| Objective: Malignant lymphoma is a kind of malignant tumor in the source of lymphaden and(or) lymph tissue. It is one of the common cancers in pediatrics and has already become a serious threat to children's health and life with an increasing incidence. The exact cause is unknown. Combination chemotherapy is still the main therapy in nowadays, but the toxicity and the drug resistance are important factors to influence long term patients'survival rate.Therefore, it is very necessary to search a kind of medicine which is safe and effective to cure lymphoma and improve the patients'prognosis and quality of life. Survivin is the new member of inhibitor of apoptosis protein family,which can inhibit cell death,regulate cell cycle and division. Caspase-3 is the factor of regulating apoptosis of cell. The most of factors which can trigger apoptosis of cell through Caspase-3 as mediated signaling pathways. Survivin can inhibit Caspase-3 activation to inhibit cell apoptosis. CyclinA is one of the positive factors of cell cycle regulating, which combines with relevant cell cyclin dependent kinase, and it's a necessary factor for cells to across R point and to complete S and G2/M period successfully. Some studies have proved that cardenolide can resist tumor action which mechanism maybe involved inhibiting tumor cell proliferation, differentiation, and inducing cell apoptosis. Digoxin is one of the common cardenolides in clinical. Now, there is few investigate and report on effects of Digoxin in human malignant lymphoma cell lines and its mechanism. In this study, in order to investigate the possible molecular mechanism of antitumor, we observed the effect of Digoxin on proliferation, apoptosis, cell cycle distribution and Survivin, Casepase-3, CyclinA expression in human malignant lymphoma cell line Raji and Jurkat. From this study we will approach about the effect of Digoxin in malignant lymphoma.And we can research possible anticancer mechanism of Digoxin though the influences about Survivin, Caspase-3 and CyclinA.Thus, theory evidence of Digoxin in clinical application will be provided. Methods: Human Burkitt's lymphoma cells Raji and T-cell lymphomaJurkat were cultivated in vitro and treated with various concentrations of Digoxin. Cell inhibitive rate was investigated by MTT assay. Apoptosis rate, cell cycle distribution in Raji and Jurkat cells were tested using Flow cytometry (FCM). Expression of Survivin, Caspase-3 and CyclinA mRNA in Raji and Jurkat cells were measured by RT-PCR. All measurement data showed by means±standard deviation ( X±S), and used SPSS 13.0 statistical software for statistical analysis. The means date between groups tested by one-way ANOVA measure, and the means date within groups tested by SNK measure. There was a statistical difference if P value less than 0.05.Results:1. MTT result showed: After Raji and Jurkat cells were treated with Digoxin of 25nmol/L,50nmol/L,100nmol/L,200nmol/L for 24, 48 and 72h, the OD value of test groups was decreased by degreed compared to the control groups. The OD value of treating Raji cells with Digoxin of 25nmol/L for 24h had no statistical significance compared to the control groups (P>0.05).The rest of test groups had statistical significance compared with the control groups (P<0.05). There was no statistical significance between groups treated using 25nmol/L and 50nmol/L digoxin for 24h(P>0.05).Test result between the rest of concentration groups had statistical significance (P<0.05). And there was statistical significance between the 24, 48, 72h time groups (P<0.05). It means, Digoxin can inhibit the proliferation of Raji and Jurkat cells as the treating concentration and time changed with limits.2. FCM showed: After Raji and Jurkat cells treated with Digoxin of 50nmol/L,100nmol/L,200nmol/L for 48h,cell cycle distributions were alteration. The cell numbers in G2/M phase were increased, while those in G0/G1 and S phase were decreased. Compared with control group, cell cycle distribution in test groups existed a significant difference (P<0.05). Cell cycle distribution were significantly different within concentration groups (P<0.05). 3. FCM showed: After Raji and Jurkat cells treateding with Digoxin of 50nmol/L,100nmol/L,200nmol/L for 48h,Apoptosis peak (AP) emerged significantly. There was no or only low apoptosis peak in control group. By one-way ANOVA, AP of Raji and Jurkat celles for treating 48h were significantly different between test groups and control groups, and AP were significantly different within concentration groups (P<0.05).4. RT-PCR showed: After Raji and Jurkat cells treateding with Digoxin of 50nmol/L,100nmol/L,200nmol/L for 48h,the expression of Survivin mRNA and CyclinA mRNA was decreased compared with control group(P<0.05), and was decreased as the treating concertration incresed. At the same time, the expression of Caspase-3 mRNA was incresed compared with control group (P<0.05), and was increased as the treating concertration increased. The correspondent expression of Survivin, Caspase-3, CyclinA mRNA in test groups was significantly different compared with control groups (P<0.05).Conclusion:1. The survival of Raji and Jurkat cells was inhibited by Digoxin in vitro with a concentration and time dependent manners from 50~200nmol/L.2. The cell numbers of Raji and Jurkat cells in G2/M phase were increased, while those in G0/G1 and S phase were decreased by the effect of Digoxin. Digoxin induced cell cycle arrest in G2/M phase and inhibited the growth of Raji and Jurkat cells, which may be one of the mechanisms of antitumor effect of Digoxin.3. Digoxin can induce Raji and Jurkat cells to apoptosis.The expression of Survivin mRNA can be decreased while the expression of Caspase-3 mRNA can be increased by Digoxin in vitro with a concentration dependent manner.And apoptosis in Raji and Jurkat cells can be induced by Digoxin through the downregulation of Survivin expression to activation of Caspase-3 expression, which may be another possible anticancer mechanism of Digoxin. |
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