title: Shikonin protects dopaminergic cell line PC12 against 6-hydroxydopamine-mediated neurotoxicity via both glutathione-dependent and independent pathways and by inhibiting apoptosis.
abstract: We have investigated the mechanism of shikonin function on protection of dopaminergic neurons against 6-OHDA-induced neurotoxicity. Treatment of rat pheochromocytoma cell line PC12 by serial dilutions of shikonin determined 10 μM of the compound as its optimum concentration for protection saving nearly 70 % of the cells against toxicity. Reverse transcription-PCR analysis of shikonin-treated cells showed threefold increase in mRNA levels of glutathione peroxidase-1 (GPX-1) as a representative component of the intracellular anti-oxidant defense system. To elucidate shikonin-GPX1 relationships and maximize protection, we transduced PC12 cells using recombinant lentivirus vectors that harbored GPX-1 coding sequence. This change upregulated GPX-1 expression, increased peroxidase activity and made neuronal cells resistant to 6-OHDA-mediated toxicity. More importantly, addition of shikonin to GPX1-overexpressing PC12 cells augmented GPX-1 protein content by eightfold leading to fivefold increase of enzymatic activity, 91 % cell survival against neurotoxicity and concomitant increases in intracellular glutathione (GSH) levels. Depletion of intracellular GSH rendered all cell groups highly susceptible to toxicity; however, shikonin was capable of partially saving them. Subsequently, GSH-independent superoxide dismutase mRNA was found upregulated by shikonin. As signs of apoptosis inhibition, the compound upregulated Bcl-2, downregulated Bax, and prevented cell nuclei from undergoing morphological changes typical of apoptosis. Also, a co-staining method demonstrated GPX-1 overexpression significantly increases the percent of live cells that is maximized by shikonin treatment. Our data indicate that shikonin as an antioxidant compound protects dopaminergic neurons against 6-OHDA toxicity and enhances their survival via both glutathione-dependent and direct anti-apoptotic pathways.
authors: National Institute of Genetic Engineering and Biotechnology (NIGEB), Pazhoohesh Blvd, Tehran-Karaj HWY Kilometer 15, PO Box 14965/161, Tehran, I. R. Iran.
authors: Emran
authors: E
authors: Esmaeilzadeh
authors: Mossa
authors: M
authors: Gardaneh
authors: Ehsan
authors: E
authors: Gharib
authors: Farzaneh
authors: F
authors: Sabouni
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001483:Base Sequence; D017931:DNA Primers; D004298:Dopamine / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D005979:Glutathione Peroxidase / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D009285:Naphthoquinones / Q000494:pharmacology*; D016627:Oxidopamine / Q000494:pharmacology*; D016716:PC12 Cells; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D009285:Naphthoquinones; C016101:shikonin; D016627:Oxidopamine; D005979:Glutathione Peroxidase; D005978:Glutathione; D004298:Dopamine
references: Jpn J Cancer Res. 1992 Dec;83(12):1273-8
references: 1483942
references: Protein Sci. 1998 Dec;7(12):2688-90
references: 9865966
references: J Mol Neurosci. 2012 Mar;46(3):654-65
references: 21969113
references: Ann Surg Oncol. 2012 Sep;19(9):3097-106
references: 22446899
references: FEBS Lett. 1992 Jul 27;307(1):108-12
references: 1322323
references: J Nat Prod. 1986 May-Jun;49(3):466-9
references: 3760886
references: J Neurochem. 1989 Jun;52(6):1830-6
references: 2723638
references: J Neural Transm Suppl. 1997;50:79-88
references: 9120427
references: Cell Mol Life Sci. 2004 Jan;61(2):192-208
references: 14745498
references: Breast Cancer Res Treat. 2010 Nov;124(2):585-91
references: 20623181
references: Diabetes. 2000 Jan;49(1):101-7
references: 10615956
references: J Med Chem. 1995 Mar 17;38(6):917-22
references: 7699708
references: Curr Med Chem. 2008;15(30):3248-67
references: 19075667
references: Anticancer Res. 1998 Mar-Apr;18(2A):783-90
references: 9615720
references: J Biol Chem. 1992 Mar 25;267(9):5949-58
references: 1556108
references: Bioorg Med Chem. 2000 Nov;8(11):2561-9
references: 11092541
references: Chin J Physiol. 2009 Nov 30;52(5 Suppl):376-83
references: 20359128
references: J Pharmacol Exp Ther. 2000 Jun;293(3):982-8
references: 10869401
references: J Bioenerg Biomembr. 2009 Dec;41(6):469-72
references: 19967436
references: Ann N Y Acad Sci. 2008 Dec;1147:93-104
references: 19076434
references: Arch Pharm (Weinheim). 2002 Jun;335(6):262-6
references: 12210768
references: FASEB J. 1999 Aug;13(11):1467-75
references: 10428770
references: Oxid Med Cell Longev. 2012;2012:781516
references: 23119122
references: Cell Tissue Res. 2011 May;344(2):313-20
references: 21437657
references: Eur J Pharmacol. 2010 Sep 25;643(2-3):211-7
references: 20599918
references: Rejuvenation Res. 2013 Jun;16(3):185-99
references: 23472740
references: J Agric Food Chem. 2004 Oct 6;52(20):6330-7
references: 15453709
references: Chin Med. 2009 Jul 11;4:14
references: 19594888
references: Rejuvenation Res. 2011 Apr;14(2):195-204
references: 21222532
references: Biochim Biophys Acta. 1998 Apr 10;1380(2):159-62
references: 9565677
references: Annu Rev Nutr. 2007;27:41-61
references: 17465855
references: J Neurol Sci. 2012 Nov 15;322(1-2):254-62
references: 22669122