Background Occupational exposure to methanol can cause damage to the central nervous system. Studies have shown that calpain can convert p35 into p25, activating calcium-dependent protein kinase 5 (CDK5) and leading to the accumulation and phosphorylation of p53 and the apoptosis of nerve cells. It is unclear whether methanol can activate calpain, initiate the p25-CDK5-p53 signaling pathway, and cause neuronal apoptosis through the classical mitochondrial pathway.
Objective This in vitro experiment is designed to investigate the role of p25-CDK5-p53 signaling pathway in the apoptosis of human neuroblastoma cells (SK-N-SH cells) induced by methanol.
Methods SK-N-SH cells were exposed to 0, 250, 750, and 1 250 mmol/L methanol (named control, low, medium, and high concentration groups, respectively) for 24 h. The apoptosis rate was detected with Annexin V-FITC/PI apoptosis kit. The relative mRNA expression levels of calpain2, CDK5, p35, p25, and p53 were measured by real-time fluorescence quantitative PCR.The protein expression levels of calpain2, CDK5, p25, p35, p53, and phosphorylated p53 were determined by Western blotting.
Results After 24 h of methanol exposure, the apoptosis rates of the control, low, medium, and high concentration groups were (8.15±1.77)%, (12.06±0.71)%, (13.81±0.67)%, and (19.88±2.40)%, respectively, and the difference among the four groups was statistically significant (F=29.05, P < 0.01). The difference of relative mRNA expression levels of calpain2, CDK5, p25, and p53 among the groups were statistically significant (F=10.02, 61.48, 28.91, and 21.12, respectively, P < 0.01), except the relative mRNA expression level of p35 (F=0.96, P>0.05). Compared with the control group, the relative mRNA expression levels of calpain2, CDK5, and p25 in the low and medium methanol concentration groups were not significantly different (P>0.05), the relative mRNA expression level of p53 in the medium methanol concentration group was increased (P < 0.01), and the relative mRNA expression levels of calpain2, CDK5, p25, and p53 in the high concentration group were increased (P < 0.01). The relative protein expression levels of calpain2, CDK5, p35, p25, phosphorylated p53, and p53 were significantly different among the designed groups (F=32.29, 17.60, 35.02, 35.92, 93.72, and 11.00, respectively, P < 0.05). Compared with the control group, the relative protein expression levels of calpain2, CDK5, p35, p25, and phosphorylated p53 were increased in the low, medium, and high concentration groups (P < 0.05), the relative protein expression levels of p53 were increased in the medium and high concentration groups (P < 0.05), and there was no significant difference in the relative protein expression level of p53 between the low concentration group and the control group (P>0.05).
Conclusion Methanol exposure may increase the expression levels of calpain2, p35, p25, and CDK5 proteins, induce p53 protein accumulation and phosphorylation, and result in the apoptosis of SK-N-SH cells.
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