Primary Studies On The Relationship Between Autophagy And Apoptosis In Bombyx Mori Bme Cells

Now more and more biologists pay attention to autophagy, particularly the relationship between autophagy and apoptosis, since it is related to the development of organism, occurrence and development of disease, the maintenance and continuation of life and so on. The relationship between autophagy and apoptosis in mammalian cells, particularly that in the cancer cell has developed greatly, and both of them can promote, inhibit or work alone, depending on different cell types, different stumuli,ect. However, it is unclear in insect cells, and furthur work is needed to be done.In this paper, high level autophagy of Bombyx mori Bme cells were induced by two different methods in order to study the influences of high level of autophagy on apoptosis. The used techniques are fluorometric analysis, electron microscope, flow cytometry, enzymology assay, agarose gel electrophoresis, and so on.Results are as follows:both the number and bulk of MDC-labeled vacuoles (autophagesome and autolysosome) in Bme cells increased significantly after the treatments with rapamycin or amino acid starvation, and the electron microscopy observation proved it, too. In addition, electron microscopy observation also showed the decrease of mitochondria number and the swelling of mitochondria volume after 4 hours treatment of rapamycin.The primary assay showed that the number of high level autophagy cells increased, from 6.1% to 15.9% after 4h starvation, while it decreased to 12.4% after 8h starvation. In the early stage, the caspase-3 activity is low in the cells treated by the rapamycin or absence of amino acid, but it increased strikingly after 12 hours of treatment, it is nearly 10 times compared with that in the control. Furthermore, the externalization of phosphatidylserine also were detected by flow cytometry with Annexin-V and propidium iodiode (PI) staining, and it showed that the rate of early apoptotic cells increased from 2% to 24% in the cell treated with rapamycin for 12 hours, from 5% to 16% in the cells starved for 12 hours. Optical microscope observation demonstrated the formation of apoptotic bodies and the condensation of nuclear chromatin after DAPI staining. The appearance of DNA ladder on the agarose gel also supported the cell apoptosis. When the high level autophagy induced by amino acids starvation was inbibited with 3-methyladenine, the apoptotic rate decreased signifincantly. However, the rate of necrocytosis increased. Inhibition of the base line autophagy of Bme cells also enhanced the cell death.Above all, high level of autophagy of silkworm Bme cells preceded apoptosis or happen simultaneously, and it may promote apoptosis under the condition of rapamycin treatment or amino acid starvation, and the base line autophagy is benefit to the cell survival. The further work is to explore the mechanism of cross-talk between the high level of autophagy and apoptosis.