A Study On Proliferation And MAGE Gene Expression Of HPB-AM Cells Affected By Topotecan

Backgroud: The melanoma antigen-encoding(MAGE) was the first tumor-associated antiges(TAA) discovered. It has been shown that the tumor rejection antigen(TRA) encoded by human MAGE gene family can produce antigen peptides which link with HLA-I and trigger tumor-specific cytotoxic T lymphocyte(CTL) proliferation thus kill tumor cells. MAGE gene has a high level expression in most malignant tumors but not in normal tissue except for placenta and testis, it can be used as the ideal vector for tumor diagnosis and immunotherapy. Preliminary clinical experiments confirmed the effect of MAGE gene in anti-tumor immunotherapy.Topotecan(TPT) is a special and effective inhibitors on Topo I. Through integrating with DNA/Topo I polymer, it comes into being a stable covalence compounds to excert an anti-tumor effect. Recently, the drug was found to be effective on many kinds of incurable non-menonoma tumor, especially on some of hematological tumors. Using topotecan, the effective rate on recurrence and incurable non-Hodgkin lymphomas reached 40%. There is little knowledge on molecular path way of TPT, although the evidence of anti- tumor effect of TPT on the malignant hematological diseases such as leukemia and lymphoma has been observed in the clinical settings. The effect of TPT on the lymphoma cells and the relationship between TPT and MAGE gene remain to be investigated. The study aims to evaluate the effectiveness of TPT on the induction of apoptosis of T cell lymphoma NK system HPB-AM cell and the relationship between the effect of TPT and MAGE gene. The study provids significant data for further understanding of molecular path way of TPT anti-tumor effects and increase the effect of TPT in the clinical settings.Methods:1. T cell lymphoma NK cell HPB-AM is employed to the study. The reversetranscription-polymerase chain reaction(RT-PCR) was also employed to detect the expression of MAGE-1 MAGE-2, MAGE-3 and MAGE-4 mRNA.2. MTT method: When the HPB-AM cells were treated with 0.05, 0.10, 0.15, 0.20μmol/L of TPT and were incubated for 4h, 8h, 12h, 16h, respectively, the inhibitory rate of cell proliferation in each group is detected by MTT assay. Then, minimum working concentration and duration was ascertained, morphology of the tumor cells was observed under microscope.3. MAGE expression of HPB-AM cells, before and after treated with TPT, at different time and concertration was detected by RT-PCT.4. SPSSIO.O statistical software was applied to analyze the data. The standard of significant level was p=0.05.Results:1. The HPB-AM cells express MAGE-3 and MAGE-4 genes(0.94±0.03 and 0.85±0.01 respectively), but did not express MAGE-1 and MAGE-2.2. Growth inhibitory effect of TPT on HPB-AM cells: Following treatment with TPT at different concentrations for the same time, the proliferation of HPB-AM cells was inhibited significantly, in comparison with that before the experiment (P<0.05). There was no difference between 0.20μmol/L and 0.15μmol/L of TPT on their inhibitory effects. The inhibitory rate of cells proliferation in the same concentration of TPT has statistical significance at different time(0h, 4h, 8h, 12h, 16h), compared with that before experiemnt (P<0.05). There was no difference between 16h and 12h treated with TPT. The results demonstated that TPT was capable of inhibiting the HPB-AM cells growth by time- and dose-dependent manners. The minimum working concentration was 0.10μmol/L and the minimum working time was 8 h among all groups.3. Effect of TPT on the expression of MAGE gene: The result of RT-PCR revealed that the change of expression level of MAGE gene effected by TPT at different concentrations on HPB-AM cells has statistical significance for a specific time point, compared with that before the experiment(P>0.05). The change of expression level of MAGE gene affected by TPT at same concentrations on HPB-AMcells has statistical significance for a series of different time, in comparison with that before the experiment(P<0.05). Conclusion:1. The HPB-AM cells express MAGE-3 and MAGE-4 genes while did not express MAGE-1 and MAGE-2.2. TPT can inhibit the proliferation of HPB-AM cells in a time-and dose-dependent manners.3. TPT is capable of inhibiting the expression of MAGE gene in HPB-AM cells in a time-and dose- dependent manners.