Indirubin-3-Oxime Prevents H2O2-Induced Neuronal Apoptosis via Concurrently Inhibiting GSK3β and the ERK Pathway
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Oxidative stress-induced neuronal apoptosis plays an important role in many neurodegenerative disorders. In this study, we have shown that indirubin-3-oxime, a derivative of indirubin originally designed for leukemia therapy, could prevent hydrogen peroxide (H2O2)-induced apoptosis in both SH-SY5Y cells and primary cerebellar granule neurons. H2O2 exposure led to the increased activities of glycogen synthase kinase 3β (GSK3β) and extracellular signal-regulated kinase (ERK) in SH-SY5Y cells. Indirubin-3-oxime treatment significantly reversed the altered activity of both the PI3-K/Akt/GSK3β cascade and the ERK pathway induced by H2O2. In addition, both GSK3β and mitogen-activated protein kinase inhibitors significantly prevented H2O2-induced neuronal apoptosis. Moreover, specific inhibitors of the phosphoinositide 3-kinase (PI3-K) abolished the neuroprotective effects of indirubin-3-oxime against H2O2-induced neuronal apoptosis. These results strongly suggest that indirubin-3-oxime prevents H2O2-induced apoptosis via concurrent inhibiting GSK3β and the ERK pathway in SH-SY5Y cells, providing support for the use of indirubin-3-oxime to treat neurodegenerative disorders caused or exacerbated by oxidative stress.
KeywordsIndirubin-3-oxime H2O2 GSK3β PI3-K ERK
Analysis of variance
Cerebellar granule neurons
Extracellular signal-regulated kinase
Fetal bovine serum
Glycogen synthase kinase 3β
Mitogen-activated protein kinase
Reactive oxygen species
Oxidative stress plays an important role in the loss of neurons in many neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and cerebral ischemia (Kim et al. 2015). Many reactive oxygen species (ROS), including hydrogen peroxide (H2O2), nitric oxide (NO), and highly reactive hydroxyl and monoxide radicals can be released immediately after injury of neurons during oxidative stress (Bhat et al. 2015). This excessive ROS further induces neuronal apoptosis via their interactions with macromolecules as well as their ability to regulate both pro-survival and pro-apoptotic signaling pathways (Yoon et al. 2002). H2O2, an uncharged and freely diffusible molecule, is widely used as a neurotoxin to establish in vitro models of oxidative stress-induced neuronal apoptosis (Lee et al. 2007). Neuronal apoptosis can be regulated by the inhibition of pro-survival signaling pathways such as the phosphoinositide 3-kinase (PI3-K)/Akt cascade and the activation of pro-apoptotic signaling pathways such as the mitogen-activated protein kinase (MAPK) pathway. Inhibition of PI3-K/Akt led to the activation of glycogen synthase kinase 3β (GSK3β), and was reported to be involved in H2O2-induced neuronal apoptosis (Gao et al. 2012; Lin et al. 2016). Moreover, inhibition of extracellular signal-regulated kinase (ERK), a key intermediate of the MAPK signaling pathway, has been proposed as a pro-apoptotic mechanism underlying H2O2-induced neuronal apoptosis (Yang et al. 2005; Lin et al. 2016).
Indirubin-3-oxime is a derivative of indirubin, an active constituent of the traditional Chinese medicine recipe Danggui Longhui Wan used to treat chronic myelocytic leukemia (Smith et al. 2006). Interestingly, a recent pharmacokinetics study has shown that indirubin-3-oxime could easily cross the blood–brain barrier, suggesting that this chemical might be used to treat brain disorders (Selenica et al. 2007). Previous studies have demonstrated that indirubin-3-oxime inhibits neuronal apoptosis induced by β-amyloid, 6-hydroxydopamine, and potassium deprivation in vitro (Zhang et al. 2009; Hu et al. 2015). Moreover, indirubin-3-oxime prevents behavioral abnormities induced by many neurotoxins in rodents, possibly by inhibiting oxidative stress-induced neuronal loss (Wang et al. 2007; Ding et al. 2010). However, the underlying molecular mechanisms by which indirubin-3-oxime protects against oxidative stress-induced neuronal apoptosis are largely unknown.
SH-SY5Y cells are rat human neuroblastoma which has been reported to be sensitive to oxidants (Li et al. 1996). Therefore, SH-SY5Y cells could be used for studying the molecular mechanisms of drugs against oxidative stress-induced neuronal apoptosis (Nirmaladevi et al. 2014; Tian et al. 2015). In addition, homogenous cerebellar granule neurons (CGNs) are easy to be acquired because more than 90 % of neurons in cerebellum are CGNs (Gonzalez-Polo et al. 2004). Therefore, CGNs could be used to investigate the effects of neuroprotective drugs on primary neurons.
In this study, we have shown that indirubin-3-oxime effectively prevents H2O2-induced neuronal apoptosis in both SH-SY5Y cells and primary CGNs. Moreover, our results have demonstrated that indirubin-3-oxime protects against H2O2-induced apoptosis via concurrent inhibiting GSK3β and the ERK pathway.
Materials and Methods
Chemicals and Reagents
H2O2 was obtained from Calbiochem (San Diego, CA, USA). Indirubin-3-oxime and SB415286 were obtained from Sigma Chemicals (St Louis, MO, USA). PD98059, U0126, Wortmannin, and LY294002 were purchased from LC Laboratories (Woburn, MA, USA). Antibodies against pSer473-Akt, Akt, pSer9 glycogen synthase kinase 3β (GSK3β), GSK3β, phospho-Thr202/Tyr204-p44/42 (pERK), and ERK were obtained from Cell Signaling Technology (Beverly, MA, USA). Unless otherwise noted, all media and supplements used for cell cultures were purchased from Invitrogen (Carlsbad, CA, USA).
SH-SY5Y Cells Culture
SH-SY5Y cells were purchased from the Shanghai Institute of Cell Biology (Chinese Academy of Sciences), and cultured in high glucose modified Eagle’s medium (DMEM) containing 10 % fetal bovine serum (FBS) and penicillin (100 U/ml)/streptomycin (100 μg/ml). The cells were cultured in an incubator at 37 °C and 5 % CO2. The medium was replaced every other day. For the experiments with H2O2, SH-SY5Y cells (1 × 105 cells/ml) in DMEM with low serum content (1 % FBS) were seeded in 6-well or 96-well plates. All experiments were carried out 24 h after the cells were seeded.
Primary CGNs Culture
CGNs were prepared from 8-day-old Sprague–Dawley rats as described in our previous publication (Luo et al. 2010). Briefly, neurons were seeded at a density of 2.7 × 105 cells/cm2 in basal modified Eagle’s medium containing 10 % FBS, 25 mM KCl, 2 mM glutamine, and penicillin (100 U/ml)/streptomycin (100 μg/ml). Cytosine arabinoside (10 μM) was added to the culture medium 24 h after plating to limit the growth of non-neuronal cells. Granule cells were identified by a combination of several criteria including their size, shape and relative proportion of the total cell population as determined by phase contrast microscopy.
Measurement of Cell Viability
Cell viability was determined by the activity of mitochondrial dehydrogenases via the 3(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) assay as previously described (Cui et al. 2013, 2014). Briefly, after treatment, 10 μl of 5 mg/ml MTT solution was added to each well. Plates were incubated at 37 °C for 4 h in a humidified incubator. 100 μl of the solvating solution (0.01 N HCl in 10 % SDS solution) was then added to each well for 16–20 h. The absorbance of the samples was measured at a wavelength of 570 nm with 655 nm as a reference wavelength. Unless otherwise indicated, the extent of MTT conversion in cells without treatment is expressed as a percentage of the control.
Measurements of Lactate Dehydrogenase (LDH) Release
Cytotoxicity was evaluated by measuring the release of LDH after drug treatment for 24 h. Cells were incubated with 2 % (v/v) Triton X-100 in culture medium for 30 min at 37 °C to obtain a maximal LDH release as the positive control with 100 % toxicity. Extracellular LDH release was further determined in the conditioned media collected from the culture dishes using the LDH assay kit (Roche Diagnostics, Indianapolis, IN, USA) according to manufacturer’s instructions. Briefly, 50 μl culture supernatants were collected from each well with the addition of 50 μl reaction buffer. Thirty minutes after mixing at room temperature, the release of LDH was measured at a wavelength of 490 nm with 655 nm as a reference wavelength.
Fluorescein Diacetate/Propidium Iodide Double Staining Assay
Viable cells were visualized by the fluorescein formed from fluorescein diacetate (FDA) by esterase activity in viable cells. Non-viable cells were stained by propidium iodide (PI) intercalation into DNA, which only penetrates the cell membranes of dead cells. Briefly, after incubation with 10 μg/ml of FDA and 5 μg/ml of PI for 15 min, the cells were examined and images were acquired using UV light microscopy for comparison with photos taken under phase contrast microscopy. To quantitative evaluate cell viability, photos of each well were taken from five random fields and the number of PI-positive cells and FDA-positive cells was counted. % of cell viability = [number of FDA-positive cells/(number of PI-positive cells + number of FDA-positive cells)] × 100 % was then averaged (Jones and Senft 1985).
Chromatin condensation was detected by staining the cell nuclei with Hoechst-33342 as described previously (Cui et al. 2012; Hu et al. 2014). After treatment, cells grown in six-well plates were washed with ice-cold phosphate-buffered saline (PBS), fixed with 4 % formaldehyde in PBS for 15 min, membrane permeabilized in 0.1 % Triton X-100 for 15 min, and blocked in 1 % bovine serum albumin (BSA) for 15 min. Cells were then stained with Hoechst-33342 (5 μg/ml) at 4 °C for 5 min. Images were acquired using a fluorescence microscope (Nikon Instruments Inc. Melville, NY, USA) at 100× magnification. Ultraviolet excitation and emission wavelengths were used to obtain images of nuclei labeled with Hoechst-33342. To quantify the percentage of apoptotic nuclei in each group, photos of each well were taken from five random fields and the number of pyknotic nuclei and total nuclei counted. The percentage of pyknotic nuclei was then averaged.
Western Blot Analysis
Western blotting was performed as previously described (Hu et al. 2013). Protein lysates were separated on SDS-polyacrylamide gels and transferred onto polyvinyldifluoride membranes. After membrane blocking, proteins were detected using primary antibodies. After incubation at 4 °C overnight, signals were obtained after binding to HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Blots were developed using the enhanced chemiluminescence plus kit (Amersham Bioscience, Aylesbury, UK) and exposed to autoradiographic film.
Results are expressed as mean ± SEM. Analysis of variance (ANOVA) followed by Dunnett’s or Tukey’s test was used for statistical comparisons. Levels of p < 0.05 were considered to be of statistical significance.
Indirubin-3-Oxime Prevents H2O2-Induced Neuronal Apoptosis in SH-SY5Y Cells
Indirubin-3-Oxime Prevents H2O2-Induced Neuronal Apoptosis in CGNs
Activation of GSK3β and the ERK Signaling are Involved in the Neurotoxicity Induced by H2O2 in SH-SY5Y Cells
Indirubin-3-Oxime Prevents the Activation of GSK3β by H2O2
Indirubin-3-Oxime Inhibits the Activation of ERK Signaling Induced by H2O2
In this study we have shown that indirubin-3-oxime effectively prevents H2O2-induced neuronal apoptosis. Moreover, our results suggest that the neuroprotective effects of indirubin-3-oxime were mediated through simultaneous inhibition of GSK3β and the ERK signaling.
H2O2-induced oxidative stress in neurons can lead to both apoptotic and necrotic death depending on the concentration of H2O2 used (Valencia and Moran 2004; Fatokun et al. 2007). Compounds with anti-apoptotic properties may have therapeutic utility in neurodegenerative diseases characterized by neuronal apoptosis (Bachis et al. 2001). In our study, we have shown that H2O2 could significantly increase the number of pyknotic bodies, suggesting that H2O2 mainly induces neuronal apoptosis rather than necrosis in SH-SY5Y cells, which is consistent with other reports that exposure of 150 μM H2O2 for 24 h produces neuronal apoptosis in SH-SY5Y cells (Tian et al. 2015).
Indirubin-3-oxime has previously been reported to have neuroprotective effects (Sharma and Taliyan 2014; 2015). It was reported that systemic administration of indirubin-3-oxime (30 mg/kg i.v. or 20 mg/kg i.p.) could easily cross the blood–brain barrier, and retain in the brain at 4 h after administration, suggesting that indirubin-3-oxime might be used in the treatment of neurological diseases (Selenica et al. 2007). Interestingly, previous studies have also shown that indirubin-3-oxime can suppress excessive ROS production in both lipopolysaccharide-induced primary microglia cultures and 6OHDA-induced PC12 cells, suggesting that its neuroprotective activity may be mediated by anti-oxidative properties (Jung et al. 2010; Hu et al. 2015). In our study, we demonstrated that indirubin-3-oxime prevented H2O2-induced neuronal death not only in SH-SY5Y cells, but also in primary CGNs, providing a strong support that indirubin-3-oxime could effectively protected against ROS-induced neuronal loss and may be used in the treatment of neurological diseases.
How does indirubin-3-oxime produce neuroprotective effects in our model? We have evaluated the effects of indirubin-3-oxime on ROS scavenge using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, and found that indirubin-3-oxime (0.1–10 μM) could not decrease DPPH radical, suggesting that the neuroprotective effects of indirubin-3-oxime might not due to ROS scavenge (Data not shown). We speculated that indirubin-3-oxime might regulate pro-survival and pro-apoptotic signaling pathways that are involved in H2O2-induced apoptosis. Indirubin-3-oxime is a potent and direct inhibitor of GSK3β with an IC50 of 22 nM (Eisenbrand et al. 2004). In our study, we found that indirubin-3-oxime prevents the H2O2-induced decrease of pSer9-GSK3β. We also showed that SB415286, another small molecule of GSK3β inhibitor, prevented H2O2-induced neuronal apoptosis, supporting the role of GSK3β inhibition in the neuroprotective effects of indirubin-3-oxime. However, indirubin-3-oxime (3 μM) could almost fully prevent H2O2-induced cell death. SB415286 could not fully reversed H2O2-induced cell death at high concentrations, suggesting that indirubin-3-oxime might act on other neuroprotective target(s) besides GSK3β.
Activation of the ERK signaling has been regarded as one of the key pathways mediating neuronal apoptosis (Jiang et al. 2005). We found that indirubin-3-oxime prevents the H2O2-induced increase of pERK. Furthermore, we show that treatment with MEK inhibitors can also prevent H2O2-induced neuronal apoptosis, suggesting that inhibition of the ERK signaling may also be involved in the neuroprotective effects of indirubin-3-oxime. How could indirubin-3-oxime act on the ERK pathway? Indirubin-3-oxime could compete with ATP for binding to the catalytic subunit of kinase (Zhang et al. 2009). Due to the similarities of ATP binding site among different kinases, indirubin-3-oxime inhibits many kinases, including GSK3β, cyclin dependent kinase (CDK) 1, CDK2, and CDK5 (Rivest et al. 2011). We speculated that indirubin-3-oxime might directly act on the ATP binding site of the upstream kinases of ERK, e.g., MEK, and inhibit the phosphorylation of ERK. To further determine which kinase(s) are directly inhibited by indirubin-3-oxime, additional experiments are being undertaken in our lab.
How could PI3-K inhibitors abolish the neuroprotective effects of indirubin-3-oxime when GSK3β was inhibited by indirubin-3-oxime? Previous studies have shown that there is a crosstalk between the PI3-K and the ERK pathways (Frebel and Wiese 2006; Yang et al. 2005; Heras-Sandoval et al. 2014). Inactivation of PI3-K triggered the activation of the ERK pathway. We speculated that PI3-K inhibitors might abolish indirubin-3-oxime-induced neuroprotective effects independent of GSK3β activation, possibly via the activation of the ERK pathway.
In summary, we have found that indirubin-3-oxime prevents H2O2-induced neuronal apoptosis via concurrent inhibition of GSK3β and the ERK signaling. Our results also provide support for the use of indirubin-3-oxime or similar compounds in the treatment neurodegenerative disorders caused or exacerbated by oxidative stress.
This work was supported by the Natural Science Foundation of Zhejiang Province (LY15H310007), Research Grants Council of Hong Kong (561011 & 15101014), the Applied Research Project on Nonprofit Technology of Zhejiang Province (2016C37110), the National Natural Science Foundation of China (U1503223, 81202150, 81471398), the Ningbo International Science and Technology Cooperation Project (2014D10019), Ningbo municipal innovation team of life science and health (2015C110026), Ningbo Natural Science Foundation (2015A610219), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, the State Education Ministry, and the K. C. Wong Magna Fund in Ningbo University.
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