Upregulation of Acetylcholinesterase Mediated by p53 Contributes to Cisplatin-Induced Apoptosis in Human Breast Cancer Cell

Abstract

Background: The expression of acetylcholinesterase (AChE) could be induced during apoptosis in various cell types. And reduced AChE expression either by siRNA could prevent apoptosis. However, the detailed mechanisms underlying the AChE regulation are largely unknown in human breast cancer cell.

Material and methods: MCF-7 cells were cultured and treated by cisplatin in the absence or presence of p53 siRNA.

Results: In this study, the regulation of AChE expression during apoptosis induced by cisplatin, a current used anticancer drug, was investigated in human breast cancer cell line MCF-7. Exposure of MCF-7 cells to cisplatin resulted in apoptosis in a time- and concentration-dependent manner. Meanwhile, the upregulated AChE and p53 were also observed during apoptosis. Silencing interfering RNA directed against p53 blocked the expression of AChE.

Conclusion: Taken together, these results suggested that AChE expression could be upregulated by the activation of p53 during apoptosis induced by cisplatin in MCF-7 cells.

Keywords: acetylcholinesterase; apoptosis; breast cancer.; cisplatin; p53.

Figure 1

Cisplatin induces cell death in MCF-7 cells. (A) Cisplatin induces cell death in a concentration-dependent manner. MCF-7 cells were exposed to cisplatin at concentration as indicated for 24 h, and then cell viability was measured by MTT assay. (B) Cisplatin induces cell death in a time-dependent manner. MCF-7 cells were exposed to 100 μM cisplatin for different times as indicated, and then cell viability was measured by MTT assay. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments, **p < 0.01 versus control group (unpaired t test).

Figure 2

Cisplatin up-regulates the expression of both AChE and p53. MCF-7 cells were treated with 100 μM cisplatin for different time as indicated, and the total proteins were extracted for the western blot assay with the specific AChE, p53 and β-actin antibodies.

Figure 3

The expression of AChE is increased in the apoptosis cell induced by cisplatin. (A) MCF-7 cells were treated with 100 μM cisplatin for different time as indicated. And then AChE immunostaining and Hoechst staining were applied in the cells. (B) Inhibition of p53 or AChE expression attenuates the apoptosis induced by cisplatin. MCF-7 cells were treated with 100μM cisplatin for 24 h in the absence or presence of p53 or AChE siRNA. Then cells were conducted by Hoechst 33342 staining assay. The amount of apoptotic nuclei with condensed chromatin were counted from representative photomicrographs and were represented as a percentage of the total number of nuclei counted. Each treatment group was compared with the other groups using unpaired t test.

Figure 4

Silencing of p53 by siRNA blocks cisplatin-induced AChE expression. MCF-7 cells were treated with100 μM cisplatin for 24 h in the absence or presence of p53 siRNA. Then the total proteins were extracted for the western blot assay with the specific AChE, p53 and β-actin antibodies.

Figure 5

Silencing of p53 by siRNA blocks the expression of AChE induced by cisplatin in the nuclear but not in cytoplasm. MCF-7 cells were treated with100 μM cisplatin for 24 h in the absence or presence of p53 siRNA. Then the proteins either from nuclear or from cytoplasm were extracted for the western blot assay with the specific AChE, β-actin and Histone H2A antibodies.