title: Thymoquinone inhibits autophagy and induces cathepsin-mediated, caspase-independent cell death in glioblastoma cells.
abstract: Glioblastoma is the most aggressive and common type of malignant brain tumor in humans, with a median survival of 15 months. There is a great need for more therapies for the treatment of glioblastoma. Naturally occurring phytochemicals have received much scientific attention because many exhibit potent tumor killing action. Thymoquinone (TQ) is the bioactive compound of the Nigella sativa seed oil. TQ has anti-oxidant, anti-inflammatory and anti-neoplastic actions with selective cytotoxicity for human cancer cells compared to normal cells. Here, we show that TQ selectively inhibits the clonogenicity of glioblastoma cells as compared to normal human astrocytes. Also, glioblastoma cell proliferation could be impaired by chloroquine, an autophagy inhibitor, suggesting that glioblastoma cells may be dependent on the autophagic pathway for survival. Exposure to TQ caused an increase in the recruitment and accumulation of the microtubule-associated protein light chain 3-II (LC3-II). TQ also caused an accumulation of the LC3-associated protein p62, confirming the inhibition of autophagy. Furthermore, the levels of Beclin-1 protein expression were unchanged, indicating that TQ interferes with a later stage of autophagy. Finally, treatment with TQ induces lysosome membrane permeabilization, as determined by a specific loss of red acridine orange staining. Lysosome membrane permeabilization resulted in a leakage of cathepsin B into the cytosol, which mediates caspase-independent cell death that can be prevented by pre-treatment with a cathepsin B inhibitor. TQ induced apoptosis, as determined by an increase in PI and Annexin V positive cells. However, apoptosis appears to be caspase-independent due to failure of the caspase inhibitor z-VAD-FMK to prevent cell death and absence of the typical apoptosis related signature DNA fragmentation. Inhibition of autophagy is an exciting and emerging strategy in cancer therapy. In this vein, our results describe a novel mechanism of action for TQ as an autophagy inhibitor selectively targeting glioblastoma cells.
authors.affiliation: Department of Radiology, The Ohio State University Wexner Medical Center, Columbus, Ohio, United States of America.
authors.forename: Ira O
authors.initials: IO
authors.lastname: Racoma
authors.forename: Walter Hans
authors.initials: WH
authors.lastname: Meisen
authors.forename: Qi-En
authors.initials: QE
authors.lastname: Wang
authors.forename: Balveen
authors.initials: B
authors.lastname: Kaur
authors.forename: Altaf A
authors.initials: AA
authors.lastname: Wani
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects*; D016227:Benzoquinones / Q000494:pharmacology*; D020169:Caspases / Q000378:metabolism*; D002403:Cathepsins / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005909:Glioblastoma / Q000378:metabolism*; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D008954:Models, Biological; D010539:Permeability / Q000187:drug effects
chemical_list: D000970:Antineoplastic Agents; D016227:Benzoquinones; D002403:Cathepsins; D020169:Caspases; C003466:thymoquinone
references.citation: Ann Intern Med. 2006 Mar 7;144(5):337-43
references.pmid: 16520474
references.citation: Cancer Res. 2008 Mar 1;68(5):1485-94
references.pmid: 18316613
references.citation: Carcinogenesis. 2008 Mar;29(3):600-9
references.pmid: 18174255
references.citation: Oncogene. 2008 Oct 27;27(50):6434-51
references.pmid: 18955971
references.citation: J Cell Biol. 1981 Sep;90(3):656-64
references.pmid: 7287819
references.citation: Int J Cancer. 2009 Mar 1;124(5):1060-71
references.pmid: 19048625
references.citation: Blood. 2011 Dec 22;118(26):6893-903
references.pmid: 22042694
references.citation: J Chem Biol. 2011 Jul;4(3):97-107
references.pmid: 22229047
references.citation: Curr Opin Investig Drugs. 2009 Dec;10(12):1269-79
references.pmid: 19943199
references.citation: Bioorg Med Chem. 2006 May 1;14(9):3218-22
references.pmid: 16413786
references.citation: J Thorac Oncol. 2012 Oct;7(10):1602-8
references.pmid: 22878749
references.citation: Cell Biol Toxicol. 2006 Nov;22(6):393-408
references.pmid: 16897440
references.citation: Phytomedicine. 2004 Jan;11(1):56-64
references.pmid: 14971722
references.citation: PLoS One. 2010 Aug 12;5(8):e12124
references.pmid: 20711342
references.citation: Exp Biol Med (Maywood). 2010 Jun;235(6):751-60
references.pmid: 20511679
references.citation: Int J Oncol. 2003 Jan;22(1):107-13
references.pmid: 12469192
references.citation: Mutat Res. 2011 Jan 10;706(1-2):28-35
references.pmid: 21040738
references.citation: J Biol Chem. 2009 Aug 14;284(33):21783-21787
references.pmid: 19473965
references.citation: Oncogene. 2001 Jun 21;20(28):3665-73
references.pmid: 11439329
references.citation: Leukemia. 2011 Jan;25(1):23-31
references.pmid: 20927132
references.citation: Autophagy. 2012 May 1;8(5):739-53
references.pmid: 22498493
references.citation: Int J Oncol. 2004 Oct;25(4):857-66
references.pmid: 15375533
references.citation: Cancer Res. 2004 Jan 1;64(1):27-30
references.pmid: 14729603
references.citation: Mol Cancer. 2010 Jun 01;9:135
references.pmid: 20515495
references.citation: Cancer Res. 2006 Sep 1;66(17):8511-9
references.pmid: 16951163
references.citation: J Neurosurg. 2003 Dec;99(6):1047-52
references.pmid: 14705733
references.citation: J Cell Biol. 1987 Sep;105(3):1253-65
references.pmid: 3308906
references.citation: Cancer Res. 2009 Dec 1;69(23):8910-7
references.pmid: 19903849
references.citation: Cancer Res. 2007 Mar 1;67(5):2217-25
references.pmid: 17332352
references.citation: Cancer Gene Ther. 2002 Mar;9(3):228-35
references.pmid: 11896438
references.citation: Cancer Cell. 2006 Jul;10(1):51-64
references.pmid: 16843265
references.citation: Hum Gene Ther. 2005 Feb;16(2):209-22
references.pmid: 15761261
references.citation: CSH Protoc. 2008 Mar 01;2008:pdb.prot4929
references.pmid: 21356782
references.citation: Int J Cancer. 2005 Nov 10;117(3):409-17
references.pmid: 15906362
references.citation: Genes Dev. 2007 Nov 15;21(22):2861-73
references.pmid: 18006683
references.citation: Int J Cancer. 2009 Aug 1;125(3):717-22
references.pmid: 19431142
references.citation: J Virol. 2007 Apr;81(8):4186-98
references.pmid: 17287256
references.citation: Autophagy. 2012 Apr;8(4):445-544
references.pmid: 22966490
references.citation: Eur J Pharmacol. 2009 Dec 25;625(1-3):220-33
references.pmid: 19836374
references.citation: Clin Cancer Res. 2006 Aug 1;12(15):4738-46
references.pmid: 16899625
references.citation: Proc Natl Acad Sci U S A. 2006 Mar 7;103(10):3604-9
references.pmid: 16505371
references.citation: Autophagy. 2008 Feb;4(2):151-75
references.pmid: 18188003
references.citation: Invest New Drugs. 2012 Oct;30(5):1813-9
references.pmid: 21881916
references.citation: Cell. 2010 Feb 5;140(3):313-26
references.pmid: 20144757
references.citation: Arch Ophthalmol. 2011 Jan;129(1):30-9
references.pmid: 21220626
references.citation: CSH Protoc. 2008 Feb 01;2008:pdb.prot4927
references.pmid: 21356764
references.citation: Apoptosis. 2010 Feb;15(2):183-95
references.pmid: 19882352
references.citation: Autophagy. 2007 Nov-Dec;3(6):542-5
references.pmid: 17611390
references.citation: Mol Cell Biol. 2005 Feb;25(3):1025-40
references.pmid: 15657430
references.citation: Int J Biochem Cell Biol. 2006;38(8):1249-53
references.pmid: 16314136
references.citation: Genes Dev. 2011 Apr 1;25(7):717-29
references.pmid: 21406549
references.citation: J Cell Sci. 2005 Jul 15;118(Pt 14):3091-102
references.pmid: 15985464
references.citation: J Biol Chem. 2011 Feb 25;286(8):6602-13
references.pmid: 21148553
references.citation: Neurol Res Int. 2012;2012:428565
references.pmid: 22530121
references.citation: Mol Cancer Ther. 2008 Jun;7(6):1461-71
references.pmid: 18566217
references.citation: Biochim Biophys Acta. 2009 Sep;1793(9):1524-32
references.pmid: 19371598
references.citation: J Clin Oncol. 2008 Jan 20;26(3):493-500
references.pmid: 18202424
references.citation: Nat Rev Cancer. 2005 Nov;5(11):886-97
references.pmid: 16239905
references.citation: Clin Cancer Res. 2005 May 1;11(9):3155-62
references.pmid: 15867207
references.citation: Food Chem Toxicol. 2009 Jan;47(1):129-33
references.pmid: 19010375
references.citation: Cell Death Differ. 2007 Mar;14(3):548-58
references.pmid: 16946731
references.citation: Cell Biol Toxicol. 2012 Jun;28(3):125-31
references.pmid: 22431173
references.citation: Clin Cancer Res. 2009 Sep 1;15(17):5308-16
references.pmid: 19706824
references.citation: Eur J Pharmacol. 2006 Aug 14;543(1-3):40-7
references.pmid: 16828080