Involvement of VDAC, Bax and ceramides in the efflux of AIF from mitochondria during curcumin-induced apoptosis.
BACKGROUND
We previously identified curcumin as a potent inducer of fibroblast apoptosis, which could be used to treat hypertrophic scar formation. Here we investigated the underlying mechanism of this process.


RESULTS
Curcumin-induced apoptosis could not be blocked by caspase-inhibitors and we could not detect any caspase-3/7 activity. Curcumin predominantly induced mitochondria-mediated ROS formation and stimulated the expression of the redox-sensitive pro-apoptotic factor p53. Inhibition of the pro-apoptotic signaling enzyme glycogen synthase kinase-3beta (GSK-3beta) blocked curcumin-induced apoptosis. Apoptosis was associated with high molecular weight DNA damage, a possible indicator of apoptosis-inducing factor (AIF) activity. Indeed, curcumin caused nuclear translocation of AIF, which could be blocked by the antioxidant N-acetyl cysteine. We next investigated how AIF is effluxed from mitochondria in more detail. The permeability transition pore complex (PTPC), of which the voltage-dependent anion channel (VDAC) is a component, could be involved since the VDAC-inhibitor DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) efficiently blocked AIF translocation. However, PTPC is not involved in AIF release since cyclosporine A, a specific inhibitor of the complex did not block apoptosis. Alternatively, the pro-apoptotic protein Bax could have formed mitochondrial channels and interacted with VDAC. Curcumin caused mitochondrial translocation of Bax, which was blocked by DIDS, suggesting a Bax-VDAC interaction. Interestingly, ceramide channels can also release apoptogenic factors from mitochondria and we found that addition of ceramide induced caspase-independent apoptosis. Surprisingly, this process could also be blocked by DIDS, suggesting the concerted action of Bax, VDAC and ceramide in the efflux of AIF from the mitochondrion.


CONCLUSIONS
Curcumin-induced fibroblast apoptosis is totally caspase-independent and relies on the mitochondrial formation of ROS and the subsequent nuclear translocation of AIF, which is released from a mitochondrial pore that involves VDAC, Bax and possibly ceramides. The composition of the AIF-releasing channel seems to be much more complex than previously thought.
Scharstuhl
Alwin
A
Department of Pharmacology and Toxicology, Radboud University Nijmegen Medical Centre, Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands.
Mutsaers
Henricus A M
HA
Pennings
Sebastiaan W C
SW
Russel
Frans G M
FG
Wagener
Frank A D T G
FA
D017209:Apoptosis / Q000187:drug effects*; D051033:Apoptosis Inducing Factor / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D002518:Ceramides / Q000502:physiology*; D003474:Curcumin / Q000494:pharmacology*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D008970:Molecular Weight; D021381:Protein Transport; D017382:Reactive Oxygen Species / Q000378:metabolism; D050994:Voltage-Dependent Anion Channels / Q000502:physiology*; D051028:bcl-2-Associated X Protein / Q000378:metabolism / Q000502:physiology*
D051033:Apoptosis Inducing Factor; D002518:Ceramides; D004791:Enzyme Inhibitors; D018840:HSP70 Heat-Shock Proteins; D017382:Reactive Oxygen Species; D050994:Voltage-Dependent Anion Channels; D051028:bcl-2-Associated X Protein; D004247:DNA; D020169:Caspases; D003474:Curcumin
Free Radic Biol Med. 2008 Nov 15;45(10):1403-12
18762247
Cell. 1996 Jul 12;86(1):147-57
8689682
Free Radic Biol Med. 2001 Sep 1;31(5):670-8
11522452
Mol Cell Biol. 2007 Jul;27(13):4844-62
17470554
Carcinogenesis. 2006 Aug;27(8):1636-44
16501251
J Appl Toxicol. 2008 May;28(4):466-74
17849503
Cell Physiol Biochem. 2008;22(1-4):177-86
18769044
J Biol Chem. 2003 Dec 5;278(49):48872-9
14523002
J Biol Chem. 2005 Nov 18;280(46):38729-39
16172114
Cell. 1995 Jan 27;80(2):293-9
7834749
Carcinogenesis. 2003 Jul;24(7):1199-208
12807727
J Biosci. 2007 Apr;32(3):595-610
17536179
Curr Opin Cell Biol. 2000 Aug;12(4):414-9
10873816
Cancer Biol Ther. 2005 Feb;4(2):139-63
15725726
Cell Death Differ. 2006 Aug;13(8):1378-86
16729025
Cell. 1997 Aug 8;90(3):405-13
9267021
EMBO J. 2003 Sep 1;22(17):4385-99
12941691
Nat Cell Biol. 2001 Sep;3(9):839-43
11533664
Science. 2002 Jul 12;297(5579):259-63
12114629
Nature. 1999 Jun 3;399(6735):483-7
10365962
Prog Neurobiol. 2006 Jul;79(4):173-89
16935409
FASEB J. 2005 Dec;19(14):1945-56
16319138
Science. 2006 Feb 10;311(5762):847-51
16469926
Mol Cancer Ther. 2006 Apr;5(4):927-34
16648563
Oncogene. 2008 Jul 10;27(30):4221-32
18362892
Biochem J. 2002 Dec 15;368(Pt 3):915-21
12193163
Nat Cell Biol. 2003 Feb;5(2):97-9
12563272
Cell. 2002 Nov 1;111(3):331-42
12419244
Nature. 1999 Feb 4;397(6718):441-6
9989411
Nat Cell Biol. 2000 Aug;2(8):476-83
10934467
Exp Cell Res. 1999 Jun 15;249(2):299-307
10366429
Nature. 2001 Jul 5;412(6842):95-9
11452314
Biophys J. 1999 Feb;76(2):725-34
9929477
FASEB J. 2003 Dec;17(15):2339-41
14563682
Neuroreport. 2003 Dec 19;14(18):2415-9
14663202
FEBS Lett. 2000 Jul 7;476(3):118-23
10913597
Science. 2004 Jul 30;305(5684):626-9
15286356
EMBO J. 1998 Apr 15;17(8):2215-23
9545235
Science. 1998 Aug 28;281(5381):1305-8
9721089
Mitochondrion. 2006 Jun;6(3):118-25
16713754
Science. 1998 Sep 25;281(5385):2027-31
9748162
Exp Cell Res. 2000 Apr 10;256(1):58-66
10739652
Science. 2004 Feb 13;303(5660):1010-4
14963330
J Cell Mol Med. 2009 Apr;13(4):712-25
18410527
Cell. 1998 Aug 21;94(4):491-501
9727492
J Bioenerg Biomembr. 2005 Jun;37(3):143-53
16167171