To study anti-cancer effect and molecular biological mechanism of bee venom for aqua-acupuncture, the effects of bee venom on cell viability, apoptosis, and cell cycle were analyzed using MTT assay, tryphan blue assay, [³H]thymidine release assay, flow cytometric analysis, activity of caspase-3 protease activity assay, and immunocytometric analysis of PCNA. To explore whether anti-cancer effects of bee venom are associated with the transcriptional control of gene expression, quantitative RT-PCR analysis of apoptosis- and cell cycle-related genes was performed.
The obtained results are summarized as follows:
1. The MTT assay demonstrated that cell viability was decreased by bee venom in a dose-dependant manner.
2. Significant induction of apoptosis was identified using tryphan blue assay, [^3H]thymidine release assay, and flow cytometric analysis of sub G₁ fraction.
3. In analysis of caspase-3 protease activity, the activity had increased significantly, in a dose-dependant manner.
4. Quantitative RT-PCR analysis of the apoptosis-related genes showed that Bcl-2 and Bcl-X_L were down-regulated whereas Bax was up-regulated by bee venom treatment.
5. In flow cytometric analysis of cell cycle and immunocytometric analysis of PCNA expression, cell numbers of G₁ phase was increased by a dose-dependant manner.
6. In quantitative RT-PCR analysis of the cell cycle-related genes, p21, p27, and p57 were increased, while Cyclin D1, CDK4, c-Myc, c-Fos, and Histone H3 were decreased. In contrast, there were no remarkable changes in expression levels of CDC2 and c-Jun.
|