Antioxidant and Antiapoptotic Effects of 1,1′-(Biphenyl-4,4′-diyl)-bis

Article pubs.acs.org/molecularpharmaceutics

Antioxidant and Antiapoptotic Effects of 1,1′-(Biphenyl-4,4′-diyl)bis(3-(dimethylamino)-propan-1-one) on Protecting PC12 Cells from Aβ-Induced Injury Yuxuan Zhu, Xun Sun, Tao Gong, Qin He, and Zhirong Zhang* Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Southern Renmin Road, No. 17, Section 3, Chengdu 610041, P. R. China S Supporting Information *

ABSTRACT: Abnormal extracellular deposition of β-amyloid (Aβ) is thought to play a key role in the pathogenesis of Alzheimer’s disease (AD). Preventing Aβ-induced neurotoxicity has become a potential therapeutic approach to improve the onset and progression of AD. Here we report the synthesis of 1,1′-(biphenyl-4,4′-diyl)-bis(3-(dimethylamino)-propan-1-one) (BDBDP) and evaluate whether it protects PC12 cells from Aβ1−42-induced cytotoxicity in PC12 cells. Treating cells with Aβ1−42 significantly reduced cell viability and mitochondrial membrane potential while also significantly increasing apoptosis and production of reactive oxygen species (ROS). Pretreating the cells with BDBDP significantly ameloriated these Aβ1−42-induced effects. Futhermore, BDBDP strongly reduced proapoptotic signaling in response to ROS by reducing levels of activated caspase-3 and increasing the ratio of Bcl-2 to Bax. These findings provide evidence that BDBDP protects against Aβ1−42-induced neurotoxicity in PC12 cells by inhibiting oxidative stress and cell apoptosis. KEYWORDS: Aβ1−42, PC12 cell, neuroprotection, oxidative stress, apoptosis

■

proposed, including anti-Aβ antibodies,15 inhibitors of the βand γ-secretases that generate Aβ from APP,16 antioxidants,17 anti-inflammatory agents,18 and estrogens.19 However, the clinical usefulness of all these treatments is severely limited by one or more factors such as stability, cost, and safety.20 Thus, identifying safer and more effective drugs for treating AD remains an important challenge in drug discovery. Extensive research into synthetic drugs that can ameloriate Aβ-induced cytotoxicity in vitro has provided valuable information about what it takes to make an effective drug against AD. It is reported that bimolecular 3-piperidyl-ethyl phenyl ketone can remarkably improve aphronesia caused by scopolamine and sodium nitrite and can enhance the ability of learning and memory.21 Most medicines that manage AD well are tertiary amine salts or quaternary ammonium salts, such as galantamine and donepezil.22,23 Moreover, numerous studies have found that N-methylated compounds are effective at preventing Aβ-induced cytotoxicity in vitro.20,24−30 In addition, two N-methylation compounds can even reverse Aβ-induced inhibition of long-term potentiation at extremely low concentrations.20,27 At the same time, the acetophenone structure has a close relationship with oxidative stress.31

INTRODUCTION Alzheimer’s disease (AD) is a neurodegenerative disease characterized by loss of cognitive ability in the elderly.1−5 The lack of effective treatments make AD a devastating illness not only for the patient but also for his or her family. The major neuropathological characteristics of AD are neurofibrillary tangles and neuronal loss due to apoptosis and senile plaques formed by pathological deposition of β-amyloid protein (Aβ), a peptide of 39−43 amino acids processed from amyloid precursor protein (APP). Deposition of Aβ in the brain is thought to play an important role in the development of AD,1,6 yet Aβ is a natural product of cellular metabolism potentially involved in various physiological activities, such as cell survival and synaptic activity.7,8 How an otherwise normal peptide can become pathological is unclear. Studies point to changes in Aβ1−42 concentration and physicochemical properties as potential triggers in this transition.7,9 How Aβ induces neurotoxicity is an area of active investigation, and several studies suggest that abnormal extracellular Aβ deposition induces oxidative stress and triggers several pro-apoptotic signaling cascades, leading to the progressive degeneration of cognitive function.10−13 Therefore, numerous studies have investigated the ability of antioxidant and antiapoptotic drugs to attenuate AD. Two types of drugs are currently used in the clinic to delay cognitive decline in AD patients: acetylcholinesterase inhibitors and glutamate modulators.13,14 Several alternative approaches have also been © 2013 American Chemical Society

Received: Revised: Accepted: Published: 428

July 9, 2013 December 17, 2013 December 18, 2013 December 18, 2013 dx.doi.org/10.1021/mp400395g | Mol. Pharmaceutics 2014, 11, 428−435

Molecular Pharmaceutics

Article

NMR (400 MHz, CDCl3): δ 8.07 (d, J = 8.4 Hz, 4 H), 7.73 (d, J = 8.4 Hz, 4 H), 3.20 (t, J = 7.2 Hz, 4 H), 2.79 (t, J = 7.2 Hz, 4 H), 2.31 (s, 12 H) (Figure S2, Supporting Information). 13C NMR (100 MHz, CDCl3): δ 37.0, 45.5, 54.3, 127.4, 128.7, 136.3, 144.2 (Figure S3, Supporting Information), 198.5. ESIHRMS (m/z): 353.22 [M + H]+ (Figure S4, Supporting Information). Cell Culture. Undifferentiated PC12 cells from the American Type Culture Collection (Rockville, MD, USA) were maintained in high-glucose DMEM supplemented with 5% FBS, 10% HS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C under a humidified atmosphere of 5% CO2. Cells seeded on poly D-lysine-coated plates were treated with 5 ng/mL NGF in DMEM containing 1% HS and 0.5% FBS, and the medium was changed every other day. Preparation of Aβ1−42 Aggregates. Aβ1−42 aggregates were prepared as described11 and analyzed by transmission electron microscopy (TecnaiG2 F-20, FEI, Holland). Aβ1−42 samples were applied onto glow-discharged copper grids and stained with 1% uranyl acetate.35 Cell Viability and LDH Release Assays. Cell viability was measured by quantitative MTT colorimetric assay as described previously.36 Briefly, PC12 cells were plated in 96-well culture plates at a density of 4 × 104 cells/well and incubated for 24 h at 37 °C for viability determination. Then the cells were treated with BDBDP at different concentrations (0, 0.1, 0.3, and 1 μg/ mL) for 12 h, after which 100 μM Aβ1−42 was added and the cells were incubated another 12 h. MTT solution (20 μL, 5 mg/mL) was added to each well and incubated at 37 °C for 4 h. The supernatants were removed, and formazan crystals were dissolved in 150 μL of DMSO. Cell viability was measured by measuring absorbance at 570 nm in a plate reader (Thermo, Varioskan Flash). The release of LDH activity was measured as an in vitro indicator of cellular toxicity. LDH activity was measured using a commercial assay kit according to the manufacturer’s instructions. In brief, 6-well plates were seeded at 4 × 105 cells/well, the cells were preincubated with BDBDP at different concentrations (0, 0.1, 0.3, and 1 μg/mL) for 12 h, and then 100 μM Aβ1−42 was added and the cells were incubated another 12 h. Aliquots (100 μL) of culture medium were used to measure extracellular LDH activity. Cells were washed with phosphate-buffered saline (PBS), scraped into 500 μL of PBS, and homogenized. The homogenate was centrifuged at 4000g for 30 min at 4 °C. Aliquots (100 μL) of the resulting supernatants were used to measure intracellular LDH activity. LDH release was quantified using the following equation:

According to these insights and our previous work, which suggest N-methylated structures may play an important role in transport across the blood−brain barrier (BBB),32 we designed and synthesized 1,1′-(biphenyl-4,4′-diyl)-bis(3-(dimethylamino)-propan-1-one (BDBDP) as a drug candidate. We have examined the ability of BDBDP to protect against Aβ-induced neurotoxicity in PC12 cells and performed initial experiments to explore the mechanism of that activity.

■

MATERIALS AND METHODS The reagents 1-(4-bromophenyl) ethanone, Pd(OAc)2, PEG 4000, paraformaldehyde, and dimethylamine hydrochloride were purchased from J&K Tech. Ltd. (Beijing, China). Dulbecco’s Modified Eagle medium (DMEM), neuron growth factor (NGF), fetal bovine serum (FBS), horse serum (HS), 6carboxy-2′-7′-dichlorofluorescein diacetate (DCFH-DA), Aβ1−42, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Inc. (St Louis, MO, USA). Kits to assay malonyldialdehyde (MDA) and lactate dehydrogenase (LDH) were purchased from Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). FragEL DNA Fragmentation Detection Kit was purchased from Calbiochem-Merck (Germany). Kits to assay reactive oxygen species (ROS) and mitochondrial membrane potential with JC-1 were purchased from the Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). Polyclonal antibodies against Bax (#2772), Bcl-2 (#2876s), and cleaved caspase-3 (#9661) were obtained from Cell Signaling Technology (Danvers, MA, USA). β-actin specific antibody (bs-0061R) was purchased from Bioss (Beijing, China). Synthesis of 1,1′-(Biphenyl-4,4′-diyl)-bis(3-(dimethylamino)-propan-1-one). Synthesis of 1,1′-(Biphenyl-4,4′diyl) Diethanone. The following components were mixed in a 50 mL round-bottom flask fitted with a magnetic stirring bar: 1(4-bromophenyl) ethanone (796 mg, 4 mmol), Pd(OAc)2 (11 mg, 0.05 mmol), K2CO3 (552 mg, 4 mmol), and PEG 4000 (4 g). The mixture was degassed three times by alternately connecting to a vacuum and an N2 supply. Then the contents were heated at 120 °C under N2 for 12 h. After cooling, the reaction mixture was poured into 250 mL of ethyl ether with vigorous agitation, and the organics were removed under reduced pressure, leaving behind a residue of 1,1′-(biphenyl4,4′-diyl) diethanon. This residue was purified by chromatography on silica gel (AcOET/PE, 20:80).33 Analysis by 1H NMR on a Bruker AMX-400 spectrometer and high-resolution mass spectrometry (HRMS) on a Bruker microTOF-QII gave the following results: 1H NMR (400 MHz, CDCl3), δ 8.07 (d, J = 8.4 Hz, 4 H), 7.73 (d, J = 8.4 Hz, 4 H), 2.66 (s, 12 H) (Figure S1, Supporting Information). Synthesis of BDBDP. Paraformaldehyde (120 mg, 4 mmol), dimethylamine hydrochloride (984 mg, 12 mmol), and 1,1′(biphenyl-4,4′-diyl) diethanone (238 mg, 1 mmol) were dissolved in ethanol (1 mL). Hydrochloric acid (35%, 0.05 mL) was added, and the mixture was refluxed for 8 h. The yellowish solution was diluted with cold acetone (50 mL) and chilled for two hours at 0 °C. The crystals were filtered, washed with acetone (2 × 20 mL), dissolved in water (20 mL), and then extracted in ethyl acetate (2 × 35 mL). The aqueous layer was treated with potassium carbonate (pH 10), and 1,1′(biphenyl-4,4′- diyl)-bis(3-(dimethylamino)-propan-1-one) was obtained as precipitate.34 The compound was analyzed by 1H and 13C NMR using a Bruker AMX-400 spectrometer, and HRMS was carried out on a Bruker microTOF-QII. 1H

LDH release rate (%) LDH activity in culture medium = × 100% LDH activity in culture medium + LDH activity in cells

TUNEL Staining. The terminal deoxynucleotidyl transferase nick end-labeling (TUNEL) assay was used to measure the extent of DNA nick formation and genomic DNA fragmentation. Briefly, PC12 cells were seeded on coverslips and treated with 1 μg/mL BDBDP followed by Aβ1−42 as described for the cell viability assays, the cells were washed with PBS three times. Then the cells were fixed in 4% paraformaldehyde for 10 min. Endogenous peroxidase was quenched by incubating the cells with 100 μL of 3% H2O2 for 5 min at room temperature, and the cells were permeabilized with 0.1% Triton X-100 in 0.1% sodium acetate for 5 min at 4 °C. The cells were labeled with the TUNEL reaction mixture for 60 min at 37 °C, then with 429

dx.doi.org/10.1021/mp400395g | Mol. Pharmaceutics 2014, 11, 428−435

Molecular Pharmaceutics

Article

1:1000), and again washed three times in TBST. Membranes were treated for chemiluminescence and scanned using a BioRad Gel Doc XR+ Molecular Imager. The grayscale intensity of bands was quantitated using Gel EQ Quantity One software (Bio-Rad). Data Analysis. Results were expressed as means ± SD. The significance of observed differences between groups was assessed by one-way ANOVA followed by Student’s t test. The threshold for significance was P < 0.05.

peroxidase-conjugated antifluorescein antibody for another 30 min. Subsequently, cells were incubated with diaminobenzidine (DAB) substrate to produce dark brown precipitate. Lipid Peroxidation Assay. The level of MDA, an index of lipid peroxidation, was measured using a commercial assay kit according to the manufacturer’s instructions. In brief, PC12 cells were seeded in 6-well culture dishes (106 per well) and incubated with BDBDP and Aβ1−42 as described for the cell viability assays. The cells were washed three times with PBS, then scraped from the plates into 0.5 mL of 0.1 M PBS containing 0.05 mM EDTA and homogenized. The homogenate was centrifuged at 4000g for 10 min at 4 °C. Aliquots (100 μL) of the resulting supernatants were used to measure the level of MDA. These levels were normalized to the protein concentration in each sample and expressed as a percentage of the level in the control samples. ROS Assay. ROS was quantified using the DCFH-DA method. Accumulation of intracellular ROS can be detected using DCFH-DA, which crosses cell membranes and is hydrolyzed into nonfluorescent DCFH by intracellular esterases. In the presence of ROS, DCFH is oxidized to highly fluorescent dichlorofluorescein (DCF), which is readily detectable by fluorescence microscopy and flow cytometry. Briefly, PC12 cells were seeded in 6-well culture dishes (106 per well) and incubated with BDBDP and Aβ1−42 as described for the cell viability assays. The cells were washed with PBS and incubated with DCFH-DA at a final concentration of 10 μM for 30 min at 37 °C in the dark. After the cells were washed three times with PBS to remove extracellular DCFH-DA, the cells were visualized by fluorescence microscopy (Carl Zeiss Shanghai Co., Ltd.), and DCF fluorescence was measured by flow cytometry (Beckman). These fluorescence intensities served as a measure of the levels of intracellular ROS and were expressed as percentages of the intensities in untreated control samples. Mitochondrial Membrane Potential Measurements. Mitochondrial membrane potential was measured using the fluorescent dye JC-1 (Molecular Probes). PC12 cells were seeded in 6-well culture dishes (106 per well) and incubated with BDBDP and Aβ1−42 as described for the cell viability assays. The cells were incubated with JC-1 for 30 min, after which cells were washed twice with PBS. The washed cells were analyzed by flow cytometry (Beckman), and the ratio of red to green signal was calculated. Western Blotting. Western blot analysis was used to assess levels of Bcl-2, Bax, and cleaved caspase-3 protein. PC12 cells were seeded in culture dishes (4 × 106) and incubated with BDBDP and Aβ1−42 as described for the cell viability assays. The cells were washed three times with cold PBS and treated with 1 mL of RIPA lysis buffer containing phenylmethylsulfonyl fluoride. Supernatants were recovered by centrifugation at 13 500g for 15 min at 4 °C. Aliquots (20 μL) of supernatant were mixed with loading buffer (5 μL) and boiled for 10 min. Aliquots (20 μL) were then electrophoresed on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel, and proteins were transferred from the gel onto polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at 25 °C. Primary antibodies were diluted in TBST (anti-Bcl-2, 1:500; anti-Bax, 1:1000; anti-cleaved caspase-3, 1:1000; anti-βactin, 1:500) and incubated with the blots overnight at 4 °C. Blots were washed three times in TBST, incubated for 1 h with peroxidase-conjugated secondary antibody (goat anti-rabbit,

■

RESULTS On the basis of structure−activity insights from previous studies of synthetic AD drugs, we designed and synthesized the drug candidate BDBDP (Figure 1). We then sought to test its

Figure 1. Chemical structure of 1,1′-(biphenyl-4,4′-diyl)-bis(3(dimethylamino)-propan-1-one) (BDBDP).

ability to prevent Aβ1−42-induced neurotoxicity in PC12 cells in vitro. Our strategy was to pretreat PC12 cultures with different concentrations of BDBDP (0−1 μg/mL) and then expose them to Aβ1−42 aggregates (Figure 2). We compared cell viability, mitochondrial membrane potential, apoptosis, and ROS production in the presence and absence of drug pretreatment. BDBDP Protects PC12 Cells against Aβ1−42-Induced Apoptosis and Increases Cell Viability. In an MTT assay of cell viability, treating PC12 cells with 100 μM Aβ1−42 for 12 h reduced mean cell viability to 65.73% of the control value in the absence of Aβ1−42 (Figure 3A). Pretreating the cells with BDBDP for 12 h prior to addition of Aβ1−42 increased cell viability in a dose-dependent manner: 0.1 μg/mL, 86.58% of the control value; 0.3 μg/mL, 89.95%; 1 μg/mL, 90.92%. In a cell viability assay based on LDH release rate, treating PC12 cells with 100 μM Aβ1−42 for 12 h increased the LDH release rate to 148% of the control value in the absence of Aβ1−42 (Figure 3B). Pretreating the cells with BDBDP for 12 h prior to addition of Aβ1−42 decreased the rate of LDH release in a dose-dependent manner: 0.1 μg/mL, 129.79% of the control value; 0.3 μg/mL, 103.66%; 1 μg/mL, 79.08%. The effects of BDBDP pretreatment on Aβ1−42-induced apoptosis were analyzed using a TUNEL assay, which takes advantage of the fact that genomic DNA is cleaved into oligonucleosome-sized fragments in apoptotic cells. PC12 cells were treated without Aβ1−42 as control (Figure 4A), and treating PC12 cells with Aβ1−42 significantly increased the level of apoptosis above that observed in the absence of Aβ1−42; however, this increase was attenuated by pretreatment with 1 μg/mL BDBDP (Figure 4B,C). These results suggest that BDBDP protects PC12 cells from Aβ1−42-induced cell apoptosis and improves cell viability. BDBDP Reduces Aβ1−42-Induced Oxidative Stress in PC12 Cells. Oxidative stress was assessed by measuring the levels of intracellular ROS (Figure 5A) and MDA (Figure 5B). Exposing cells to 100 μM Aβ1−42 for 12 h increased ROS and MDA levels to 121.69% and 132.21% of control values, respectively. Pretreating the cells with different concentrations of BDBDP for 12 h ameliorated these Aβ1−42-induced increases in a dose-dependent manner. 430

dx.doi.org/10.1021/mp400395g | Mol. Pharmaceutics 2014, 11, 428−435

Molecular Pharmaceutics

Article

Figure 2. Transmission electron micrographs of Aβ1−42 aggregates used in PC12 neurotoxicity assays. (A) Commercially available Aβ1−42 was dissolved in deionized distilled water at a concentration of 1 mM and incubated at 37 °C for 7 days to induce aggregation. (B) Aβ1−42 without treatment.

Figure 3. Ability of BDBDP to protect PC12 cells from Aβ1−42-induced cytotoxicity. Cells were treated with no Aβ1−42 (control) or with 100 μM Aβ1−42 after pretreatment with the indicated concentrations of BDBDP. Cell viability was measured using assays based on MTT (A) and LDH release (B). Results are expressed as percentages of the control value (mean ± SD, n = 5). #P < 0.01 compared with the control group (no Aβ1−42); *P < 0.05 and **P < 0.01 compared with the Aβ1−42-treated group.

Figure 4. Ability of BDBDP to protect PC12 cells from Aβ1−42-induced apoptosis. Cells were treated with no Aβ1−42 (control) or with 100 μM Aβ1−42 after pretreatment with different concentrations of BDBDP. The TUNEL assay was used to assess apoptosis; brown nuclei are TUNEL positive and apoptotic cells. (A) Control (no Aβ1−42); (B) 100 μM Aβ1−42 only; (C) 1 μg/mL BDBDP + 100 μM Aβ1−42; and (D−F) higher magnification images of the boxed areas in panels A−C.

BDBDP Partially Reverses Aβ1−42-Induced Changes in the Mitochondrial Membrane Potential in PC12 Cells. Treating cells with 100 μM Aβ1−42 for 12 h decreased the mitochondrial membrane potential to 33.71% of the value in the absence of Aβ1−42, based on the JC-1 assay (Figure 6).

Pretreating the cells with different concentrations of BDBDP for 12 h ameliorated this Aβ1−42-induced reduction in a dosedependent manner. BDBDP Partially Reverses Aβ1−42-Induced Changes in Apoptotic Protein Expression and Activation in PC12 431

dx.doi.org/10.1021/mp400395g | Mol. Pharmaceutics 2014, 11, 428−435

Molecular Pharmaceutics

Article

Figure 5. Ability of BDBDP to protect PC12 cells from Aβ1−42-induced oxidative stress. Cells were treated with no Aβ1−42 (control) or with 100 μM Aβ1−42 after pretreatment with different concentrations of BDBDP. (A) ROS production was measured by fluorescence microscopy and flow cytometry (insets): (a) control (no Aβ1−42), (b) 100 μM Aβ1−42 only, (c) 0.1 μg/mL BDBDP + 100 μM Aβ1−42, (d) 0.3 μg/mL BDBDP + 100 μM Aβ1−42, and (e) 1 μg/mL BDBDP + 100 μM Aβ1−42. Results are expressed as percentages of control values in the control group (mean ± SD, n = 3). (B) Levels of ROS were measured using the DCFH-DA assay. (C) Levels of MDA were measured using a standard assay. Results are expressed as percentages of control values (mean ± SD, n = 5). #P < 0.01 compared with the control group (no Aβ1−42); *P < 0.05 and **P < 0.01 compared with the Aβ1−42-treated group.

Cells. The activation of caspase-3 and the relative levels of antiapoptotic protein Bcl-2 and pro-apoptotic protein Bax play a key role in apoptotic cell death. Therefore, we used Western blotting to measure levels of cleaved caspase-3 and the Bcl-2/ Bax ratio in PC12 cells after pretreating them or not with BDBDP and then exposing them to Aβ1−42 (Figure 7). Exposing the cells to 100 μM Aβ1−42 for 12 h significantly increased the level of activated caspase-3 to 154.9% of the control value in the absence of Aβ1−42, and it decreased the Bcl2/Bax ratio to 81.35% of the control value. Pretreating the cells with different concentrations of BDBDP for 12 h partially reversed these Aβ1−42-induced changes in a dose-dependent manner.

■

DISCUSSION Here we describe the design, synthesis, and characterization of BDBDP, and we provide evidence that it can partially protect PC12 cells against Aβ1−42-induced toxicity, mitochondrial membrane depolarization, and apoptosis. BDBDP was designed based on previous studies of Nmethylated structures that play an important role in transport across the BBB32 and anti-AD drug bimolecular 3-piperidylethyl phenyl ketone.21 Our drug candidate was designed to contain two tertiary amines, given that most AD drugs that

Figure 6. Ability of BDBDP to protect PC12 cells from Aβ1−42induced depolarization of the mitochondrial membrane. Cells were treated with no Aβ1−42 (control) or with 100 μM Aβ1−42 after pretreatment with different concentrations of BDBDP. Mitochondrial membrane potential was measured using JC-1 staining. Results are expressed as percentages of control values (mean ± SD, n = 3). #P < 0.01 compared with the control group (no Aβ1−42); *P < 0.05 and **P < 0.01 compared with the Aβ1−42-treated group.

432

dx.doi.org/10.1021/mp400395g | Mol. Pharmaceutics 2014, 11, 428−435

Molecular Pharmaceutics

Article

Figure 7. Ability of BDBDP to partially reverse Aβ1−42-induced changes in caspase-3 activation and the ratio of Bcl-2 to Bax expression in PC12 cells. Cells were treated with no Aβ1−42 (control) or with 100 μM Aβ1−42 after pretreatment with different concentrations of BDBDP. (A) Levels of cleaved caspase-3, Bax, and Bcl-2 were assayed by Western blotting. (B) Quantitation of levels of active caspase-3. Results are expressed as percentages of control values (mean ± SD, n = 5). ##P < 0.01 compared with the control group (no Aβ1−42); *P < 0.05 and **P < 0.01 compared with the Aβ1−42-treated group. (C) Quantitation of the Bcl-2/Bax ratio. Results are expressed as percentages of control values (mean ± SD, n = 5). # P < 0.05 compared with the control group (no Aβ1−42); *P < 0.05 and **P < 0.01 compared with the Aβ1−42-treated group.

effects revealed that BDBDP pretreatment ameliorated Aβ1−42induced oxidative stress and apoptosis. Oxidative stress, which occurs when the production of ROS and the natural antioxidant defense system are no longer balanced, plays an important role in the cellular damage and neurodegeneration that occur in AD.42 Numerous studies in vitro and in vivo indicate that Aβ1−42 treatment significantly increases ROS production. This overproduction damages major macromolecules in cells, including proteins, lipids, and DNA, causing neuronal dysfunction and depression.36,43,44 Consistent with previous studies, we found that treating PC12 cells with Aβ1−42 not only led to ROS overproduction but also increased levels of MDA. Pretreating cells with BDBDP mitigated these changes in a dose-dependent manner, suggesting that the antioxidant activity of BDBDP may make it useful for attenuating and preventing apoptosis in AD. Mitochondrial membrane potential is essential for cell survival, particularly when the cell is under oxidative stress.13,45 Mitochondrial dysfunction is a prominent feature in Aβinduced neuronal toxicity in patients with AD,13,46 and cells treated with Aβ1−42 suffer irreversible depolarization of the mitochondrial membrane. This depolarization then allows ROS produced in the mitochondria to leak into the cytoplasm, causing oxidative stress via activation of apoptosis signaling. We found that BDBDP partially reversed Aβ1−42-induced depolarization of the mitochondrial membrane in PC12 cells. Our findings provide further evidence that BDBDP exerts neuroprotective effects by preventing oxidative stress and apoptosis.

perform well are tertiary amine salts or quaternary ammonium salts.22,23 Our compound is N-methylated, which appears to help prevent Aβ-induced cytotoxicity in vitro,24−30 and it contains the acetophenone structure, which has a close relationship with oxidative stress.31 To test the clinical potential of our drug candidate, we assessed its ability to inhibit Aβ1−42-induced changes in PC12 cells. The PC12 cell line is widely used as a cellular model to investigate cytotoxicity and neuronal injury in AD. PC12 cells are relatively easy to culture and survive longer than primary neuronal cultures.37 We conducted our experiments in PC12 cultures that had differentiated the following treatment with NGF. NGF-differentiated PC12 cells are more similar to neurons than are undifferentiated PC12 cells, and they are sensitive to Aβ peptides.38 Several studies in PC12 cells suggest that Aβ1−42 not only induces cellular toxicity and apoptosis but also excessive ROS production and mitochondrial dysfunction.39−41 We also observed these effects in our experimental system. We then found that pretreating the cells with BDBDP ameliorated these changes, and these changes are not based on affection Aβ1−42 aggregation performed by BDBDP (Figure S5 and S6, Supporting Information). We used both MTT and LDH release assays to evaluate whether BDBDP can protect cells against Aβ1−42-induced decreases in cell survival. We also visualized Aβ1−42-induced apoptosis directly in the presence and absence of BDBDP pretreatment. Our results provide evidence that BDBDP can protect PC12 cells from Aβ1−42-induced cytotoxicity. Further experiments to explore the mechanism of these protective 433

dx.doi.org/10.1021/mp400395g | Mol. Pharmaceutics 2014, 11, 428−435

Molecular Pharmaceutics

Article

(3) Nordberg, A. PET imaging of amyloid in Alzheimer’s disease. Lancet Neurol. 2004, 3 (9), 519−527. (4) Li, G.; Ma, R.; Huang, C.; Tang, Q.; Fu, Q.; Liu, H.; Hu, B.; Xiang, J. Protective effect of erythropoietin on-amyloid-induced PC12 cell death through antioxidant mechanisms. Neurosci. Lett. 2008, 442 (2), 143−147. (5) Li, R. C.; Pouranfar, F.; Lee, S. K.; Morris, M. W.; Wang, Y.; Gozal, D. Neuroglobin protects PC12 cells against beta-amyloidinduced cell injury. Neurobiol. Aging 2008, 29 (12), 1815−1822. (6) Hardy, J.; Selkoe, D. J. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 2002, 297 (5580), 353−356. (7) Khodadadi, S.; Riazi, G. H.; Ahmadian, S.; Hoveizi, E.; Karima, O.; Aryapour, H. Effect of N-homocysteinylation on physicochemical and cytotoxic properties of amyloid beta-peptide. FEBS Lett. 2012, 586 (2), 127−131. (8) Pearson, H. A.; Peers, C. Physiological roles for amyloid β peptides. J. Physiol. 2006, 575 (1), 5−10. (9) Selkoe, D. J. Soluble oligomers of the amyloid β-protein impair synaptic plasticity and behavior. Behav. Brain Res. 2008, 192 (1), 106− 113. (10) Hu, J.-F.; Chu, S.-F.; Ning, N.; Yuan, Y.-H.; Xue, W.; Chen, N.H.; Zhang, J.-T. Protective effect of (−) Clausenamide against Aβinduced neurotoxicity in differentiated PC12 cells. Neurosci. Lett. 2010, 483 (1), 78−82. (11) Li, G.; Ma, R.; Huang, C.; Tang, Q.; Fu, Q.; Liu, H.; Hu, B.; Xiang, J. Protective effect of erythropoietin on beta-amyloid-induced PC12 cell death through antioxidant mechanisms. Neurosci. Lett. 2008, 442 (2), 143−147. (12) Zhang, H.-Y.; Liu, Y.-H.; Wang, H.-Q.; Xu, J.-H.; Hu, H.-T. Puerarin protects PC12 cells against β-amyloid-induced cell injury. Cell Biol. Int. 2008, 32 (10), 1230−1237. (13) Xian, Y. F.; Lin, Z. X.; Mao, Q. Q.; Ip, S. P.; Su, Z. R.; Lai, X. P. Protective effect of isorhynchophylline against beta-amyloid-induced neurotoxicity in PC12 cells. Cell. Mol. Neurobiol. 2012, 32 (3), 353− 360. (14) Knopman, D. S. Current treatment of mild cognitive impairment and Alzheimer’s disease. Curr. Neurol. Neurosci. Rep. 2006, 6 (5), 365−371. (15) Schenk, D. Hopes remain for an Alzheimer’s vaccine. Nature 2004, 431 (7007), 398−398. (16) Frisardi, V.; Solfrizzi, V.; Imbimbo, B. P.; Capurso, C.; D’Introno, A.; Colacicco, A. M.; Vendemiale, G.; Seripa, D.; Pilotto, A.; Capurso, A.; Panza, F. Towards disease-modifying treatment of Alzheimer’s disease: Drugs targeting beta-amyloid. Curr. Alzheimer Res. 2010, 7 (1), 40−55. (17) Yamada, K.; Nabeshima, T. Animal models of Alzheimer’s disease and evaluation of anti-dementia drugs. Pharmacol. Ther. 2000, 88 (2), 93−113. (18) Thomas, T.; Nadackal, T. G.; Thomas, K. Aspirin and nonsteroidal anti-inflammatory drugs inhibit amyloid-[beta] aggregation. Neuroreport 2001, 12 (15), 3263−3267. (19) Frydman-Marom, A.; Levin, A.; Farfara, D.; Benromano, T.; Scherzer-Attali, R.; Peled, S.; Vassar, R.; Segal, D.; Gazit, E.; Frenkel, D.; Ovadia, M. Orally Administrated Cinnamon Extract Reduces betaAmyloid Oligomerization and Corrects Cognitive Impairment in Alzheimer’s Disease Animal Models. PLoS One 2011, 6 (1), e16564. (20) Wang, F.; Zhou, X. L.; Yang, Q. G.; Xu, W. H.; Chen, Y. P.; Chen, G. H. A peptide that binds specifically to the beta-amyloid of Alzheimer’s disease: selection and assessment of anti-beta-amyloid neurotoxic effects. PLoS One 2011, 6 (11), e27649. (21) Liu, A. L.; Gong, L. L.; Du, G. H.; Wang, L.; Gao, M. Compound with dual restraining activities to acetyl cholinesterase and butyryl cholinesterase and use thereof. CN101311171A, 2007. (22) Burns, A.; Rossor, M.; Hecker, J.; Gauthier, S.; Petit, H.; Möller, H.-J.; Rogers, S.; Friedhoff, L. The effects of donepezil in Alzheimer’s disease: Results from a multinational trial. Dementia Geriatr. Cognit. Disord. 1999, 10 (3), 237−244.

The key to explaining these neuroprotective effects may lie in how BDBDP influences the expression of Bcl-2 family proteins. The Bcl-2 family includes several homologous proteins such as the antiapoptotic Bcl-2, which inhibits mitochondrial depolarization and ROS production, and the pro-apoptotic Bax, which induces both these processes.13 Thus, the balance between Bax and Bcl-2 is an important determinant of cell survival or death. We found that Aβ1−42 significantly decreased the ratio of Bcl-2 to Bax, consistent with previous studies in PC12 cells showing that these proteins play a pivotal role in mitochondrial apoptosis caused by oxidative stress.47−49 We then showed that pretreating cells with BDBDP increased the ratio of Bcl-2 to Bax. Changing the Bcl-2/Bax ratio influences the activity of caspase-3, a key apoptotic effector. ROS overproduction can cause the release of mitochondrial proteins from the intermembrane space, leading to the proteolytic activation of caspase-3. Thus, levels of activated caspase-3 are an indicator of ROS-induced apoptosis. In our study, treating cells with Aβ1−42 increased the level of cleaved caspase-3, consistent with the fact that Aβ1−42 decreased the ratio of Bcl-2 to Bax. Pretreating cells with BDBDP partially reversed these Aβ1−42-induced changes, suggesting that the neuroprotective effects of BDBDP in vitro depend, at least in part, on its interaction with the Bcl-2 family of proteins. Taken together, our experiments suggest that BDBDP holds promise as a candidate AD drug. These in vitro studies suggest that the drug exerts neuroprotective effects by inhibiting oxidative stress and neuronal apoptosis. Furthermore, we are now verifying and extending these findings in vivo in our laboratory.

■

ASSOCIATED CONTENT

S Supporting Information *

Additional experimental details and figures. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*(Z.Z.) Tel: +86-28-85501566. Fax: +86-28-85501615. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work were supported by National Basic Research Program of China (No. 2013CB932504) and the National Natural Science Foundation of China (No.81130060)

■

ABBREVIATIONS AD, Alzheimer’s disease; Aβ, β-amyloid; APP, amyloid precursor protein; ROS, reactive oxygen species; MDA, malonyldialdehyde; LDH, lactate dehydrogenase

■

REFERENCES

(1) Zheng, W.-H.; Bastianetto, S.; Mennicken, F.; Ma, W.; Kar, S. Amyloid β peptide induces tau phosphorylation and loss of cholinergic neurons in rat primary septal cultures. Neuroscience 2002, 115 (1), 201−211. (2) Forman, M. S.; Trojanowski, J. Q.; Lee, V. M. Neurodegenerative diseases: a decade of discoveries paves the way for therapeutic breakthroughs. Nat. Med. 2004, 10 (10), 1055−1063. 434

dx.doi.org/10.1021/mp400395g | Mol. Pharmaceutics 2014, 11, 428−435

Molecular Pharmaceutics

Article

(23) Erkinjuntti, T.; Kurz, A.; Gauthier, S.; Bullock, R.; Lilienfeld, S.; Damaraju, C. V. Efficacy of galantamine in probable vascular dementia and Alzheimer’s disease combined with cerebrovascular disease: a randomised trial. Lancet 2002, 359, 1283−1290. (24) Gordon, D. J.; Tappe, R.; Meredith, S. C. Design and characterization of a membrane permeable N-methyl amino acidcontaining peptide that inhibits A beta(1−40) fibrillogenesis. J. Pept. Res. 2002, 60 (1), 37−55. (25) Cruz, M.; Tusell, J. M.; Grillo-Bosch, D.; Albericio, F.; Serratosa, J.; Rabanal, F.; Giralt, E. Inhibition of beta-amyloid toxicity by short peptides containing N-methyl amino acids. J. Pept. Res. 2004, 63 (3), 324−328. (26) Hughes, E.; Burke, R. M.; Doig, A. J. Inhibition of toxicity in the beta-amyloid peptide fragment beta-(25−35) using N-methylated derivatives: a general strategy to prevent amyloid formation. J. Biol. Chem. 2000, 275 (33), 25109−25115. (27) Amijee, H.; Madine, J.; Middleton, D.; Doig, A. Inhibitors of protein aggregation and toxicity. Biochem. Soc. Trans. 2009, 37 (4), 692. (28) Kokkoni, N.; Stott, K.; Amijee, H.; Mason, J. M.; Doig, A. J. Nmethylated peptide inhibitors of beta-amyloid aggregation and toxicity. Optimization of the inhibitor structure. Biochemistry 2006, 45 (32), 9906−9918. (29) Doig, A. Peptide inhibitors of beta-amyloid aggregation. Curr. Opin. Drug Discovery Dev. 2007, 10 (5), 533. (30) Sciarretta, K. L.; Boire, A.; Gordon, D. J.; Meredith, S. C. Spatial separation of beta-sheet domains of beta-amyloid: Disruption of each beta-sheet by N-methyl amino acids. Biochemistry 2006, 45 (31), 9485−9495. (31) Mardanov, R. G.; Orlov, O. I.; Markin, A. A.; Tsar’kov, D. S.; Mukhamedieva, L. N. Molecular markers of oxidative stress in the expired air of healthy humans. Hum. Physiol. 2012, 38 (7), 794−797. (32) Zhang, X.; Liu, X.; Gong, T.; Sun, X.; Zhang, Z.-R. In vitro and in vivo investigation of dexibuprofen derivatives for CNS delivery. Acta Pharmacol. Sin. 2012, 33 (2), 279−288. (33) Wang, L.; Zhang, Y.; Liu, L.; Wang, Y. Palladium-catalyzed homocoupling and cross-coupling reactions of aryl halides in poly (ethylene glycol). J. Org. Chem. 2006, 71 (3), 1284−1287. (34) Chung, F.; Tisné, C.; Lecourt, T.; Dardel, F.; Micouin, L. NMRguided fragment-based approach for the design of tRNALys3 ligands. Angew. Chem., Int. Ed. 2007, 46 (24), 4489−4491. (35) Ngo, S.; Guo, Z. Key residues for the oligomerization of Abeta42 protein in Alzheimer’s disease. Biochem. Biophys. Res. Commun. 2011, 414 (3), 512−516. (36) Mao, Q.-Q.; Xian, Y.-F.; Ip, S.-P.; Tsai, S.-H.; Che, C.-T. Protective effects of peony glycosides against corticosterone-induced cell death in PC12 cells through antioxidant action. J. Ethnopharmacol. 2011, 133 (3), 1121−1125. (37) Tan, J.; Town, T.; Placzek, A.; Kundtz, A.; Yu, H.; Mullan, M. Bcl-xL inhibits apoptosis and necrosis produced by Alzheimer’s βamyloid-1−40 peptide in PC12 cells. Neurosci. Lett. 1999, 272 (1), 5− 8. (38) Harper, J. D.; Lansbury, P. T., Jr. Models of amyloid seeding in Alzheimer’s disease and scrapie: mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins. Annu. Rev. Biochem. 1997, 66 (1), 385−407. (39) Behl, C.; Davis, J.; Lesley, R.; Schubert, D. Hydrogen peroxide mediates amyloid β protein toxicity. Cell 1994, 77 (6), 817−827. (40) Butterfield, D. A.; Castegna, A.; Lauderback, C. M.; Drake, J. Evidence that amyloid beta-peptide-induced lipid peroxidation and its sequelae in Alzheimer’s disease brain contribute to neuronal death. Neurobiol. Aging 2002, 23 (5), 655−664. (41) Hensley, K.; Carney, J.; Mattson, M.; Aksenova, M.; Harris, M.; Wu, J.; Floyd, R.; Butterfield, D. A model for beta-amyloid aggregation and neurotoxicity based on free radical generation by the peptide: relevance to Alzheimer disease. Proc. Natl. Acad. Sci. 1994, 91 (8), 3270−3274.

(42) Smith, M. A.; Rottkamp, C. A.; Nunomura, A.; Raina, A. K.; Perry, G. Oxidative stress in Alzheimer’s disease. Biochim. Biophys. Acta: Mol. Basis Res. 2000, 1502 (1), 139−144. (43) Zhao, Z.; Wang, W.; Guo, H.; Zhou, D. Antidepressant-like effect of liquiritin from Glycyrrhiza uralensis in chronic variable stress induced depression model rats. Behav. Brain Res. 2008, 194 (1), 108− 113. (44) Niebrój-Dobosz, I.; Dziewulska, D.; Kwieciński, H. Oxidative damage to proteins in the spinal cord in amyotrophic lateral sclerosis (ALS). Folia Neuropathol. 2004, 42 (3), 151. (45) Pérez, M. J.; Cederbaum, A. I. Adenovirus-mediated expression of Cu/Zn−or Mn−superoxide dismutase protects against CYP2E1dependent toxicity. Hepatology 2003, 38 (5), 1146−1158. (46) Chen, J. X.; Du Yan, S. Pathogenic role of mitochondrial amyloid-β peptide. Expert Rev. Neurother. 2007, 7 (11), 1517−1525. (47) Pias, E.; Aw, T. Early redox imbalance mediates hydroperoxideinduced apoptosis in mitotic competent undifferentiated PC-12 cells. Cell Death Differ. 2002, 9 (9), 1007−1016. (48) Saito, Y.; Nishio, K.; Ogawa, Y.; Kinumi, T.; Yoshida, Y.; Masuo, Y.; Niki, E. Molecular mechanisms of 6-hydroxydopamine-induced cytotoxicity in PC12 cells: Involvement of hydrogen peroxidedependent and-independent action. Free Radical Biol. Med. 2007, 42 (5), 675−685. (49) Yu, Y.; Du, J. R.; Wang, C. Y.; Qian, Z. M. Protection against hydrogen peroxide-induced injury by Z-ligustilide in PC12 cells. Exp. Brain Res. 2008, 184 (3), 307−312.

435

dx.doi.org/10.1021/mp400395g | Mol. Pharmaceutics 2014, 11, 428−435