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[The toxic effects of high-dose Bcl-2 antisense phosphorothioate oligodeoxynucleotides incubation on cell line]
1Union Hospital Affiliate to Fujian Medical University, Fujian Insititute of Hematology, Fuzhou 350001, China.
Objective:
To investigate the toxic effects of high-dose Bcl-2 antisense phosphorothioate oligodeoxynucleotides (AS-PS-ODN, ASPO) incubation on HL60 cells and to understand the relationship between the dose of ASPO and the toxicity.
Methods:
Cellular viability and toxicity were detected by trypan blue exclusion, colony-forming unit HL60 cells and MTT assay. The expression of Bcl-2 protein was determined by immunocytochemistry and flow cytometry analysis. The proportion of apoptosis cells was tested by acridine orange (AO) standing. The sense (PS-ODN, SPO) was chosen for the toxic control of independent sequence. The curves of dose-effect and dose-toxicity were transfered into straight line and the reguession analysis was done by computer with the SPSS soften-ware.
Results:
When the dose of Bcl-2 PS-ODN was higher than 20 micromol/L, the non-specific effect of suppressing cell proliferation could occur. When the dose of Bcl-2 PS-ODN was more than 160 micromol/L, the non-specific effect (toxic effect) increased significantly. When the dose of Bcl-2 ASPO was higher than 80 micromol/L, the specific effect of inhibitting the expression of Bcl-2 proteion increased slowly. When the dose of PS-ODN reached 262 micromol/L, the toxic effect had no significant differences between Bcl-2 ASPO and Bcl-2 SPO. In the presence of ASPO or SPO at 160 micromol/L, the expression of Bcl-2 protein in the two groups of HL60 was decreased to 28% and 78% respectively, after 72 hours of incubation. The expression of Bcl-2 protein in the groups of HL60 which were passaged after incubation with ASPO or SPO was reinereased to 99.6% and 97.8%, respectively.
Conclusion:
Although ASPO is a kind of low-toxic drug, the toxic effects of Bcl-2 ASPO seem to be dependent on the dose increasing to a certain extent. It is clear that it can be used over a wide range of doses, however. Our results may also provide some referring concentrations of Bcl-2 ASPO for the pharmacodynamic and toxicological study in vivo in future.