Influences Of Proteasome Inhibitor MG132 On Apoptosis Of Hela Cell And Expression Of Caspase-3

Objective: To observe the apoptosis of Hela cells induced by MG132; To explore the effect of MG132 on the expression of caspase-3 of Hela cells.Methods: Human cervical cancer cell line,Hela ,was cultured in vitro and Cells in logarithmic growth phase were selected and examined.MG132 was added in treated group,but not in control group.1.The morphological features of apoptotic Hela cells induced by MG132 will be observed through inverted microscope.2.Acridine orange combine with Ethidium bromide staining: Hela cells were treated with 10μmol/L MG132. And then there were collected 48 hours later and observed after acridine orange combine with EB staining by fluorescence microscope.3.Transmission electron microscope was used to observe the morphological alteration of Hela cells treated with 10μmol/L MG132 for 48 hours.4.After treated with 10μmol/L MG132 for 48 h , Hela cells were collected and DNA was abstracted for gel electrophorosis .5. Hela cells were cultured in drug-free medium or in the presence of MG 132 at various concentrations (2.5,5,10,20μmol/L) for 48h. The apoptosis rate was detected by PI single staining flow cytometry. The apoptosis rate and alteration of cell cycle of Hela cell treated with 10μmol/L MG132 was detected at 24h,48h,72h by the same way.6.The expression of caspase-3 protein was detected by SABC of immunocytochemistry in cells treated with MG132 at different concentration for 48 h .Average IOD of single cell in each group was measured by IPP for further analyse. Results:1.The morphological features of apoptotic cells induced by MG132 including cell shrinkage, membrane blebbing and so on.2.Acridine orange combine with Ethidium bromide staining showed that most of the cells in control group appeared fluorescent green uniform.But in experement group,most cells were yellow-green fluorescence.3.DNA ladder exist in experiment group.4.The typical ultrastructure of apoptosis cell was found by electron microscope after the cells treated with MG132 : cell shrinkage, nucleus pycnosis, chromatin margination and apoptotic body can be seen.5.Flow cytometry test showed that apoptotic rates of control group and treated groups were 0.96%, 7.47%, 12.15%, 19.35%, 28.94%, among MG132 groups and the control group were statistically significant (P<0.001).There was a distinct G2/M phase arrest in groups exposure to 10μmol/L MG132,but in control groups there were no obvious change.6. Expression of caspase-3 was increased in treated groups,and the difference among control group and treated groups were statistically significant (P<0.001) .Conclusion: 1.MG132 induces the apoptosis of Hela cells in a dose and time-dependent manner. Hela cell could be arrestted at G2/M phase by in present of MG132.2. Expression of caspase-3 was gradually increased in experiment groups, caspase-3 may play an important role in the procedure of apoptosis induced by MG132.