Establishment of apoptosis model in mouse cone cell line 661W cells and the primary research on protective effects of autophagy

Objective

To establish the apoptosis model in mouse cone cell line 661W cells and to investigate the viability of 661W cells in the conditions of different levels of autophagy.

Methods

Different concentrations of anti-Fas antibody were added to establish the apoptosis model of 661W cells, the expression of caspase-3 was detected by Western blot and the appropriate concentration of anti-Fas antibody was screened.Different concentrations of the autophagy inhibitor 3-methyladenine (3-MA) or autophagy inducer rapamycin were added to inhibit or induce autophagy, the expression of microtubule-associated protein 1 light chain 3 (LC-3)Ⅱ/LC-3Ⅰwere detected by Western blot and the appropriate concentrations were also screened.The cultured cells were divided into 6 groups: control group, simple 3-MA group, simple rapamycin group, model group, model+ 3-MA group and model+ rapamycin group.Western blot was adopted to detect the expression of caspase-3, caspase-8, autophagy related genes 5 (Atg-5) and LC-3 Ⅱ/LC-3 Ⅰ at 0 hour, 3, 6, 12, 24 and 48 hours after induction.Flowcytometer was adopted to detect the apoptosis rate of 661W cells.

Results

The apoptosis model of 661W cells was successfully established, and the appropriate concentration of anti-Fas antibody was 2.0 μg/ml.After stimulated by the anti-Fas antibody, the expression of caspase-3 and caspase-8 of 661W cells increased from the time point of 6 hours, peaked at 12 hours, and sustained to 48 hours.However, the expression of Atg-5 and LC-3Ⅱ/LC-3Ⅰraised from the time point of 3 hours, peaked at 24 hours, and decreased to the basic level at 48 hours.In addition, the appropriate concentrations of 3-MA and rapamycin were 20 nmol/L and 2.0 nmol/L, respectively.There was no statistical difference among the control group, simple 3-MA group and simple rapamycin group on the expression of caspase-3 and caspase-8 and the apoptosis rate of 661W cells at different time points (all at P>0.05). The expressions of Atg-5 and LC-3Ⅱ/LC-3Ⅰin the simple rapamycin group were significantly higher than those in the control group at different time points (all at P<0.05). The expressions of caspase-3 and caspase-8 and the apoptosis rate in the model+ 3-MA group were significantlly higher than those in the model group at 3, 6, 12, 24 and 48 hours after induction, while the expressions of Atg-5 and LC-3Ⅱ/LC-3Ⅰwere obviously lower than those in the model group at 3, 6, 12 and 24 hours after induction (all at P<0.05). The expressions of caspase-3 and caspase-8 and the apoptosis rate at 6, 12, 24 and 48 hours after induction in the model+ rapamycin group were significantly lower than those in the model group, while the expressions of Atg-5 and LC-3Ⅱ/LC-3Ⅰat the time points of 3, 6, 12 and 24 hours after induction were obviously higher than those in the model group (all at P<0.05).

Conclusions

Under the condition of anti Fas antibody inducing apoptosis, enhancing autophagy can reduce the apoptosis rate of cells, inhibiting autophagy can increase the apoptosis rate.Autophagy may play a protective role in 661W cells.