Novel SMAC-mimetics synergistically stimulate melanoma cell death in combination with TRAIL and Bortezomib.
BACKGROUND
XIAP (X-linked inhibitor of apoptosis protein) is an anti-apoptotic protein exerting its activity by binding and suppressing caspases. As XIAP is overexpressed in several tumours, in which it apparently contributes to chemoresistance, and because its activity in vivo is antagonised by second mitochondria-derived activator of caspase (SMAC)/direct inhibitor of apoptosis-binding protein with low pI, small molecules mimicking SMAC (so called SMAC-mimetics) can potentially overcome tumour resistance by promoting apoptosis.


METHODS
Three homodimeric compounds were synthesised tethering a monomeric SMAC-mimetic with different linkers and their affinity binding for the baculoviral inhibitor repeats domains of XIAP measured by fluorescent polarisation assay. The apoptotic activity of these molecules, alone or in combination with tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and/or Bortezomib, was tested in melanoma cell lines by MTT viability assays and western blot analysis of activated caspases.


RESULTS
We show that in melanoma cell lines, which are typically resistant to chemotherapeutic agents, XIAP knock-down sensitises cells to TRAIL treatment in vitro, also favouring the accumulation of cleaved caspase-8. We also describe a new series of 4-substituted azabicyclo[5.3.0]alkane monomeric and dimeric SMAC-mimetics that target various members of the IAP family and powerfully synergise at submicromolar concentrations with TRAIL in inducing cell death. Finally, we show that the simultaneous administration of newly developed SMAC-mimetics with Bortezomib potently triggers apoptosis in a melanoma cell line resistant to the combined effect of SMAC-mimetics and TRAIL.


CONCLUSIONS
Hence, the newly developed SMAC-mimetics effectively synergise with TRAIL and Bortezomib in inducing cell death. These findings warrant further preclinical studies in vivo to verify the anticancer effectiveness of the combination of these agents.
Lecis
D
D
Department of Experimental Oncology, Fondazione IRCCS Istituto Nazionale Tumori, Via G Venezian 1, 20133 Milano, Italy.
Drago
C
C
Manzoni
L
L
Seneci
P
P
Scolastico
C
C
Mastrangelo
E
E
Bolognesi
M
M
Anichini
A
A
Kashkar
H
H
Walczak
H
H
Delia
D
D
D051017:Apoptosis Regulatory Proteins; D001897:Boronic Acids / Q000494:pharmacology*; D000069286:Bortezomib; D053181:Caspase 8 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects*; D045744:Cell Line, Tumor; D015536:Down-Regulation; D004347:Drug Interactions; D004357:Drug Synergism; D055785:Gene Knockdown Techniques; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000008:administration & dosage / Q000494:pharmacology*; D008545:Melanoma / Q000188:drug therapy*; D024101:Mitochondrial Proteins / Q000008:administration & dosage / Q000494:pharmacology*; D011719:Pyrazines / Q000494:pharmacology*; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000008:administration & dosage / Q000494:pharmacology*; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; D001897:Boronic Acids; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D011719:Pyrazines; D053221:TNF-Related Apoptosis-Inducing Ligand; D051636:X-Linked Inhibitor of Apoptosis Protein; D000069286:Bortezomib; D053181:Caspase 8
Cancer Res. 2007 Dec 15;67(24):11493-8
18089776
Cell. 2007 Nov 16;131(4):669-81
18022362
J Immunol. 2005 Dec 15;175(12):8226-35
16339562
EMBO J. 1997 Sep 1;16(17):5386-97
9311998
Cell. 2007 Nov 16;131(4):682-93
18022363
Cancer Res. 2001 Oct 1;61(19):7339-48
11585775
EMBO J. 1998 Apr 15;17(8):2215-23
9545235
Anal Biochem. 2008 Mar 1;374(1):87-98
18023397
Biochem Biophys Res Commun. 2009 Jan 9;378(2):162-7
18992220
Cell Death Differ. 2004 Jul;11 Suppl 1:S86-96
15105837
Mol Cancer Ther. 2009 Jun;8(6):1515-25
19509255
J Biol Chem. 1998 Apr 3;273(14):7787-90
9525868
J Mol Biol. 2009 Sep 25;392(3):630-44
19393243
Clin Cancer Res. 2007 Jun 1;13(11):3403-12
17545549
Blood. 2007 Feb 1;109(3):1220-7
17032924
Cancer Res. 2007 Jun 15;67(12):5880-8
17575157
J Mol Biol. 2008 Dec 19;384(3):673-89
18851976
Int J Cancer. 2007 Mar 1;120(5):1081-6
17154176
Curr Opin Immunol. 1998 Oct;10(5):573-80
9794839
Nature. 2000 Dec 21-28;408(6815):1008-12
11140638
Nature. 2000 Dec 21-28;408(6815):1004-8
11140637
Oncogene. 2007 May 17;26(23):3364-77
17160022
J Clin Invest. 2008 Jun;118(6):1979-90
18523647
Cell. 2000 Jul 7;102(1):33-42
10929711
Nat Med. 1999 Feb;5(2):157-63
9930862
Cell. 2008 May 16;133(4):693-703
18485876
Cancer Res. 2004 May 1;64(9):3006-8
15126334
Cancer Res. 2009 Mar 15;69(6):2425-34
19258513
Apoptosis. 2008 Apr;13(4):523-30
18297401
Blood. 2001 Mar 1;97(5):1378-87
11222383
J Med Chem. 2006 Dec 28;49(26):7916-20
17181177
Clin Cancer Res. 2006 Apr 15;12(8):2640-6
16638878
Bioorg Med Chem. 2009 Aug 15;17(16):5834-56
19620011
Cell Death Differ. 2008 Apr;15(4):762-72
18202702
Cancer Res. 2008 Feb 1;68(3):834-42
18245485
Carcinogenesis. 2007 Nov;28(11):2328-36
17652336
Mol Cell. 2008 Jun 20;30(6):689-700
18570872
Science. 2004 Sep 3;305(5689):1471-4
15353805
Cancer Cell Int. 2005 Jun 01;5(1):18
15929791
J Biol Chem. 2006 Feb 10;281(6):3254-60
16339151
Cancer Res. 2008 Oct 1;68(19):7956-65
18829553
Cancer Res. 2008 Nov 15;68(22):9384-93
19010913
Nature. 1997 Jul 17;388(6639):300-4
9230442
Cancer Cell. 2007 Nov;12(5):445-56
17996648
Blood. 2007 May 1;109(9):3982-8
17185461
Biochem J. 2009 Feb 1;417(3):765-71
18851715
Anal Biochem. 2004 Sep 15;332(2):261-73
15325294
Apoptosis. 2009 Apr;14(4):607-23
19194800
J Invest Dermatol. 2005 Nov;125(5):1010-9
16297203
J Med Chem. 2004 Aug 26;47(18):4417-26
15317454