Planta Medica Letters 2015; 2(1): e15-e18
DOI: 10.1055/s-0035-1545936
Letter
Georg Thieme Verlag KG Stuttgart · New York

Neuroprotective Effect of Demethylsuberosin, a Proteasome Activator, against MPP+-induced Cell Death in Human Neuroblastoma SH-SY5Y Cells

Bo-Hyung Kim
1   Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul, Republic of Korea
,
Jaeyoung Kwon
2   Department of Biosystems and Biotechnology, Korea University, Seoul, Republic of Korea
,
Dongho Lee
2   Department of Biosystems and Biotechnology, Korea University, Seoul, Republic of Korea
,
Woongchon Mar
1   Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul, Republic of Korea
› Author Affiliations
Further Information

Correspondence

Prof. Dr. Woongchon Mar
Natural Products Research Institute, College of Pharmacy, Seoul National University
599 Gwanangro, Gwanak-Gu
Seoul 151–742
Republic of Korea
Phone: +82 28 80 24 73   
Fax: +82 28 88 91 22   

 

Prof. Dr. Dongho Lee
Korea University, Department of Biosystems and Biotechnology
145 Anam-ro, Seongbuk-gu
Seoul 136-701
Republic of Korea
Phone: +82 2 32 90 30 17   
Fax: +82 29 53 07 37   

Publication History

received 21 December 2014
revised 13 March 2015

accepted 22 March 2015

Publication Date:
23 April 2015 (online)

 

Abstract

Demethylsuberosin isolated from the roots of Cudrania tricuspidata demonstrated a potent proteasome activator by enhancing all three chymotrypsin-like, trypsin-like, and caspase-like proteasome activities in a 20S proteasome activity assay. It also attenuated the 1-methyl-4-phenylpyridinium-induced dysfunction of the chymotrypsin-like and caspase-like activities of proteasome in SH-SY5Y cells with EC50 values of 0.76 µM and 0.82 µM, respectively. Additionally, demethylsuberosin protected neuronal cells against 1-methyl-4-phenylpyridinium-induced cell death with an EC50 value of 0.17 µM, while the EC50 value of betulinic acid was 4.29 µM. We are reporting that demethylsuberosin is a potent proteasome activator with a neuroprotective effect, suggesting a possible candidate for the protection or treatment of neurodegenerative diseases such as Parkinsonʼs disease.


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Cudrania tricuspidata (Carr.) Bureau ex Lavallee, belonging to the Moraceae family, is a small thorny tree widely distributed in Korea, Japan, and China, and its neuroprotective [1], anti-inflammatory [2], and antioxidant [3] effects have been reported. Recently it was reported that xanthones from the root bark [4] and isoflavones from the fruit [5] of C. tricuspidata reveleaed neuroprotective effects against 6-hydroxydopamine (6-OHDA)-induced cell death in SH-SY5Y cells. Demethylsuberosin, a prenylated coumarin, was isolated from the root of C. tricuspidata, and it was reported that demethylsuberosin showed a significant feeding deterrence effect against instars of Spodoptera exigua [6] and anti-inflammatory activity [7]. While many naturally occurring or synthetic proteasome inhibitors like epoxomicin and carfilzomib [8], [9] have been substantially reported, proteasome activators have been rarely reported except for betulinic acid. Betulinic acid, as such, is a naturally occurring compound and it activates the chymotrypsin-like property of proteasome [10]. Neurodegenerative diseases such as Parkinsonʼs disease are characteristic of the failure of the ubiquitin proteasome system (UPS) [11]. It was reported that proteasome activator 700 (PA700) and proteasome activator 28 (PA28), the cellular proteasome activators, showed decreased activity in the pars compacta of the substantia nigra in sporadic Parkinsonʼs diseases [12]. 1-Methyl-4-phenylpyridinium (MPP+) is a Parkinsonism-inducing neurotoxin and it causes specific cell death in dopaminergic neurons. The rapid accumulation of MPP+ in the mitochondrial matrix inhibits mitochondrial respiratory chain complex I (NADH : ubiquinone oxidoreductase), resulting in the depletion of adenosine triphosphate (ATP) synthesis [13]. The depletion of ATP synthesis generates reactive oxygen species (ROS) and inhibits the function of ATP-dependent UPS, leading to the disruption of the unfolded protein response (UPR), and finally causes neuronal cell death [14]. We are reporting that demethylsuberosin potently activates proteasome and it has a neuroprotective effect.

Demethylsuberosin ([Fig. 1]) (> 95 % purity) was isolated from the root of C. tricuspidata, and its structure was characterized by previously reported spectroscopic data [15]. As shown in [Fig. 2], demethylsuberosin dose-dependently activated the chymotrypsin-like, trypsin-like, and caspase-like proteasome activities in a 20S proteasome activity assay, whereas the control compound, betulinic acid (> 97 % purity, Enzo Life Science), revealed a less potent proteasome activity than demethylsuberosin. As shown in [Fig. 3], demethylsuberosin attenuated the MPP+-induced dysfunction of the chymotrypsin-like and caspase-like activities of proteasome in SH-SY5Y cells (Human neuroblastoma; ATCC No. CRL-2266) with EC50 values of 0.76 µM and 0.82 µM, respectively, but not the trypsin-like activity. Betulinic acid revealed a less potent proteasome activity with an EC50 value of 3.56 µM (chymotrypsin-like activity) and 3.66 µM (caspase-like activity). As shown in [Fig. 4], demethylsuberosin protected neuronal cells against MPP+-induced cell death with an EC50 value of 0.17 µM, while the EC50 value of betulinic acid was 4.29 µM.

Zoom Image
Fig. 1 Chemical structure of demethylsuberosin.
Zoom Image
Fig. 2 Effects of demethylsuberosin on proteasome activities. Fluorogenic peptides used as substrates were Suc-LLVY-AMC (at 40 µM), Boc-LRR-AMC (at 40 µM), and Z-LLE-MCA (at 80 µM) for chymotrypsin-like (A), trypsin-like (B), and caspase-like (C) proteases activities, respectively. Betulinic acid was used as a control compound. The activity is given as a percentage of that of the control, and data represent the mean ± SD of three independent experiments; p < 0.01 and **p < 0.005, compared with the control group, respectively.
Zoom Image
Fig. 3 Effects of demethylsuberosin against MPP+-induced dysfunction of proteasome activities in SH-SY5Y cells. Cells were cultured in 48-well plates for 24 h and samples were simultaneously treated with MPP+ (2 mM) for 48 h. Betulinic acid was used as a control compound. The activity is given as a percentage of that of the control, and data represent the mean ± SD of three independent experiments; ##p < 0.005, compared with the control group; *p < 0.01 and **p < 0.005, compared with the MPP+-induced group, respectively.
Zoom Image
Fig. 4 Neuroprotective effects of demethylsuberosin against MPP+-induced cell death in SH-SY5Y cells. Cells were cultured in 96-well plates for 24 h and samples were simultaneously treated with MPP+ (2 mM) for 48 h. Betulinic acid was used as a control compound. The cell viability is given as a percentage of that of the control, and data represent the mean ± SD of three independent experiments; p < 0.005, compared with the control group; *p < 0.01 and **p < 0.005, compared with the MPP+-induced group, respectively.

UPS is critical for the degradation of damaged and aberrant proteins, and the accumulation of ubiquitinated proteins is a hallmark of many neurodegenerative diseases, such as Parkinsonʼs disease. Recent studies suggest that proteasomal impairment plays an important role in these diseases [14], [16]. In dopaminergic neurons, the rapid accumulation of MPP+ occurs in the mitochondrial matrix and it inhibits NADH dehydrogenase, resulting in the depletion of ATP synthesis and decreasing the function of ATP-dependent UPS [17]. It was reported that MPP+ upregulates and aggregates α-synuclein by dysfunction of the UPS and, finally, causes neuronal cell death [18]. It was also reported that an antioxidant, a keto-cartenoid astaxanthin [19], and verbascoside [20] protected neuronal cells against MPP+-induced cell death in SH-SY5Y cells. Based on our data, the neuroprotective effect of demethylsuberosin is partly due to the activation of proteasome, even though further research is needed in relation to the neuroprotective effect of demethylsuberosin and the UPS.

Materials and Methods

The root bark of C. tricuspidata was collected by the Korea Forest Research Institute, Southern Forest Research Center, Jinju, Korea, in September 2008 and authenticated by Dr. Hak Ju Lee (Korea Forest Research Institute, Seoul, Korea). A voucher specimen (accession number KH1–4–090 814) was deposited at the Department of Biosystems and Biotechnology, Korea University, Seoul, Korea.

The dried root bark of C. tricuspidata (13.0 kg) was ground and extracted with MeOH (48 L, 20 L, and 18 L) at room temperature, and the extracts were concentrated in vacuo at 35 °C. The dark brown residue (702.1 g) was suspended in H2O (4.0 L) and partitioned with n-hexane (4.0 × 5 L) and EtOAc (4.0 × 6 L), sequentially. The EtOAc-soluble fraction (213.0 g) was applied to a silica gel column (15 × 60 cm, mesh 230–400) using CHCl3/MeOH (1 : 0 to 1 : 1, 6 L for each eluent) to afford seven fractions (F1, 6 L; F2, 6 L; F3, 6 L; F4, 12 L; F5, 18 L; F6, 24 L; F7, 18 L). F4 (36.0 g) was fractionated on a silica gel column (10 × 60 cm, mesh 230–400) with n-hexane/EtOAc (30 : 1 to 0 : 1, 4 L for each eluent) to give eight fractions (F4.1, 7 L; F4.2, 3 L; F4.3, 4 L; F4.4, 4 L; F4.5, 3 L; F4.6, 7 L; F4.8, 9 L). F4.5 (7.2 g) was chromatographed on a silica gel column (10 × 60 cm, mesh 230–400) using CHCl3/MeOH (1 : 0 to 5 : 1, 2 L for each eluent) to afford seven fractions (F4.5.1 to F4.5.7, each 2 L), and F4.5.2 (4.6 g) was then subjected to a C18 reversed-phase silica gel column (6 × 70 cm, 75 µm) with MeOH/H2O (4 : 1 to 1 : 0, 5 L for each eluent) to give 15 fractions (F4.5.2.1 to F4.5.2.15; each 1 L). F4.5.2.2 (37.1 mg) was purified by preparative HPLC (YMC Pack ODS-A, 250 × 20 mm i. d., 5 µm, 50–85 % MeOH in H2O, flow rate 8.0 mL/min) to afford demethylsuberosin (2.9 mg, > 95 %).

SH-SY5Y cells in Dulbecoʼs modified Eagleʼs medium (DMEM) media were cultured in 96-well plates (5 × 104 cells/200 µL/well) for 24 h, and samples were simultaneously treated with MPP+ (2 mM) for 48 h. Cell viability was performed using the MTT assay by measuring at 540 nm using a microplate reader (SpectraMax Plus 384, Molecular Devices), as described by Carmichael [21].

The proteolytic activities of the proteasome were measured by using a 20S proteasome activity kit (APT 280, Millipore), as described by the manufacturerʼs instructions. Briefly, 10 µL of test compounds (final 1 % DMSO) were incubated in a provided buffer (final 100 µL) containing 40 µg of 20S proteasome and a substrate for 2 h at 37 °C. Fluorogenic peptides used as substrates were succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (Suc-LLVY-AMC at 40 µM), t-butyloxycarbonyl-Leu-Arg-Arg-7-amido-4-methylcoumarin (Boc-LRR-AMC at 40 µM), and benzyloxycarbonyl-Leu-Leu-Glu-4-methyl-coumaryl-7-amide (Z-LLE-MCA at 80 µM) for chymotrypsin-like, trypsin-like, and caspase-like proteases activities, respectively. Suc-LLVY-AMC included in the kit was used and Boc-LRR-AMC and Z-LLE-MCA were purchased from Enzo Life Sciences. Reaction mixtures containing substrates and test samples without 20S proteasome were used as blanks, and proteasome activities were measured by quantification of relative fluorescent units from the release of the fluorescent-cleaved products aminomethylcoumarin (AMC) (at 380/460 nm) or methylcoumarylamide (MCA) (at 380/440 nm) using a microplate reader (SpectraMax Plus 384, Molecular Devices).

Cell-based proteasome activity was determined using MPP+-treated SH-SY5Y cells, as described by Henrik Lovborg and coworkers [22]. Briefly, cells (1.0 × 105 cells/300 µL/well) were cultured in 48-well plates for 24 h in DMEM media supplemented with 10 % FBS. Samples and MPP+ (2 mM) were simultaneously treated for 48 h in DMEM media supplemented with 5 % FBS. The proteolytic activity of the proteasome was evaluated in cell lysates by using a proteasome activity kit (APT 280; Millipore). In brief, 40 µg of cell lysate were incubated for 2 h at 37 °C in the provided buffer with fluorophore-linked peptide substrates. We used Suc-LLVY-AMC, Boc-LRR-AMC, and Z-LLE-MCA as the same substrates. Reaction mixtures without cell lysates were used as blanks and AMC or MCA fluorescence was measured at excitation/emission wavelengths of 380/460 and 380/440 nm, respectively.

Data obtained are expressed as the mean ± standard deviation (SD). Statistical significance was determined using GraphPad Prism (GraphPad Software). The differences among groups were evaluated by one-way analysis of variance (ANOVA) with Bonferroniʼs multiple comparison method. A p value less than 0.05 was considered to be statistically significant. All the data were obtained from at least three independent experiments.

Supporting information

Spectroscopic data of demethylsuberosin including an HPLC chromatogram and microscopy images of the cells are available as Supporting Information.


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Acknowledgements

Plant materials and structural elucidation were supported by Korea University, and the research fund was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) (Grant no. NRF-2013R1A1A2008111). This work was supported by the BK21 Plus Program in 2014.


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Conflict of Interest

The authors declare no conflict of interest.

Supporting Information

  • References

  • 1 Jeong CH, Choi GN, Kim JH, Kwak JH, Jeong HR, Kim DO, Heo HJ. Protective effects of aqueous extract from Cudrania tricuspidata on oxidative stress-induced neurotoxicity. Food Sci Biotechnol 2010; 19: 1113-1117
  • 2 Seo WG, Pae HO, Oh GS, Chai KY, Yun YG, Kwon TO, Chung HT. Inhibitory effect of ethyl acetate fraction from Cudrania tricuspidata on the expression of nitric oxide synthase gene in RAW 264.7 macrophages stimulated with interferon-gamma and lipopolysaccharide. Gen Pharmacol 2000; 35: 21-28
  • 3 Lee BW, Lee JH, Lee ST, Lee HS, Lee WS, Jeong TS, Park KH. Antioxidant and cytotoxic activities of xanthones from Cudrania tricuspidata . Bioorg Med Chem Lett 2005; 15: 5548-5552
  • 4 Kwon J, Hiep NT, Kim DW, Hwang BY, Lee HJ, Mar W, Lee D. Neuroprotective Xanthones from the Root Bark of Cudrania tricuspidata . J Nat Prod 2014; 77: 1893-1901
  • 5 Hiep NT, Kwon J, Kim DW, Hwang BY, Lee HJ, Mar W, Lee D. Isoflavones with neuroprotective activities from fruits of Cudrania tricuspidata . Phytochemistry 2015; 111: 141-148
  • 6 Trumble JT, Millar JG. Biological activity of marmesin and demethylsuberosin against a generalist herbivore, Spodoptera exigua (Lepidoptera: Noctuidae). J Agric Food Chem 1996; 44: 2859-2864
  • 7 Ma Y, Jung JY, Choi JH, Jeong WS, Song YS, Kang JS, Bi K, Kim MJ. Anti-inflammatory activities of coumarins isolated from Angelica gigas Nakai on LPS-stimulated RAW 264.7 cells. J Food Sci Nutr 2009; 14: 179-187
  • 8 Sin N, Kim KB, Elofsson M, Meng L, Auth H, Kwok BH, Crews CM. Total synthesis of the potent proteasome inhibitor epoxomicin: a useful tool for understanding proteasome biology. Bioorg Med Chem Lett 1999; 9: 2283-2288
  • 9 Kuhn DJ, Chen Q, Voorhees PM, Strader JS, Shenk KD, Sun CM, Demo SD, Bennett MK, van Leeuwen FW, Chanan-Khan AA, Orlowski RZ. Potent activity of carfilzomib, a novel, irreversible inhibitor of the ubiquitin-proteasome pathway, against preclinical models of multiple myeloma. Blood 2007; 110: 3281-3290
  • 10 Huang L, Ho P, Chen CH. Activation and inhibition of the proteasome by betulinic acid and its derivatives. FEBS Lett 2007; 581: 4955-4959
  • 11 McNaught KS, Olanow CW, Halliwell B, Isacson O, Jenner P. Failure of the ubiquitin-proteasome system in Parkinsonʼs disease. Nat Rev Neurosci 2001; 2: 589-594
  • 12 McNaught KS, Belizaire R, Isacson O, Jenner P, Olanow CW. Altered proteasomal function in sporadic Parkinsonʼs disease. Exp Neurol 2003; 179: 38-46
  • 13 Mizuno Y, Suzuki K, Sone N, Saitoh T. Inhibition of ATP synthesis by 1-methyl-4-phenylpyridinium ion (MPP+) in isolated mitochondria from mouse brains. Neurosci Lett 1987; 81: 204-208
  • 14 Lim KL. Ubiquitin-proteasome system dysfunction in Parkinsonʼs disease: current evidence and controversies. Expert Rev Proteomics 2007; 4: 769-781
  • 15 Jiang H, Hamada Y. Highly enantioselective synthesis of angelmarin. Org Biomol Chem 2009; 7: 4173-4176
  • 16 Jansen AH, Reits EA, Hol EM. The ubiquitin proteasome system in glia and its role in neurodegenerative diseases. Front Mol Neurosci 2014; 7: 73
  • 17 Ramsay RR, Salach JI, Singer TP. Uptake of the neurotoxin 1-methyl-4-phenylpyridine (MPP+) by mitochondria and its relation to the inhibition of the mitochondrial oxidation of NAD+-linked substrates by MPP+. Biochem Biophys Res Commun 1986; 134: 743-748
  • 18 Chiba K, Trevor AJ, Castagnoli jr. N. Active uptake of MPP+, a metabolite of MPTP, by brain synaptosomes. Biochem Biophys Res Commun 1985; 128: 1228-1232
  • 19 Lee DH, Kim CS, Lee YJ. Astaxanthin protects against MPTP/MPP+-induced mitochondrial dysfunction and ROS production in vivo and in vitro . Food Chem Toxicol 2011; 49: 271-280
  • 20 Sheng GQ, Zhang JR, Pu XP, Ma J, Li CL. Protective effect of verbascoside on 1-methyl-4-phenylpyridinium ion-induced neurotoxicity in PC12 cells. Eur J Pharmacol 2002; 451: 119-124
  • 21 Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res 1987; 47: 936-942
  • 22 Lovborg H, Oberg F, Rickardson L, Gullbo J, Nygren P, Larsson R. Inhibition of proteasome activity, nuclear factor-KappaB translocation and cell survival by the antialcoholism drug disulfiram. Int J Cancer 2006; 118: 1577-1580

Correspondence

Prof. Dr. Woongchon Mar
Natural Products Research Institute, College of Pharmacy, Seoul National University
599 Gwanangro, Gwanak-Gu
Seoul 151–742
Republic of Korea
Phone: +82 28 80 24 73   
Fax: +82 28 88 91 22   

 

Prof. Dr. Dongho Lee
Korea University, Department of Biosystems and Biotechnology
145 Anam-ro, Seongbuk-gu
Seoul 136-701
Republic of Korea
Phone: +82 2 32 90 30 17   
Fax: +82 29 53 07 37   

  • References

  • 1 Jeong CH, Choi GN, Kim JH, Kwak JH, Jeong HR, Kim DO, Heo HJ. Protective effects of aqueous extract from Cudrania tricuspidata on oxidative stress-induced neurotoxicity. Food Sci Biotechnol 2010; 19: 1113-1117
  • 2 Seo WG, Pae HO, Oh GS, Chai KY, Yun YG, Kwon TO, Chung HT. Inhibitory effect of ethyl acetate fraction from Cudrania tricuspidata on the expression of nitric oxide synthase gene in RAW 264.7 macrophages stimulated with interferon-gamma and lipopolysaccharide. Gen Pharmacol 2000; 35: 21-28
  • 3 Lee BW, Lee JH, Lee ST, Lee HS, Lee WS, Jeong TS, Park KH. Antioxidant and cytotoxic activities of xanthones from Cudrania tricuspidata . Bioorg Med Chem Lett 2005; 15: 5548-5552
  • 4 Kwon J, Hiep NT, Kim DW, Hwang BY, Lee HJ, Mar W, Lee D. Neuroprotective Xanthones from the Root Bark of Cudrania tricuspidata . J Nat Prod 2014; 77: 1893-1901
  • 5 Hiep NT, Kwon J, Kim DW, Hwang BY, Lee HJ, Mar W, Lee D. Isoflavones with neuroprotective activities from fruits of Cudrania tricuspidata . Phytochemistry 2015; 111: 141-148
  • 6 Trumble JT, Millar JG. Biological activity of marmesin and demethylsuberosin against a generalist herbivore, Spodoptera exigua (Lepidoptera: Noctuidae). J Agric Food Chem 1996; 44: 2859-2864
  • 7 Ma Y, Jung JY, Choi JH, Jeong WS, Song YS, Kang JS, Bi K, Kim MJ. Anti-inflammatory activities of coumarins isolated from Angelica gigas Nakai on LPS-stimulated RAW 264.7 cells. J Food Sci Nutr 2009; 14: 179-187
  • 8 Sin N, Kim KB, Elofsson M, Meng L, Auth H, Kwok BH, Crews CM. Total synthesis of the potent proteasome inhibitor epoxomicin: a useful tool for understanding proteasome biology. Bioorg Med Chem Lett 1999; 9: 2283-2288
  • 9 Kuhn DJ, Chen Q, Voorhees PM, Strader JS, Shenk KD, Sun CM, Demo SD, Bennett MK, van Leeuwen FW, Chanan-Khan AA, Orlowski RZ. Potent activity of carfilzomib, a novel, irreversible inhibitor of the ubiquitin-proteasome pathway, against preclinical models of multiple myeloma. Blood 2007; 110: 3281-3290
  • 10 Huang L, Ho P, Chen CH. Activation and inhibition of the proteasome by betulinic acid and its derivatives. FEBS Lett 2007; 581: 4955-4959
  • 11 McNaught KS, Olanow CW, Halliwell B, Isacson O, Jenner P. Failure of the ubiquitin-proteasome system in Parkinsonʼs disease. Nat Rev Neurosci 2001; 2: 589-594
  • 12 McNaught KS, Belizaire R, Isacson O, Jenner P, Olanow CW. Altered proteasomal function in sporadic Parkinsonʼs disease. Exp Neurol 2003; 179: 38-46
  • 13 Mizuno Y, Suzuki K, Sone N, Saitoh T. Inhibition of ATP synthesis by 1-methyl-4-phenylpyridinium ion (MPP+) in isolated mitochondria from mouse brains. Neurosci Lett 1987; 81: 204-208
  • 14 Lim KL. Ubiquitin-proteasome system dysfunction in Parkinsonʼs disease: current evidence and controversies. Expert Rev Proteomics 2007; 4: 769-781
  • 15 Jiang H, Hamada Y. Highly enantioselective synthesis of angelmarin. Org Biomol Chem 2009; 7: 4173-4176
  • 16 Jansen AH, Reits EA, Hol EM. The ubiquitin proteasome system in glia and its role in neurodegenerative diseases. Front Mol Neurosci 2014; 7: 73
  • 17 Ramsay RR, Salach JI, Singer TP. Uptake of the neurotoxin 1-methyl-4-phenylpyridine (MPP+) by mitochondria and its relation to the inhibition of the mitochondrial oxidation of NAD+-linked substrates by MPP+. Biochem Biophys Res Commun 1986; 134: 743-748
  • 18 Chiba K, Trevor AJ, Castagnoli jr. N. Active uptake of MPP+, a metabolite of MPTP, by brain synaptosomes. Biochem Biophys Res Commun 1985; 128: 1228-1232
  • 19 Lee DH, Kim CS, Lee YJ. Astaxanthin protects against MPTP/MPP+-induced mitochondrial dysfunction and ROS production in vivo and in vitro . Food Chem Toxicol 2011; 49: 271-280
  • 20 Sheng GQ, Zhang JR, Pu XP, Ma J, Li CL. Protective effect of verbascoside on 1-methyl-4-phenylpyridinium ion-induced neurotoxicity in PC12 cells. Eur J Pharmacol 2002; 451: 119-124
  • 21 Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res 1987; 47: 936-942
  • 22 Lovborg H, Oberg F, Rickardson L, Gullbo J, Nygren P, Larsson R. Inhibition of proteasome activity, nuclear factor-KappaB translocation and cell survival by the antialcoholism drug disulfiram. Int J Cancer 2006; 118: 1577-1580

Zoom Image
Fig. 1 Chemical structure of demethylsuberosin.
Zoom Image
Fig. 2 Effects of demethylsuberosin on proteasome activities. Fluorogenic peptides used as substrates were Suc-LLVY-AMC (at 40 µM), Boc-LRR-AMC (at 40 µM), and Z-LLE-MCA (at 80 µM) for chymotrypsin-like (A), trypsin-like (B), and caspase-like (C) proteases activities, respectively. Betulinic acid was used as a control compound. The activity is given as a percentage of that of the control, and data represent the mean ± SD of three independent experiments; p < 0.01 and **p < 0.005, compared with the control group, respectively.
Zoom Image
Fig. 3 Effects of demethylsuberosin against MPP+-induced dysfunction of proteasome activities in SH-SY5Y cells. Cells were cultured in 48-well plates for 24 h and samples were simultaneously treated with MPP+ (2 mM) for 48 h. Betulinic acid was used as a control compound. The activity is given as a percentage of that of the control, and data represent the mean ± SD of three independent experiments; ##p < 0.005, compared with the control group; *p < 0.01 and **p < 0.005, compared with the MPP+-induced group, respectively.
Zoom Image
Fig. 4 Neuroprotective effects of demethylsuberosin against MPP+-induced cell death in SH-SY5Y cells. Cells were cultured in 96-well plates for 24 h and samples were simultaneously treated with MPP+ (2 mM) for 48 h. Betulinic acid was used as a control compound. The cell viability is given as a percentage of that of the control, and data represent the mean ± SD of three independent experiments; p < 0.005, compared with the control group; *p < 0.01 and **p < 0.005, compared with the MPP+-induced group, respectively.