The Study On Autophagic Apoptosis Induced By Vincristine In Human Liver Cell Line L02

We have developed the study on relationship between autophagy and apoptosis in domestic field firstly . We put forward the concept of 'autophagic apoptosis' for the first time, and hold that autophagic apoptosis is a specific type of apoptosis. We term apoptosis with distinct autophagic characteristic as 'autophagic apoptosis' Autophagy maybe relate with the regulative mechanism of apoptosis. A model of autophagic apoptosis was successfully established by the treatment with vincristine (VCR) in human liver cell line L02. With measures of morphology, cytobiology and molecular biology we have made a systematic study on changes in morphology, biophysics , biochemistry and relative genic regulation about autophagic apoptosis of L02 cell line. Many characteristics about origination and development of autophagic apoptosis have been revealed. In the course of L02 autophagic apoptosis induced by VCR the autophagic stage is prior to executional stage of apoptosis, and run through the whole course of autophagic apoptosis of L02 cells. Autophagy can ~be observed with monodansylcadaverin (MDC) staining under optical microscope .We have firstly used the method of MDC staining and Flow Cytometry to quantitatively analyze autophagy , and consider the method more objective and more accurate. 3-methyladenine (3MA) can specifically inhibit the formation of autophagic vacuoles and can partially prevent the L02 autophagic apoptosis. Autophagy is needful to autophagic apoptosis of L02 cells induced by VCR. Cathepsins B and D are involved in the autophagic apoptotic cascade of L02 cells following VCR induction, and cathepsin D maybe acts as a positive mediators of autophagic apoptosis. Our data provide 5 the evidence that cytochrome c was released to the cytosol from mitochondrir in L02 cells after treatment with VCR and that the release preceded DNA fragmentation. These findings suggest cytochrome c involvement in the autophagic stage of the autophagic apoptosis. The release of cytochrome c can be prevented by the using of 3MA. L02 autophagic apoptosis has relation to the increased of lipid peroxide (LPO) levels and lipid peroxide levels . 3MA can also prevent the production of LPO. In the L02 cells sensitive to VCR-induced autophagic apoptosis, the mitochondrial transmembrane potential (Δψm) was disrupted. Autophagic apoptosis could closely associate with the Δψm collapse. mitochondrial permeability transition (MPT) maybe is the common event of VCR-induced autophagy and apoptosis. The increase of [Ca2+] ii i in VCR-treated L02 cells may be related to autophagy and apoptosis induced by VCR. 3MA may reduce VCR-induced apoptosis by inhibiting increase of [Ca2+] i and by inhibiting autophagy in L02 cells. The fact that ubiquitin level is increased in L02 cells treated with VCR suggest that ubiquitin-proteasome pathway also is a way of protein degradation in the process of autophagic apoptosis. lJbiquitin-proteasome inhibition can enhances VCR-induced autophagic apoptosis in L02 cells. We also confirmed that ubiquitin-proteasome pathway is involved in regulating the levels of Bcl-2, which might have a role in mediating autophagic apoptosis in L02 cells, The expression and translocation of NF- K B to nuclei was closely relative to autophagy. Caspase-3 was isolated and identified from VCR-induced autophagic autophagy model . It was proved that Caspases is involve in VCR...