Protective effects of proanthocyanidins on beta-amyloid peptide (25-35)-induced PC12 cell apoptosis by blocking S-phase and increasing p53 gene expression*☆
Hanfang Mei1, 2, Zhaoyang Xie3, Qifeng Zhu3

1Department of Biochemistry and Molecular Biology, Guangdong Pharmaceutical University, Guangzhou  510006, Guangdong Province, China
2School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou  510515, Guangdong Province, China
3Institute of Biochemistry and Molecular Biology, Guangdong Medical College, Zhanjiang  524023, Guangdong Province, China

Hanfang Mei☆, Studying for doctorate, Lecturer, Department of Biochemistry and Molecular Biology, Guangdong Pharmaceutical University, Guangzhou  510006, Guangdong Province, China; School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou  510515, Guangdong Province, China

Corresponding author: Qifeng Zhu, Professor, Master's supervisor, Institute of Biochemistry and Molecular Biology, Guangdong Medical College, Zhanjiang  524023, Guangdong Province, China
zhuqifeng3@163.com

Supported by: Key Discipline Key Projects in Guangdong Province (9808)*

Abstract
BACKGROUND: Current studies related to the effects of proanthocyanidins on Alzheimer’s disease have focused primarily on the signal transduction pathway of cellular apoptosis. However, the in-fluence of p53 gene expression on cell cycle regulation, with regard to the protective mechanisms of proanthocyanidins, has not been reported.
OBJECTIVE: To observe the effect of proanthocyanidins on cell cycle distribution, cellular apoptosis, and p53 gene expression in β-amyloid peptide (25-35) (Aβ25-35)-induced PC12 cells cultured in se-rum-free media, and to investigate the molecular neuroprotective mechanisms of proanthocyanidins with regard to cell cycle regulation.
DESIGN, TIME AND SETTING: A parallel, controlled, cellular, and molecular study was performed at the Institute of Biochemistry and Molecular Biology, Guangdong Medical College from July 2006 to July 2008.
MATERIALS: Proanthocyanidins were provided by Nanjing Xuezi Medical and Chemical Research Center, China; Aβ25-35 was provided by Sigma, USA; PC12 cells were provided by the Institute of Basic Medical Science, Academy of Military Medical Sciences; and rabbit anti-p53 polyclonal anti-body was provided by Santa Cruz Biotechnology, USA.
METHODS: PC12 cells were cultured in serum-free media for 24 hours. Cells from the model group were treated with 25 μmol/L Aβ25-35 for 24 hours. Cells in the drug protection group were pre-treated with 30 mg/L proanthocyanidins for 1 hour and then treated with 25 μmol/L Aβ25-35 for 24 hours. The control group was not treated.
MAIN OUTCOME MEASURES: Flow cytometry was used to detect cell cycle distribution and rate of apoptosis; reverse-transcriptase polymerase chain reaction was used to detect p53 mRNA ex-pression; and Western blot was used to detect p53 protein expression.
RESULTS: After treating with 25 μmol/L Aβ25-35 for 24 hours, the rate of apoptosis and the per-centage of cells in S phase were significantly increased (P < 0.01), and p53 mRNA and protein ex-pressions were decreased. Pretreatment with proanthocyanidins for 1 hour blocked the increase in apoptosis and the percentage of cells in S phase in Aβ25-35-induced PC12 cells (P < 0.01) and in-creased p53 mRNA and protein expressions.
CONCLUSION: Proanthocyanidins blocked apoptosis and S-phase arrest in Aβ25-35-induced PC12 cells cultured in serum-free media. The protective mechanism could be related to increased p53 mRNA and protein expressions.
Key Words: proanthocyanidins; β-amyloid peptide (25-35); Alzheimer’s disease; PC12 cells; p53 gene; neural regeneration

INTRODUCTION
  
The pathogenesis of neuronal mitotic signal activation in Alzheimer’s disease is currently receiving much attention. Expression of cell cycle-related molecules, such as proliferating cell nuclear antigen (PCNA), cyclin D, cyclin E, cyclin B, CDK4, p16, and p21, are increased in Alzheimer’s disease patients[1], suggesting that the cell cycle could be involved in the pathogenesis of Alzheimer’s disease.
In 1999, Copani et al[2] demonstrated that β-amyloid peptide (25-35) (Aβ25-35) induces cell cycle S-phase arrest in primary cultured rat cortical neurons. However, inactivation of cyclin-dependent protein kinase 4 or 2, which are cell cycle regulation molecules, inhibits S-phase arrest and apoptosis induced by Aβ25-35. These results suggest that cell cycle signal transduction is related to cellular apoptosis. The majority of studies have indicated that cell cycle-related incidents in Alzheimer’s disease patients occur prior to the formation of typical pathological changes, such as senile plaques. This suggests that damaged neurons attempting to re-enter cell cycle is an early manifestation of neuronal stress[3]. Therefore, the development of effective drugs to inhibit neuronal re-entry into the cell cycle could potentially control Alzheimer’s disease during the early phase.
Proanthocyanidins are a type of polyphenol, antioxidant compound widely produced by plants. Results from our laboratory have shown that proanthocyanidins have a significant protective effect on Aβ25-35-induced apoptosis in PC12 cells, based on levels of apoptosis-related molecules, such as bax and bcl-2[4]. The present apoptosis-related study further explored the neuroprotective mechanisms of proanthocyanidins from the perspective of cell cycle regulation.
p53 is involved in the regulation of apoptosis[5], but also plays a role in cell cycle[6-7]. This study hypothesized that proanthocyanidins provide neuroprotection by regulating p53 gene expression, thereby influencing the cell cycle distribution of neurons in each phase.
To test this hypothesis, our experiments utilized PC12 cells cultured in serum-free medium so that cell cycle was synchronized to the G1/G0 phase to simulate a neuronal in vivo state. The study induced an Alzheimer’s disease cellular model via exposure to Aβ25-35 and measured cell cycle distribution, cellular apoptosis, and p53 gene and protein expressions with or without intervention of proanthocyanidins.

MATERIALS AND METHODS

Design
A parallel, controlled, cellular, and molecular study.
Time and setting
This study was performed at the Institute of Biochemistry and Molecular Biology, Guangdong Medical College from July 2006 to July 2008.
Materials
Proanthocyanidins were provided by Nanjing Xuezi Medical and Chemical Research Center, with a purity > 95%. Proanthocyanidins were dissolved in a DMSO concentration less than 0.1% (V/V), which did not affect PC12 cell growth. Aβ25-35 was provided by Sigma, USA and was dissolved in PBS (pH 7.4). PC12 cells were provided by the Institute of Basic Medical Science Academy of Military Medical Sciences.
Instruments and reagents are as follows: 

Methods
Cell culture
PC12 cells were cultured in DMEM containing 10% heat-inactivated horse serum and 5% fetal bovine serum with 100 U/mL penicillin and 100 μg/mL streptomycin in a 5% CO2 incubator at 37 °C and saturated humidity.
Grouping and intervention
PC12 cells were plated at a density of 2 × 108/L in 6-well plates, 2 mL per well, cultured for 24 hours, and then incubated in serum-free culture media for another 24 hours.
The model group was exposed for 24 hours to a final concentration of 25 μmol/L Aβ25-35[8].
The drug protection group was preconditioned with a final concentration of 30 mg/L proanthocyanidins for    1 hour prior to co-culture with a final concentration of  25 μmol/L Aβ25-35 for 24 hours[9].
The control group was cultured at the same time, but was not treated with any drugs.
Cellular apoptosis and cell cycle distribution, as detected by FCM
Cells from each group were collected into 1.5 mL Eppendorf tubes, washed twice with phosphate-buffered saline (PBS), centrifuged at 800 r/min for 10 minutes, and fixed with 70% ice-cold ethanol for 24 hours. Cells were then centrifuged at 1 200 r/min for 2 minutes to remove the ethanol, washed once with PBS, adjusted to 500 μL PBS to resuspend cells, and incubated in propidium iodide (50 mg/L) in the dark for 30 minutes, followed by infiltration prior to FCM analysis[10]. A total of 10 000 cells from each sample were measured, and an average was obtained following three replicated measurements.
p53 mRNA expression, as determined by RT-PCR
Cells were harvested from each group to extract total RNA. An ultraviolet spectrophotometer was used to measure A260/A280, which was between 1.7–2.0. RNA concentration was calculated, cDNA was synthesized using reverse transcription, and the transcripts were amplified. GAPDH was used as an internal reference.
The primer sequences are as follows:

Reaction parameters are as follows. p53: pre-denaturation at 95 °C for 5 minutes, denaturation at 94 °C for 1 minute, annealing at 50 °C for 45 seconds, and extension at 72 °C for 1 minute for 30 cycles in total, followed by extension at 72 °C for 10 minutes. GAPDH: pre-denaturation at 95 °C for 5 minutes, denaturation at 94 °C for 1 minute, annealing at 59.1 °C for 45 seconds, and extension at 72 °C for 1 minute for 30 cycles in total, followed by extension at 72 °C for 10 minutes. PCR products were detected by 2% agarose gel electrophoresis[11]. The experiment was repeated three times.
p53 protein expression, as determined by Western blot
Cells were harvested from each group for total protein extraction, which was measured using the Bradford method[12]. A total of 50 μg protein from each sample was fractionated by 12% SDS-PAGE, transferred to PVDF membrane, blocked, incubated in primary antibody (rabbit anti-p53 polyclonal antibody 1: 500, goat anti-β-actin polyclonal antibody 1: 500) for 2 hours, washed, and incubated with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG 1: 4 000, rat anti-goat IgG 1: 5 000) for 1.5 hours at room temperature. The samples were then washed, incubated with enhanced chemiluminescence for 1 minute, and visualized on X-ray films[10]. The experiment was repeated three times.
Main outcome measures
Cellular apoptosis, cell cycle distribution, and p53 mRNA and protein expression.
Statistical analysis
SPSS 10.0 for Windows statistics software package (SPSS, Chicago, IL, USA) was employed for statistical analysis. The experimental results were expressed as Mean ± SD and compared among groups using one-way variance analysis, F, and Bonferroni or Tamhane’s T2 test. A P value < 0.05 was considered statistically significant.

RESULTS

Results of FCM analysis 
Compared with the control group, the percentage of cells in S phase and the rate of apoptosis were significantly increased in the model group (P < 0.01). Compared with the model group, the percentage of cells in S phase and the rate of apoptosis significantly decreased in the drug protection group (P < 0.01) (Figure 1, Table 1).

p53 mRNA expression in each group
Results from RT-PCR revealed significantly decreased p53 mRNA expression in the model group, compared with the control group. However, treatment with proanthocyanidins blocked the decrease in p53 mRNA expression (Figure 2).

p53 protein expression in each group
Results from Western blot revealed decreased p53 protein expression in the model group, compared with the control group. However, treatment with proanthocyanidins blocked the reduction in p53 protein expression (Figure 3).

DISCUSSION

In recent years, studies have shown that nanomolar concentrations of soluble Aβ polymers can induce hippocampal neuronal death[13]. Aβ includes Aβ1-42/43 and Aβ1-40, as well as the polypeptide between 25 and 35, which includes the Gly-Ala-Ile-Ile-Gly-Leu-Met (GAIIGLM) sequence, a hydrophobic peptide termed Aβ25-35. The Aβ25-35 molecule is stably assembled in aqueous solution, and this peptide assembling is directly related to its neurotoxicity. Aβ25-35 has been commonly used to establish in vitro models of neurotoxicity for the study of Alzheimer’s disease[14-20].
PC12 cells are derived from a cell line established from a transplantable rat adrenal pheochromocytoma[21].  When cultured under normal conditions, these cells extend branches and exhibit morphological features similar to neurons. PC12 cells have been widely used for in vitro neurobiology studies, including Alzheimer’s disease-related research, due to ease of culture and homogeneity[4, 14-15].
The present study utilized an in vitro PC12 cell model. Initially, serum-free culture methods were used to synchronize PC12 cell cycle to the G1/G0 phase. This was used to simulate a state of neuronal terminal differentiation. Subsequently, the cells were co-cultured in 25 μmol/L Aβ25-35 for another 24 hours, resulting in a higher apoptotic rate and a greater percentage of cells in the S phase (compared with the control group), which suggested that PC12 apoptosis and cell cycle arrest in the S phase took place at the same time. These results were consistent with previously reported results[2].
Proanthocyanidins’ antioxidant activity is 20 times greater than vitamin E and 50 times greater than vitamin C[22]. Previous studies have shown that the natural grape seed polyphenol extract (proanthocyanidin) inhibits Aβ aggregation in vitro, reduces cognitive impairment in Alzheimer’s disease model mice (Tg2576 mice), and plays a role in the prevention and treatment of Alzheimer’s disease[23]. The present study analyzed the neuroprotective effects of proanthocyanidins from the perspective of cell cycle regulation. Results demonstrated that 30 mg/L proanthocyanidins pretreatment for 1 hour resulted in a decreased rate of apoptosis, as well as decreased percentage of Aβ25-35-induced PC12 cells in S phase. These results suggested that proanthocyanidins blocking of S-phase arrest played a role in the protective effect against cellular apoptosis. It is highly probably that proanthocyanidins protected cells cultured in serum-free medium from Aβ25-35-induced damage by inhibiting cell cycle re-entry. However, the exact molecular targets require further study.
p53 is an important cell cycle regulatory protein, which can arrest cell cycle in the G1 or G2 phase when stimulated by a signal, such as DNA damage, to allow for repair and subsequent mitosis[24]. When DNA injury is serious enough that a cell cannot repair by itself, p53 can promote cell apoptosis[25]. Results from the present study demonstrated that Aβ25-35 induced S phase arrest in PC12 cells cultured in serum-free medium, as well as decreased p53 mRNA and protein expression. Proanthocyanidins preconditioning blocked S-phase arrest and promoted p53 mRNA and protein expression, suggesting that proanthocyanidins reversed cell cycle re-entry by promoting p53 mRNA expression.
The present study was not able to identify the precise signal transduction pathway involved in PC12 cell cycle re-entry under serum-free conditions or the mechanisms of proanthocyanidins involved in blocking the above-mentioned phenomena. In addition, p53 mRNA and protein expressions were not quantified in the present study.
In summary, proanthocyanidins preconditioning protected PC12 cells cultured in serum-free medium from Aβ25-35-induced apoptosis by blocking S-phase cell cycle arrest and promoting p53 gene expression.

Acknowledgments
PC12 cell was a generous gift from Dr. Ma, Institute of Basic Medical Science Academy of Military Medical Sciences.

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 (Edited by Li SJ/Ji H/Song LP)