanshinone II A on MKN-45 cells. The effect of Tanshinone II A on the cell cycle and apoptosis of MKN-45 cells were examined by propidium iodide (PI) staining and flow cytometry. Semi-quantitative RT-PCR was used to further verify the expression of p53 and bcl-2 gene after exposure to Tanshinone A in MKN-45 cells. The results showed that Tanshinone A significantly inhibited the growth and proliferation of MKN-45 cells in a dose-and time-dependent manner (P<0.05). Tanshinone A arrested MKN-45 cells in G(2)/M phase which led to an obvious accumulation of G(2)/M phase cells while decreased number of G(0)/G(1) phase cells. This resulted in apoptosis of MKN-45 cells and the apoptosis rate was as high as 43.91% after treatment with 2.0 microg/mL Tanshinone II A for 96 h. It was also found that Tanshinone II A up-regulated expression of p53 gene and down-regulated expression of bcl-2 gene. The cytostatic and antiproliferative effect of Tanshinone II A makes it a promising anticancer agent for the treatment of gastric carcinoma.

Transcription Factor E2F-1 Is Upregulated in Human Gastric Cancer Tissues and Its Overexpression Suppresses Gastric Tumor Cell Proliferation

The E2F family members play a critical role in cell cycle regulation and other biological processes in the cell. To better understand the involvement of E2F-1 in the development and progression of gastric tumors, we investigated the mutation and expression of E2F-1 in human gastric cancer tissues and the effect of E2F-1 overexpression on the proliferation of gastric carcinoma cells. In this study, 80 pairs of gastric cancer specimens and paratumor tissues from different patients and 40 stomach mucosa specimens from healthy individuals were examined. PCR-SSCP analysis demonstrated that mutations were not detected in any of the gastric cancer and normal tissue specimens. In addition, the results of an immunohistochemistry assay revealed higher expression rates of E2F-1 (P < 0.01) in gastric cancer tissues (72.5%) than in paratumor tissues (30.0%) of the same individuals and stomach mucosa from healthy individuals (22.5%). However, no correlation was observed between the E2F-1 levels and patients’ clinical features, such as sex, age, histological types, lymph node metastasis, and clinical stages (P >0.05). Finally, the influence of E2F-1 overexpression on the growth of human gastric carcinoma MKN-45 cells in vitro was assessed by measuring colony formation, cell survival, and cell cycle progression. Our data clearly showed that cell growth and proliferation were significantly inhibited in MKN-45 tumor cells transfected with the expression vector encoding E2F-1 in comparison with nontransfected cells or cells transfected with empty vector. These findings suggest that E2F-1, a stable and conservative gene during the oncogenesis and progression of stomach cancers, may potentially serve as a biomarker for clinical diagnosis of gastric carcinomas and as a target for the development of novel therapeutic interventions to treat this disease.

CDX2 does not suppress tumorigenicity in the human gastric cancer cell line MKN45

CDX2 is a Drosophila caudal-related homeobox transcription factor that is expressed specifically in the intestine. In mice, ectopic expression of CDX2 in the gastric mucosa gives rise to intestinal metaplasia and in one model, gastric carcinoma. In humans, increased CDX2 expression is associated with gastric intestinal metaplasia and tubular adenocarcinomas. These patterns of expression have shown that CDX2 is important for the initiation of intestinal metaplasia in the gastric mucosa, but the role of CDX2 in established gastric cancer remains unclear. We sought to determine whether CDX2 contributes to tumorigenic potential in established gastric cancer. The CDX2 gene in MKN45 gastric carcinoma cells was disrupted using targeted homologous recombination. The resulting CDX2-/- cells are essentially identical to their parental cells, with the exception of CDX2 ablation. We found no significant differences in the proliferation of CDX2-/- cells compared to CDX2+/+ cells, in vitro or in vivo. Molecular analyses show that loss of CDX2 predominantly altered the expression of genes involved in intestinal glandular differentiation and adhesion. However, there were no microscopic differences in tumor differentiation. We conclude that disruption of CDX2 in MKN45 cells does not significantly affect their tumorigenic potential.

Chemosensitization by STI571 targeting the platelet‐derived growth factor/platelet‐derived growth factor receptor‐signaling pathway in the tumor progression and angiogenesis of gastric carcinoma

Autocrine and paracrine growth mediated by the platelet-derived growth factor (PDGF)/PDGF receptor (PDGFR)-signaling pathway plays an important role in the progression of solid tumors. The authors assessed the effect of STI571 on the tumor growth of gastric carcinoma in combination with 5-fluorouracil (5-FU) or paclitaxel targeting the PDGF/PDGFR-signaling pathway. In MKN-45 gastric carcinoma cells, the cytotoxic effect was evaluated by 3-(4,5 dimethiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and the in vivo antitumor effect was evaluated in a nude mouse xenograft. Both STI571 and an antitumor drug were administered intraperitoneally. Gene expression was assessed by Western blot analysis and immunohistochemical staining. Apoptotic cell death was evaluated by the terminal deoxyuridine triphosphate-biotin nick-end labeling assay, and tumor angiogenesis was evaluated by microvessel density analysis. Treatment with STI571 alone was not effective in vitro, as assessed by a 50% inhibitory concentration value of 24.3 microM. Combination treatment with STI571 and 5-FU or paclitaxel enhanced the cytotoxic effect somewhat when the concentration of STI571 was increased to 10 microM. Combination treatment with STI571 and 5-FU or paclitaxel enhanced the antitumor effect of the antitumor drug significantly in vivo. The enhanced antitumor effect was associated with increased apoptotic cell death and inhibition of tumor angiogenesis. Treatment with STI571 down-regulated the expression of PDGF-BB and PDGFR-beta in tumor cells and decreased the production of phosphorylated PDGFR-beta and phosphorylated Akt. Furthermore, treatment with STI571 inhibited the expression of PDGFR-beta in stromal cells. STI571 was an effective chemosensitizer of antitumor drugs, such as 5-FU and paclitaxel for gastric carcinoma, targeting the PDGF/PDGFR-signaling pathway of tumor cells and stromal cells in disease progression and angiogenesis.

Enhancement of therapeutic effect in combination treatment with imatinib and anticancer drug in gastric carcinoma

4163 Background: Imatinib (STI571, Gleevec) is a novel molecular targeting agent, which inhibits the activity of c-kit and PDGFR in gastrointestinal stromal tumors. We conducted preclinical evaluation of therapeutic effect of imatinib and potential synergism in combination with imatinib and anticancer drug in gastric carcinoma cells, in terms of growth inhibition by targeting PDGF/PDGFR signaling pathway. Materials and Methods: MKN-45 gastric carcinoma cells were employed, and the cells were transplanted into nude mice. When the tumor size was reached approximately 100 mm3 after transplantation, the mice were arbitrarily divided in four groups (n=8/group), according to no treatment, treatment with anticancer drug alone, treatment with imatinib alone, and combination treatment with imatinib and anticancer drug, respectively. Imatinib was administered intraperitoneally at 50 mg/kg for consecutive 28 days, whereas anticancer drug was administered at LD50/3 four times in qwk. Results: The treatment with imatinib of MKN-45 gastric carcinoma xenograft showed antitumor effect to a greater extent than the treatment with 5-fluorouracil (5-FU) or docetaxel (TXT) alone. Further, antitumor effect was significantly enhanced in the combination treatment with imatinib and 5-FU or TXT, compared to the treatment with imatinib, 5-FU or TXT alone (P<0.01, Student t-test). Enhanced antitumor effect in combination treatment with imatinib and anticancer drug was associated with both induction of apoptosis and inhibition of tumor angiogenesis, which was explained with the decreased expression in pAkt, and PDGF-betaR, and decreased phosphorylation of PDGF-betaR. Conclusions: Combination treatment with imatinib and anticancer drugs such as 5-FU and TXT may a new strategy for enhancing therapeutic effect in the treatment of gastric carcinoma, targeting inhibition of PDGFR-mediated paracrine tumor growth. No significant financial relationships to disclose.

Enhancement of therapeutic effect in combination treatment with imatinib and anticancer drug in gastric carcinoma

4163 Background: Imatinib (STI571, Gleevec) is a novel molecular targeting agent, which inhibits the activity of c-kit and PDGFR in gastrointestinal stromal tumors. We conducted preclinical evaluation of therapeutic effect of imatinib and potential synergism in combination with imatinib and anticancer drug in gastric carcinoma cells, in terms of growth inhibition by targeting PDGF/PDGFR signaling pathway. Materials and Methods: MKN-45 gastric carcinoma cells were employed, and the cells were transplanted into nude mice. When the tumor size was reached approximately 100 mm3 after transplantation, the mice were arbitrarily divided in four groups (n=8/group), according to no treatment, treatment with anticancer drug alone, treatment with imatinib alone, and combination treatment with imatinib and anticancer drug, respectively. Imatinib was administered intraperitoneally at 50 mg/kg for consecutive 28 days, whereas anticancer drug was administered at LD50/3 four times in qwk. Results: The treatment with imatinib of MKN-45 gastric carcinoma xenograft showed antitumor effect to a greater extent than the treatment with 5-fluorouracil (5-FU) or docetaxel (TXT) alone. Further, antitumor effect was significantly enhanced in the combination treatment with imatinib and 5-FU or TXT, compared to the treatment with imatinib, 5-FU or TXT alone (P<0.01, Student t-test). Enhanced antitumor effect in combination treatment with imatinib and anticancer drug was associated with both induction of apoptosis and inhibition of tumor angiogenesis, which was explained with the decreased expression in pAkt, and PDGF-betaR, and decreased phosphorylation of PDGF-betaR. Conclusions: Combination treatment with imatinib and anticancer drugs such as 5-FU and TXT may a new strategy for enhancing therapeutic effect in the treatment of gastric carcinoma, targeting inhibition of PDGFR-mediated paracrine tumor growth. No significant financial relationships to disclose.

463. Conditionally Replicative Adenovirus Expressing Degradation-Resistant p53 for Enhanced Oncolysis of MDM2 Overexpressing Cancers

Conditionally replicative adenoviruses (CRAds) are novel anti-cancer agents, designed to selectively replicate in and to destroy cancer cells. A critical determinant for the oncolytic potency of CRAds is their capacity to induce cancer cell death. In this respect, dysfunctional cell death pathways in cancer cells reduce CRAd efficacy. Previously, we showed that expressing p53 tumor suppressor protein from the genome of the CRAd AdΔ24 (with a deletion in the pRb-binding E1A-CR2 domain) resulted in more effective cytotoxicity on most cancer cell lines (Cancer Res., 62:6165–6171, 2002). More recently, we confirmed this finding on primary tumor cells in vitro and in vivo. However, in approximately 20% of tested cancer cell lines AdΔ24-p53 was not more effective than AdΔ24. In search for molecular determinants of resistance against the oncolysis enhancing effect of exogenous p53 we analyzed these cell lines for expression of the major negative p53 regulator MDM2. Western analysis on 5 resistant cell lines revealed strong MDM2 expression in 4 cases, suggesting that MDM2-mediated inhibition and degradation of exogenous p53 could have limited the oncolysis of these cells by AdΔ24-p53. To develop a CRAd for more effective treatment of MDM2 overexpressing cancers, we made the new AdΔ24-derivative AdΔ24-p53(14/19). This CRAd expresses a variant of p53 with amino acid substitutions L14Q and F19S that abrogate p53 binding to MDM2 (Genes Dev., 8:1235–1246, 1994). AdΔ24-p53 and AdΔ24-p53(14/19) expressed similar levels of p53 protein, which peaked at 24 hours after infection, as tested by Western analysis on infected p53-null SaOs-2 cells. Transactivation of a p53-dependent reporter construct transfected into SaOs-2 cells by AdΔ24-p53(14/19) was approximately 40% of transactivation by AdΔ24-p53. The oncolytic potency of AdΔ24, AdΔ24-p53 and AdΔ24-p53(14/19) was compared on MDM2-overexpressing MKN45 gastric carcinoma cells and MG-63, MNNG-HOS and CAL-72 osteosarcoma cells. To this end, cells were infected at a range of MOI and cultured to allow replication and lateral spread. After culture, remaining viable cells were stained with crystal violet. Consistent with previous observations, AdΔ24 and AdΔ24-p53 were similarly oncolytic. In contrast, AdΔ24-p53(14/19) was on average 10-times more effective than AdΔ24 and AdΔ24-p53. Hence, abrogating binding of exogenous p53 to MDM2 overcame resistance to p53-mediated oncolysis enhancement in MDM2 overexpressing cancer cells, confirming our hypothesis that CRAd oncolysis enhancement by exogenous p53 can be improved by preventing its degradation. Furthermore, our findings suggest that knowledge on molecular defects in cancer cells can be used to design tailored CRAds with enhanced oncolytic potency.

Recombinant ricin toxin A chain cytotoxicity against carcinoembryonic antigen expressing tumour cells mediated by a bispecific monoclonal antibody and its potentiation by ricin toxin B chain

A bispecific monoclonal antibody recognising both carcinoembryonic antigen (CEA) and ricin toxin A chain (RTA) was tested for its ability to target recombinant RTA (r-RTA) to CEA-expressing tumour cells, alone and in combination with ricin B chain (RTB). The antibody, 636 (Robins et al., 1990), induced significant RTA cytotoxicity against MKN45 gastric carcinoma cells which express high levels of CEA, using the r-RTA at a concentration below that known to be intrinsically cytotoxic. The addition of ricin toxin B chain (RTB) also potentiated cytotoxicity of r-RTA, and there was an additive increase in potentiation against CEA-positive cells when both RTB and 636 were included. The bispecific antibody restored potentiation by RTB after blocking of its binding site with excess galactose, and also the cytotoxic activity of whole ricin which had been blocked with galactose. It was concluded that the 636 bispecific antibody was highly effective in targeting the toxic moiety of the molecule to CEA-expressing cells, and allowed exploitation of the additional ability of the B chain to facilitate cellular incorporation. The facilitating function of the B chain was equally effective whether or not its lectin site was active.