A novel assay for high-throughput screening of anti-Alzheimer's disease drugs to determine their efficacy by real-time monitoring of changes in PC12 cell proliferation

Abstract

Alzheimer's disease (AD) is a neurodegenerative disease that is characterized by the accumulation of senile plaque and neurofibrilary tangle formation in the brain, including the cerebral cortex and hippocampus. Nowadays, the first-line treatment for AD is the application of acetylcholinesterase inhibitors. However, acetylcholinesterase inhibitors are basically anti-symptomatic for a limited aspect of AD pathology and are associated with serious side-effects. With the advantage of multiple targets, pathways and systems, Chinese herbal compounds hold promising potential for the development of drugs for the treatment of AD. Over the past few years, with the development of Chinese herbal compounds and in vitro pharmacological studies, cell-based disease models are one of the main methods used to screen Chinese herbal compounds for potential efficacy. Testing the efficacy of possible anti-Alzheimer's disease drugs and the development of new drugs are hindered by the lack of objective high-throughput screening methods. Currently, the assessment of the effects of drugs is usually made by MTT assays, involving laborious, subjective, low-throughput methods. Herein, we suggest a novel application for a real-time cell monitoring device (xCELLigence) that can simply and objectively assess the effective composition of Chinese herbal compounds by assessing amyloid-β peptide Aβ1-42-induced apoptosis in PC12 cells. We detected the proliferation and motility of the cells using a fully automated high-throughput and real-time system. We quantitatively assessed cell motility and determined the real-time IC50 values of various anti-AD drugs that intervene in several developmental stages of Aβ1-42-induced apoptosis in PC12 cells, Then, we identified the optimal time phase by curative efficacy. Our data indicate that this technique may aid in the discovery and development of novel anti-Alzheimer's disease drugs. It is possible to utilize a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance of 9-12. The impedance is displayed as a dimensionless para-meter termed the cell index, which is directly proportional to the total area of tissue culture well that is covered by the cells. Hence, the cell index can be used to monitor cell adhesion, spreading, morphological variation and cell density.

Figure 1

Real-time cell electronic sensing (RT-CES) system.

Figure 2

Cell quantification on the real-time cell electronic sensing (RT-CES) system. The top panels show the RT-CES growth curves of PC12 cells at different starting numbers ranging from 3,125 to 100,000. The slope of the growth curve indicates the different cell-specific growth rates. The optimal inoculation density is 50,000 cells per well.

Figure 3

Dynamic monitoring of cytotoxic response to Aβ1-42. PC12 cells were treated with Aβ1-42 at concentrations of 100, 50, 25 and 12.5 μM in serum-free medium. At the concentration of 50 μM, Aβ1-42 reduced the viability of PC12 cells to approximately 55%.

Figure 4

Toxicity of the eight fractions of Bu-Shen-Yi-Zhi prescription (BSYZ) on PC12 cells. (a–h) PC12 cells were treated with eight fractions of BSYZ at concentrations of 100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 μg/ml in serum-free medium for 48 h.

Figure 4

Toxicity of the eight fractions of Bu-Shen-Yi-Zhi prescription (BSYZ) on PC12 cells. (a–h) PC12 cells were treated with eight fractions of BSYZ at concentrations of 100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 μg/ml in serum-free medium for 48 h.

Figure 4

Toxicity of the eight fractions of Bu-Shen-Yi-Zhi prescription (BSYZ) on PC12 cells. (a–h) PC12 cells were treated with eight fractions of BSYZ at concentrations of 100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 μg/ml in serum-free medium for 48 h.

Figure 4

Toxicity of the eight fractions of Bu-Shen-Yi-Zhi prescription (BSYZ) on PC12 cells. (a–h) PC12 cells were treated with eight fractions of BSYZ at concentrations of 100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 μg/ml in serum-free medium for 48 h.

Figure 4

Toxicity of the eight fractions of Bu-Shen-Yi-Zhi prescription (BSYZ) on PC12 cells. (a–h) PC12 cells were treated with eight fractions of BSYZ at concentrations of 100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 μg/ml in serum-free medium for 48 h.

Figure 4

Toxicity of the eight fractions of Bu-Shen-Yi-Zhi prescription (BSYZ) on PC12 cells. (a–h) PC12 cells were treated with eight fractions of BSYZ at concentrations of 100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 μg/ml in serum-free medium for 48 h.

Figure 4

Toxicity of the eight fractions of Bu-Shen-Yi-Zhi prescription (BSYZ) on PC12 cells. (a–h) PC12 cells were treated with eight fractions of BSYZ at concentrations of 100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 μg/ml in serum-free medium for 48 h.

Figure 4

Toxicity of the eight fractions of Bu-Shen-Yi-Zhi prescription (BSYZ) on PC12 cells. (a–h) PC12 cells were treated with eight fractions of BSYZ at concentrations of 100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 μg/ml in serum-free medium for 48 h.

Figure 5

Protective effects of eight fractions of Bu-Shen-Yi-Zhi prescription (BSYZ) against Aβ1-42-induced toxicity in PC12 cells. BSYZ extracts at concentrations of 100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 μg/ml was tested to examine its protective effects against Aβ1-42-induced toxicity at 50 μM. The time intervals for induction were 24 h. Data are the means ± SD of triplicate experiments compared with the group treated with Aβ1-42 for 24 h.

Figure 5

Protective effects of eight fractions of Bu-Shen-Yi-Zhi prescription (BSYZ) against Aβ1-42-induced toxicity in PC12 cells. BSYZ extracts at concentrations of 100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 μg/ml was tested to examine its protective effects against Aβ1-42-induced toxicity at 50 μM. The time intervals for induction were 24 h. Data are the means ± SD of triplicate experiments compared with the group treated with Aβ1-42 for 24 h.

Figure 5

Protective effects of eight fractions of Bu-Shen-Yi-Zhi prescription (BSYZ) against Aβ1-42-induced toxicity in PC12 cells. BSYZ extracts at concentrations of 100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 μg/ml was tested to examine its protective effects against Aβ1-42-induced toxicity at 50 μM. The time intervals for induction were 24 h. Data are the means ± SD of triplicate experiments compared with the group treated with Aβ1-42 for 24 h.

Figure 5

Protective effects of eight fractions of Bu-Shen-Yi-Zhi prescription (BSYZ) against Aβ1-42-induced toxicity in PC12 cells. BSYZ extracts at concentrations of 100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 μg/ml was tested to examine its protective effects against Aβ1-42-induced toxicity at 50 μM. The time intervals for induction were 24 h. Data are the means ± SD of triplicate experiments compared with the group treated with Aβ1-42 for 24 h.

Figure 5

Protective effects of eight fractions of Bu-Shen-Yi-Zhi prescription (BSYZ) against Aβ1-42-induced toxicity in PC12 cells. BSYZ extracts at concentrations of 100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 μg/ml was tested to examine its protective effects against Aβ1-42-induced toxicity at 50 μM. The time intervals for induction were 24 h. Data are the means ± SD of triplicate experiments compared with the group treated with Aβ1-42 for 24 h.

Figure 5

Protective effects of eight fractions of Bu-Shen-Yi-Zhi prescription (BSYZ) against Aβ1-42-induced toxicity in PC12 cells. BSYZ extracts at concentrations of 100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 μg/ml was tested to examine its protective effects against Aβ1-42-induced toxicity at 50 μM. The time intervals for induction were 24 h. Data are the means ± SD of triplicate experiments compared with the group treated with Aβ1-42 for 24 h.

Figure 5

Protective effects of eight fractions of Bu-Shen-Yi-Zhi prescription (BSYZ) against Aβ1-42-induced toxicity in PC12 cells. BSYZ extracts at concentrations of 100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 μg/ml was tested to examine its protective effects against Aβ1-42-induced toxicity at 50 μM. The time intervals for induction were 24 h. Data are the means ± SD of triplicate experiments compared with the group treated with Aβ1-42 for 24 h.

Figure 5

Protective effects of eight fractions of Bu-Shen-Yi-Zhi prescription (BSYZ) against Aβ1-42-induced toxicity in PC12 cells. BSYZ extracts at concentrations of 100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 μg/ml was tested to examine its protective effects against Aβ1-42-induced toxicity at 50 μM. The time intervals for induction were 24 h. Data are the means ± SD of triplicate experiments compared with the group treated with Aβ1-42 for 24 h.