Astaxanthine Secured Apoptotic Death of PC12 Cells Induced by β-Amyloid Peptide 25

Abstract

Astaxanthine (ASTx) is a novel carotenoid nutraceutical occurring in many crustaceans and red yeasts. It has potent antioxidant, photoprotective, hepatodetoxicant, and anti-inflammatory activities. Documented effect of ASTx on treatment of neurodegenerative disease is still lacking. We used the β-amyloid peptide (Aβ) 25–35-treated PC12 model to investigate the neuron-protective effect of ASTx. The parameters examined included cell viability, caspase activation, and various apoptotic biomarkers that play their critical roles in the transduction pathways independently or synergistically. Results indicated that Aβ25–35 at 30 μM suppressed cell viability by 55%, whereas ASTx was totally nontoxic below a dose of 5.00 μM. ASTx at 0.1 μM protected PC12 cells from damaging effects of Aβ25–35 in several ways: (1) by securing the cell viability; (2) by partially down-regulating the activation of caspase 3; (3) by inhibiting the expression of Bax; (4) by completely eliminating the elevation of interleukin-1β and tumor necrosis factor-α; (5) by inhibiting the nuclear translocation of nuclear factor κB; (6) by completely suppressing the phosphorylation of p38 mitogen-activated protein kinase; (7) by completely abolishing the calcium ion influx to effectively maintain calcium homeostasis; and (8) by suppressing the majority (about 75%) of reactive oxygen species production. Conclusively, ASTx may have merit to be used as a very potential neuron protectant and an anti–early-stage Alzheimer's disease adjuvant therapy.

Introduction

A ll-trans-astaxanthine (ASTx) (Fig. 1), the isomer commonly meant when using the term ASTx, is a novel carotenoid widely occurring in many crustaceans, including shrimps and crabs. Recently, the mass production of ASTx using red yeast fermentation in natural seawater bioreactors has been commercialized in Hawaii.^1
–3 In general, ASTx is added to animal feeds for the sake of pigmentation, growth, and proliferation. ASTx possesses a very strong antioxidative effect, especially against the most active reactive oxygen species (ROS) like singlet oxygen, superoxide anions, hydrogen peroxides, and hydroxyl free radicals. Biochemically it serves as a strong anti-lipid peroxidative agent.^1
–3 Alternatively, as often cited, it has long been considered to be good at photoprotection, anti-inflammation, and antihepatotoxicity and as an immune enhancer as well as an excellent protective agent for neurodegenerative and cardiovascular diseases.^1
–3

FIG. 1.

Major isomers of ASTx.

β-Amyloid peptide (Aβ) normally is secreted by neurons in low concentrations and transported into cerebrospinal fluid and plasma, where it associates with lipoproteins and protects the lipid from peroxidation.⁴ In brain, it plays an essential role in synapse and neuronal plasticity that underlie learning and memory.⁵ However, pathologically the neurological deficits of Alzheimer's disease (AD) are ultimately characteristic of neuronal loss or death and oligomeric Aβ deposits in distinct anatomical regions of brain.^6
–8 Consequently, the action mechanism of Aβ to induce neuronal death has become extremely controversial. Accumulating biochemical findings have revealed that a diversity of pathological outcomes may be induced by Aβ. In this regard, the pathogenesis of AD may include (1) plaque formation,^6
–8 (2) biochemical hampering,⁸ (3) reversibly impairing hippocampal synaptic plasticity and spatial learning,⁹ (4) change of electrochemical potential due to loss of calcium homeostasis,^10,11 and (5) oxidative attacks exerted by the secondary ROS.^12
–14

Aβ has been demonstrated to affect signal transmission across N-methyl-d-aspartate receptor synapses, resulting in modified synaptic plasticity.^15,16 In addition, Aβ induces memory loss by altering the potassium channels or eliciting different transmembranous ion transport through different, specific channel sites.^17,18 Recently, studies of Song et al.¹⁹ and Shen et al.²⁰ pointed out that ROS are actually mediating the Aβ25–35 toxicity in PC12 cells, a cell that would undergo neuron-like differentiation. Eckert et al.²¹ and other researchers¹¹ have demonstrated that extremely serious mitochondrial deficits could be induced by soluble Aβ. Biopharmacologically, Aβ25–35 elicited effects similar to those of Aβ1–42, which involved the binding to the receptors of advanced glycation end products,²² the down-regulation of acetylcholine receptors,²³ and fibril and nonfibril formation.²⁴ Furthermore, a variety of signaling pathways like the mitogen-activated protein kinase (MAPK) cascade^20,25
–27 and the Akt dephosphorylation/inactivation-related PTEN pathways²⁸ could be up-regulated by Aβ.

Interestingly, Holscher et al. ⁹ suggested that the damages induced by low concentration of soluble Aβ species might be reversible in the very early stage of AD, which indeed strongly inspired this investigation. Because evidence on the neuroprotective bioactivity of ASTx against the adverse effects of Aβ25–35 is still lacking, we first designed this Aβ–PC12 cell–ASTx model to mimic the neurotoxic effects of Aβ in PC12 cells and concomitantly used ASTx to study its amelioration effects.

Materials and Methods

Chemicals and reagents

Dulbecco's modified Eagle's medium was provided by Hylone Co. (Logan, UT, USA). Aβ25–35, N,N,N,N-tetramethylethylenediamine, and ASTx were products of Sigma-Aldrich Co. (St. Louis, MO, USA). Protein assay kits were provided by Bio-Rad Co. (Hercules, CA, USA). The enhanced chemiluminescence assay kit was purchased from PerkinElmer Life Sciences, Inc. (Boston, MA, USA). Re-Blot™ Plus-Mild was the product of Chemicon International (Temecula, CA, USA). The Endogen rat tumor necrosis factor (TNF)-α enzyme-linked immunosorbent assay (ELISA) kit, Endogen rat interleukin (IL)-1β ELISA kit, and the NE-PER™ nuclear and cytoplasmic extraction reagents were manufactured by Pierce Biotechnology Co. (Rockford, IL, USA). 2',7'-Dichlorofluorescein diacetate (H₂DCFDA) and Fluo-3 acetoxymethyl ester were manufactured by Invitrogen Co. (Grand Island, NY, USA). All antibodies used were products of Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cell culture

The PC12 cells were obtained from the Bioresource Collection and Research Center (Food Industry Research and Development Institute, Hsinchu, Taiwan). PC12 cells were incubated at 37°C under a 5% CO₂ atmosphere in Dulbecco's modified Eagle's medium to which had been added 10% heat-inactivated fetal bovine serum, 1% antibiotics (100 IU of penicillin and 100 μg of streptomycin), and 50 μL of 2 mM glutamine.

Toxicity test of Aβ25–35

Synthetic Aβ25–35 was prepared at a concentration of 1 mM in water and aggregated at 4°C for 60 hours and then at 37°C for 8 hours before use.²⁹

PC12 cells were cultivated in 96-well plates at a density of 2 × 10⁴ cells per well. After adhesion for 24 hours, the medium was replaced with serum-free medium at 100 μL per well. Aβ25–35 was added at concentrations as indicated. The cultivation was continued and determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 24 or 48 hours. In brief, 10 μL of MTT solution (5 mg/mL) was added to each culture well containing 100 μL of medium. After incubation for 1 hour at 37°C, the medium was carefully removed, and the formazan crystals formed were dissolved in 200 μL of dimethyl sulfoxide with gentle shaking. Absorbance was determined at 570 nm with a spectrophotometer against a reference measured at 630 nm.²⁰

Toxicity test of ASTx

PC12 cells were seeded onto a 96-well plate at a density of 2 × 10⁴ cells per well. On adhesion for 24 hours, the medium was replaced with serum-free medium at 100 μL per well. ASTx was dissolved in dimethyl sulfoxide and added at concentrations as indicated. The cultivation was continued and assessed by MTT assay at 24 or 48 hours, as mentioned above.

Effect of ASTx against the damaging effect of Aβ25–35

PC12 cells were seeded onto a 96-well plate at a density of 2 × 10⁴ cells per well. After complete adhesion for 24 hours, the medium was replaced with serum-free medium at 100 μL per well. ASTx was added at concentrations as indicated. The cultivation was continued for an additional 24 hours. The medium was replaced with serum-free medium, and stock 30 μM Aβ25–35 was added at concentrations as indicated. The cultivation was continued, and MTT assays were performed at 24 or 48 hours, as mentioned above.

Assay for intracellular ROS

To monitor intracellular ROS accumulation, the fluorescent probe H₂DCFDA was used. Following the treatment with 30 μM Aβ25–35 for 18, 24, and 48 hours in the presence or absence of 0.10 μM ASTx, PC12 cells (1.5 × 10⁵ cells/mL) were centrifuged, collected, and resuspended in warm phosphate-buffered saline (PBS). The suspension was seeded onto a 96-well plate (195 μL for each well). Five microliters of H₂DCFDA was added to give a final concentration of 10 μM. The culture solution was incubated in the dark at 37°C for 30 minutes. The fluorescence intensity was measured with a fluorescence spectrophotometer (LUMIstar OPTIMA, BMG Labtech, Offenburg, Germany) using an excitation wavelength of 485 nm and an emission wavelength of 538 nm. The data were expressed in relative fluorescence units.

Determination of intracellular calcium ion concentration

After treatment with 30 μM Aβ25–35 for 24 hours in the presence or absence of ASTx (0.10 μM), the PC12 cells (1 × 10⁵ cells/mL) were collected and resuspended in warm PBS. One hundred ninety-nine microliters of the suspension was seeded onto a 96-well plate, and 1 μL of Fluo-3 acetoxymethyl ester was added to obtain a final concentration of 1 μM. The mixture was kept in the dark to avoid direct sun light radiation. After incubation for 30 minutes at 37°C, the intensity of fluorescence was determined as mentioned above.¹⁰

Analysis of released TNF-α and IL-1β

The expression of cytokines was assessed by Endogen rat TNF-α and IL-1β ELISA kits to measure the protein levels of the respective cytokine in the culture having a cell density of 1 × 10⁶ cells per well. On treatment with 30 μM Aβ25–35 for 24 hours in the presence or absence of ASTx (0.10 μM), the culture medium was assayed according to the manufacturer's protocol and quantified against a standard curve.

Western blot and analysis for cytosolic and nuclear protein extract

PC12 cells were seeded onto a six-well plate at a density of 1 × 10⁶ cells per well and incubated at 37°C. After cell adhesion for 24 hours, the medium was replaced with serum-free medium at 2 mL per well with or without 0.10 μM ASTx. To the culture 0.10 μM ASTx was added. On cultivation for 24 hours, the medium was again replaced with fresh medium. To the culture 30 μM Aβ25–35 was added, and the incubation was continued for 48 hours. The cells were washed and collected in ice-cold PBS. The cell pellets were resuspended in 100 μL of lysis buffer and incubated in an ice bath for 10 minutes. After centrifugation at 12,000 g for 5 minutes, the supernatant was separated and stored at −70°C. To prepare nuclear protein extract, the PC12 cells were treated with 30 μM Aβ25–35. The cultivation was continued for an additional 24 hours, and NE-PER reagent was added. The protein concentration was then determined using a protein assay kit. In brief, an equivalent amount (15 μg) of each sample was electrophoresed in a 12.5% sodium dodecyl sulfate-polyacrylamide gel. The proteins were transferred onto polyvinylidene fluoride (PVDF) membranes, which were saturated by co-incubation with 0.05% nonfat dry milk in PBS with Tween 20 overnight at 4°C. After agitation for 1 hour, the membrane was washed four times with ice-cooled PBS with Tween 20, each time for 5 minutes. Primary antibodies (including β-actin, Bcl-2, Bax, pro-caspase 3, caspase 3, p65, p38, and phosphorylated p38 [p-p38] MAPK) were added and left to react for 1 hour. The primary antibodies were removed by rinsing four times with PBS with Tween 20, each time for 5 minutes. The secondary antibodies were applied, and the reaction was left to proceed at room temperature for 1 hour. On rinsing four times with ice-cooled PBS with Tween 20, the PVDF membranes were treated with enhanced chemiluminescence reagent, and the reaction was further facilitated at room temperature for 2 minutes. X-ray film (Kodak, Tokyo, Japan) was used to print and develop the images for examination of the protein spots. All the data obtained were shown from the same PVDF membrane. To examine an alternate protein spot, the PVDF membrane has to be first rinsed with RE-Blot Plus to remove the previous protein spots and then treated with the secondary antibodies. The rinse time requires approximately 15 minutes. The remaining procedure to follow was entirely the same as that mentioned above.

Statistical analysis

Data were analyzed statistically by unpaired Student's t test. Results were expressed as mean ± SD values. A level of P < .05 was considered to be statistically significant.

Results

Effect of Aβ25–35 on cell viability

Aβ25–35 suppressed cell viability in a dose-responsive manner. During the initial 24 hours, the cell growth was slightly stimulated (Fig. 2A). However, a prominent suppressive effect appeared at 48 hours. At the concentration of 30 μM, Aβ25–35 reduced the viability of PC12 cells to approximately 55% (Fig. 2A).

FIG. 2.

The toxicity of Aβ25–35 and ASTx on PC12 cells. (A) PC12 cells were treated with Aβ25–35 at concentrations of 0.0, 10, 15, and 30 μM in serum-free medium for 24 or 48 hours. (B) PC12 cells were treated with ASTx at concentrations of 0.0, 0.10, 0.15, 0.20, and 0.25 μM in serum-free medium for 24 or 48 hours. Data are mean ± SD values from triplicate experiments. *P < .05, **P < .01 compared with the control at 48 hours. ^## P < .01 compared with the control at 24 hours.

Effect of ASTx on cell viability

ASTx stimulated the proliferation of PC12 cells in a dose-responsive manner. The cells were shown to grow steadily within the dose range of 0.10–5.00 μM. Dosages ≥5.00 μM were seen to exert slight inhibition on the cell viability (Fig. 2B).

Effect of ASTx on Aβ25–35-induced cytotoxicity

On incubation for 48 hours, 0.10 μM ASTx totally reversed the viability-suppressive effect of Aβ25–35 (Fig. 3). However, increased dosages of ASTx failed to show any better effects (Fig. 3).

FIG. 3.

Protective effect of ASTx against Aβ25–35 in PC12 cells. ASTx at 0.10, 0.15, 0.20, and 0.25 μM was tested to examine its protective effect against Aβ25–35 at 30 μM for each. The time intervals for induction were 24 or 48 hours. Data are mean ± SD values of triplicate experiments. **P < .01 compared with the 48-hour-Aβ(25–35)-treated group.

Effect of ASTx on caspase 3 activation induced by Aβ25–35

Figure 4 indicates that Aβ25–35 up-regulated caspase 3 and simultaneously down-regulated pro-caspase 3 to yield a 60% increase of the caspase 3/pro-caspase 3 ratio. Such an adverse effect was seen partially inhibited (about 15%) in the presence of 0.10 μM ASTx (Fig. 4). In addition, the total amount of caspase protein was slightly decreased in the ASTx-treated group. These data suggested the anti-apoptotic ability of ASTx. In the following experiments, we observed whether ASTx could regulate the expression of biomarkers involved in the apoptotic signals induced by Aβ25–35.

FIG. 4.

Effect of ASTx on inhibiting the expression of caspase 3/pro-caspase 3 induced by Aβ25–35. ASTx (0.10 μM) was used to examine the suppressive effect on the expression of caspase-3 and pro-capase-3 induced by Aβ25–35 (30 μM) in PC12 cells after incubation for 48 hours. Three independent experiments were conducted that showed a similar pattern of changes. The bar graph shows the ratio of expressed caspase–3/pro-capase-3 proteins in PC12 cell homogenates as indicated above. Data are mean ± SD values. *P < .05 compared with the Aβ(25–35)-treated group.

Effect of ASTx on Aβ25–35-induced expression of Bax

Although Aβ25–35 did not alter the expression of Bcl-2, it up-regulated Bax amounts by about 60%. Treatment of ASTx showed to exert a protective effect to suppress this change induced by Aβ25–35 (Fig. 5).

FIG. 5.

Effect of ASTx on inhibiting the expression of Bax/Bcl-2. ASTx (0.10 μM) was used to examine its effect against the ability of Aβ25–35 (30 μM) to affect the expressions of Bax (20 kDa) and Bcl-2 (28 kDa) in PC12 cells after incubation for 48 hours. Three independent experiments were conducted that showed a similar pattern of changes.

Effect of ASTx on Aβ25–35-induced expression of IL-1β and TNF-α

As shown in Figure 6, Aβ25–35 increased the expression of IL-1β and TNF-α about 75% and 180%, respectively, compared with the control, whereas ASTx at 0.10 μM was shown to have apparently suppressed their expressions. IL-1β was inhibited even to a level much lower than the control (Fig. 6).

FIG. 6.

Effect of ASTx on inhibiting the expression of (A) IL-1β and (B) TNF-α. ASTx (0.10 μM) was used to retard the inducing effect of Aβ25–35 (30 μM) for expression of IL-1β and TNF-α in PC12 cells. Incubation time was 24 hours. Data are mean ± SD values from triplicate experiments. **P < .01 compared with the Aβ(25–35)-treated group.

Effect of ASTx on p65 translocation induced by Aβ25–35

The nuclear protein extract was analyzed with western blotting. The nuclear/cytosol p65 ratio was elevated about 40% by Aβ25–35 (Fig. 7). It was demonstrated that the translocation of p65 induced by Aβ25–35 was significantly reduced in the presence of 0.10 μM ASTx.

FIG. 7.

Effect of ASTx on inhibiting the translocation of p65. ASTx (0.10 μM) suppressed the activation of p65 induced by Aβ25–35 (30 μM) with incubation for 24 hours. Three independent experiments were conducted that showed a similar pattern of changes. The bar graph shows the ratio of expressed nuclear extract (NE)/cytosol p65 proteins as indicated above. Data are mean ± SD values. **P < .01 compared with the Aβ-treated group.

Effect of ASTx on p38 MAPK activation induced by Aβ25–35

The up-regulation of p-p38 MAPK induced by Aβ25–35 was about 50%. ASTx at 0.10 μM was shown to apparently suppress the activation of p38 MAPK to a level 20% lower than the control (Fig. 8).

FIG. 8.

Effect of ASTx on inhibiting the phosphorylation of p38 MAPK. ASTx (0.10 μM) was used to examine its suppressive effect on p-p38 up-regulation induced by Aβ25–35 (30 μM). Incubation time was 24 hours. Three independent experiments were conducted that showed a similar pattern of changes.

Effect of ASTx to suppress the calcium influx induced by Aβ25–35

The influx of Ca²⁺ ions into PC12 cells was significantly enhanced by 80% after treatment with Aβ25–35 (Fig. 9A). Concomitantly, ASTx at 0.10 μM was seen to almost abolish the influx of calcium after incubation for 18 hours. Nevertheless, a longer time of incubation did not show any better effect (Fig. 9A).

FIG. 9.

The (A) calcium influx retardative capability and (B) antioxidative capability of ASTx. (A) Calcium mobilization was induced by Aβ25–35 (30 μM). AST (0.10 μM) was used to retard such mobilization on incubation for 24 hours with PC12 cells. Data are mean ± SD values. **P < .01 compared with the Aβ(25–35)-treated group. (B) ASTx (0.10 μM) was used to test its antioxidative effect against 30 μM Aβ25–35. The oxidative capability was measured by the fluorescence intensity. Data taken at intervals of 18, 24, and 48 hours are expressed in mean ± SD values from triplicate experiments. **P < .01 compared with the 18-hour-Aβ(25–35)-treated group.

Effect of ASTx against ROS induced by Aβ25–35

A tremendous amount of ROS was evoked by 30 μM Aβ25–35, reaching a net elevation of 20% at 18 hours. After treatment with 0.1 μM ASTx, approximately 15% of ROS generated was reduced (Fig. 9B). However, this was not a time-dependent response (Fig. 9B).

Discussion

Aβ25–35 suppressed PC12 cell viability in a dose-responsive manner, although its growth stimulatory effect was most obvious during the first 24 hours of incubation. A significant toxic effect appeared at 48 hours, implicating a problem due to either the limited mass transfer rate of Aβ25–35 across the cell membranes or the aggregation of toxic Aβ25–35 requiring a duration longer than 48 hours. As shown in Figure 2A, approximately half of the viability suppression was achieved with treatment with 30 μM Aβ25–35; hence the 50% lethal dose value of Aβ25–35 was estimated to be 30 μM. This dose was thus selected to use in the following experiments.

Literature indicated that Aβ actually is an important physiological antioxidant for lipoproteins in cerebrospinal fluid and plasma. At very low concentrations, e.g., 0.1–1.0 nM, Aβ(1–40) strongly inhibits autooxidation of cerebrospinal fluid and plasma low-density lipoprotein.⁴ However, at higher concentrations, the antioxidant action of this peptide disappeared. Instead, it acted as an ROS inducer.⁸

In contrast, ASTx at a dose within the range of 0.10–5.00 μM apparently stimulated PC12 cell proliferation in a dose-responsive manner. However, at dosages higher than 5.00 μM, the cell viability was slightly suppressed, a result that could be ascribed to the “tar-coating” effect of the excess ASTx on the cell surface (Fig. 2B). Considering the solubility, practical application, and cost efficiency, a dosage of 0.10 μM was preferably adopted in further experiments, which showed that 0.10 μM ASTx completely inhibited the toxicity evoked by Aβ25–35 (Fig. 3). From this result the titer of ASTx was estimated to be (30/0.1 =) 300 with respect to Aβ25–35, implicating the highly effective anti-Aβ25–35 nature of ASTx.

To further examine the cause of reduced cell viability, we examined the levels of apoptotic caspase 3 and pro-caspase 3 in cells treated with Aβ25–35. Figure 4 indicates that Aβ25-35 up-regulated caspase 3 and concomitantly down-regulated its pro-caspase, whereas ASTx was able to reverse this effect (Fig. 4). To obtain such an effect, ASTx at a dose higher than 0.10 μM would be required. Furthermore, Vaisid et al.³⁰ demonstrated that caspase-8 was also activated by Aβ25–35.

The phosphorylation and subsequent degradation of nuclear factor κB (NFκB) are closely related with IL-1β and TNF-α.³¹ ASTx significantly inhibited the expression of IL-1β and TNF-α (Fig. 6). There are seven IκB family members—IκBα, IκBβ, BCl-3, IκBe, IκBγ, and the precursor proteins p100 and p105—that assemble to bind the dimerization domain of NFκB dimers.^32,33 IκB proteins mask only the nuclear localization sequence of p65, whereas the nuclear localization sequence of p50 remains accessible.^34,35 Hence p65 was used as the marker in this experiment. As shown, ASTx effectively inhibited the translocation of p65 from cytoplasm to nuclear fraction (Fig. 7). Jang and Surh³¹ suggested that the Aβ-induced apoptosis of PC12 cells took the way of NFκB activation, which was mediated by the upstream kinases, including extracellular signal-regulated kinase and p38 MAPK. A dynamic balance between cytosolic and nuclear location was altered upon the nuclear export sequence of IκBα and exposure of the masked nuclear localization sequence of p65. As a result, predominant nuclear localization of NFκB occurred.³¹ A similar consequence had been observed in AD.¹¹

Literature elsewhere indicated that an increase of the activity of ROS like H₂O₂ would subsequently activate c-Jun N-terminal kinase 1/2 MAPKs, leading to lethal damage to PC12 cells.¹⁹ Consistent with this, ASTx ameliorated the damaging signal of p38 MAPK by inhibiting the phosphorylation of p38 MAPK (Fig. 8).

As recently cited, the selective reduction of Na⁺, K⁺-ATPase activity in vivo usually precedes the loss of calcium homeostasis and cell degeneration in cells exposed to Aβ1–40 or Aβ25–35,²⁹ suggesting the feasibility of ASTx to prevent such a cascade of molecular events. On the other hand, overstimulation of glutamate receptors produced ROS, which might evoke programmed cell death cascades.^16,34 Increased levels of intracellular Ca²⁺ might initiate a signal transduction pathway in which Ca²⁺ and diacylglycerol acted together to activate Ca²⁺-sensitive protein kinases. Release of intracellular Ca²⁺ stores promoted influx of extracellular Ca²⁺, which in turn acted as a positive feedback mechanism to enhance the excitation and release of more glutamate until this cycle became toxic to the cell.¹⁶ However, immunohistochemical studies revealed that this excitatory mechanism only acts as an important role in late stages of AD.^16,34

As often cited, oxidative cell death caused by accumulation of intracellular ROS and calcium ions has been implicated in pathophysiology of degenerative neuronal disease such as stroke, Parkinson's disease, and AD.²⁹ In addition, the molecular pathway of nitric oxide might also be affected, leading to the suppression of hippocampal synaptic plasticity.²⁸ As is well known, the molecular oxygen level and its reactive metabolites contribute to the major source of genomic instability.^35,36 Endoplasmic reticulum stress and ROS resulting from excitotoxicity and Aβ-induced toxicity are all damaging to DNA. The exposure of PC12 cells to Aβ25–35 could increase intracellular ROS generation, impair Na⁺, K⁺-ATPase activity, and elevate the intracellular calcium ion concentration, thus enhancing the neurotoxicity.^20,29,35 Obviously, the strong antioxidative behavior of ASTx would virtually contribute to the intensive protective effect.

To explore the molecular mechanisms involved in the protective effect of ASTx against the Aβ25–35-induced cell apoptosis, we further examined the expression of Bcl-2 and Bax to see whether the regulation of these cell death-associated proteins might respond to ASTx. As well cited, Bcl-2 family members such as Bax and Bcl-2 could be implicated in the process of apoptosis induced by ROS-generating agents, including Aβ25–35.¹⁹ In the present investigation, although ASTx did not change the expression of Bcl-2, it reduced the Bax expression induced by Aβ25–35, indicating that the intrinsic apoptotic pathway could be modulated by ASTx (Fig. 5).

To summarize, ASTx was entirely nontoxic in nature (Fig. 2B). In contrast, the oligomerized Aβ25–35 species revealed prominent neurotoxic effects in PC12 cells (Fig. 2A). ASTx successfully protected the cell viability from damages caused by Aβ25–35 (Fig. 3). To maintain cell survival, ASTx inhibited the expression of IL-1β and TNF-α (Fig. 6). Furthermore, much of the literature has demonstrated that ROS activate NFκB through the degradation of IκB.³¹ Compared with the results shown in Figures 7 and 9B, ASTx might prevent the ROS-transduced damage via inhibiting the nuclear translocation of p65. In addition, Aβ25–35 had been reported to affect the intracellular calcium level in PC12 cells. Calcium ion elevation could induce a huge production of ROS (Fig. 9B). Because caspase 3 activation is a redox modulator of cell death,^20,31 ASTx attenuated the biochemical alterations associated with Aβ25–35-induced apoptotic cell death by up-regulating caspase 3 and down-regulating pro-caspase 3 (Fig. 4), consistent with the report of Song et al.¹⁹

In conclusion, ASTx has shown an amazingly potent protective effect against the damaging effects elicited by Aβ25–35 in PC12 cells, which probably happens through a diversity of action mechanisms (Fig. 10) that involves:

blocking the direct lethal effect of oligomerized Aβ25–35 peptides

suppressing the calcium ion influx that acts as an ROS-generating pump

down-regulation of the Bax/Bcl-2 ratio and through the caspase 9 → caspase 3 pathway

down-regulating the cytokine pathway of TNF-α/IL-1β and through the caspase 8 → caspase 3 pathway

suppressing the cytokine pathway of TNF-α/IL-1β and increasing the translocation of NFκB into the nucleus

down-regulating the p-p38 MAPK pathway

FIG. 10.

Major neuronal cell apoptotic signals induced by Aβ25–35. The native Aβ species are nontoxic to the neuron; instead, they are required for neural plasticity in brain and normal antioxidative protection of low-density proteins in the cerebrospinal fluid. The toxicity of Aβ species is initiated when they are oligomerized or aggregated. The extrinsic apoptotic pathways exerted by the toxicity of oligomerized Aβ species involve cytokine receptor pathways, which proceed in two pathways: the first pathway is induced directly by the toxic Aβ oligomers, probably going from Ras → Raf → MAPK kinase 3 (MKK3)/p21-activated kinase (PAK) → p38 to induce neuronal death. The second pathway is regulated by the cytokine receptor pathway, which involves two subpathways: one triggered by TNF-α and IL-1β → IκB → NFκB → neuronal death; the other proceeding from TNF-α and IL-1β → Fas-associated death domain (FADD) → Fas-CD95 → caspase 8 → caspase 9 → caspase 3 to induce DNA damage. Alternatively, the glutamate receptor pathway may be triggered by the toxic Aβ oligomers (at the right side). In this pathway, the influx of Ca²⁺ is triggered. The increased Ca²⁺ further up-regulates the ROS (nitric oxide [NO]) production, which in turn initiates in the intrinsic pathway a huge production of free radicals (such as H₂O₂ elsewhere cited). Subsequently Bax is up-regulated (or Bcl-2 down-regulated), and mitochondria are damaged, leading to cytochrome c release → up-regulation of caspase 9 → caspase 3 and finally DNA damage. The molecules marked by a thick box denote the data resulting from this study. APAF, apoptotic protease-activating factor; CHOP, CCAAT/enhancer-binding protein homologous protein; GADD, growth arrest and DNA damage-inducible transcription factor; Fas L, Fas ligand; IKK, inhibitor κB kinase; TRADD, TNF receptor-associated death domain.

In summary, ASTx could be used as a potent anti-AD adjuvant therapy, in particular in the early stage of AD.

Footnotes

Acknowledgment

Special thanks are due for the grant by project HK-KTOH-96-01 offered by the Joint Research Program of Hungkuang University and Kuan-Tien Community Complex Hospital.

Author Disclosure Statement

The authors declare that there are no conflicts of interest.

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Astaxanthine Secured Apoptotic Death of PC12 Cells Induced by β-Amyloid Peptide 25–35: Its Molecular Action Targets

Abstract

Introduction

Materials and Methods

Chemicals and reagents

Cell culture

Toxicity test of Aβ25–35

Toxicity test of ASTx

Effect of ASTx against the damaging effect of Aβ25–35

Assay for intracellular ROS

Determination of intracellular calcium ion concentration

Analysis of released TNF-α and IL-1β

Western blot and analysis for cytosolic and nuclear protein extract

Statistical analysis

Results

Effect of Aβ25–35 on cell viability

Effect of ASTx on cell viability

Effect of ASTx on Aβ25–35-induced cytotoxicity

Effect of ASTx on caspase 3 activation induced by Aβ25–35

Effect of ASTx on Aβ25–35-induced expression of Bax

Effect of ASTx on Aβ25–35-induced expression of IL-1β and TNF-α

Effect of ASTx on p65 translocation induced by Aβ25–35

Effect of ASTx on p38 MAPK activation induced by Aβ25–35

Effect of ASTx to suppress the calcium influx induced by Aβ25–35

Effect of ASTx against ROS induced by Aβ25–35

Discussion

Footnotes

Acknowledgment

Author Disclosure Statement

References