Smac mimetic reverses resistance to TRAIL and chemotherapy in human urothelial cancer cells.
OBJECTIVE
inhibitors of apoptosis proteins (IAPs) have been shown to contribute to resistance of neoplastic cells to chemotherapy and to biologic antineoplastic agents. Consequently, new agents are being developed targeting this family of proteins. In a panel of bladder cancer cell lines, we evaluated a Smac mimetic that antagonizes several IAPs for its suitability for bladder cancer therapy. Experimental design: A panel of seven bladder cancer cell lines were evaluated for sensitivity to the Smac mimetic compound-A alone, TRAIL alone, chemotherapy alone, compound-A plus TRAIL, and compound-A plus chemotherapy by DNA fragmentation analysis. IAP levels and caspase activation were examined by western blotting. Release of caspase-3 from X-linked inhibitor of apoptosis protein (XIAP), the most effective IAP, was assessed by immunoprecipitation and western blotting. Finally, siRNA knockdown of XIAP was correlated with the sensitivity of cells to apoptosis induced by compound-A plus TRAIL by DNA fragmentation and western blotting.


RESULTS
single-agent compound-A had little effect, but compound-A augmented TRAIL- and chemotherapy-induced apoptosis. Immunoblotting showed that combination treatment with compound-A and TRAIL resulted in cleavage of procaspase-3 and procaspase-7, activation of which irreversibly commits cells to apoptosis. Immunoprecipitation of XIAP showed displacement of active caspase-3 fragments from XIAP, supporting the proposed mechanism of action. Furthermore, siRNA-mediated silencing of XIAP similarly sensitized these cells to apoptosis.


METHODS
a panel of seven bladder cancer cell lines were evaluated for sensitivity to the Smac mimetic compound-Alone, TRAIL alone, Chemotherapy alone, compound-A plus TRAIL and compound-A plus chemotherapy by DNA fragmentation analysis. IAP levels and caspase activation were examined by western blotting. Release of caspase-3 from X-linked inhibitor of apoptosis protein (XIAP), the most effective IAP, was assessed by immunoprecipitation and western blotting. Finally siRNA knockdown of XIAP was correlated with the sensitivity of cells to apoptosis induced by compound-A plus TRAIL by DNA fragmentation and western blotting.


CONCLUSIONS
our results suggest that targeting of XIAP with the Smac mimetic compound-A has the potential to augment the effects of a variety of chemotherapeutic and biologic therapies in bladder cancer.
Metwalli
Adam R
AR
Department of Urology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA.
Khanbolooki
Sanaz
S
Jinesh
Goodwin
G
Sundi
Debasish
D
Shah
Jay B
JB
Shrader
Marissa
M
Choi
Woonyoung
W
Lashinger
Laura M
LM
Chunduru
Srinivas
S
McConkey
David J
DJ
McKinlay
Mark
M
Kamat
Ashish M
AM
D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology* / Q000627:therapeutic use; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D045744:Cell Line, Tumor; D053938:DNA Fragmentation / Q000187:drug effects; D019008:Drug Resistance, Neoplasm; D004354:Drug Screening Assays, Antitumor; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology* / Q000627:therapeutic use; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000627:therapeutic use*; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D019459:Urothelium / Q000473:pathology; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
D000970:Antineoplastic Agents; D009842:Oligopeptides; D034741:RNA, Small Interfering; C532637:SMAC peptide; D053221:TNF-Related Apoptosis-Inducing Ligand; D051636:X-Linked Inhibitor of Apoptosis Protein; D053148:Caspase 3; D053179:Caspase 7
Urol Int. 2005;75(1):80-7
16037714
Oncogene. 2005 Nov 10;24(49):7381-8
16044155
Genes Dev. 1999 Feb 1;13(3):239-52
9990849
Oncogene. 2004 Oct 21;23(49):8105-17
15378029
Cancer Res. 2005 Jun 1;65(11):4902-8
15930312
Nature. 2000 Oct 12;407(6805):770-6
11048727
Cancer Res. 2004 May 1;64(9):3006-8
15126334
Cell. 2007 Nov 16;131(4):682-93
18022363
FEBS Lett. 2005 Nov 7;579(27):6217-22
16246331
Cancer Cell. 2003 Jan;3(1):17-22
12559172
Nat Rev Mol Cell Biol. 2002 Jun;3(6):401-10
12042762
Cancer Gene Ther. 2004 May;11(5):309-16
15031723
J Immunol Methods. 1991 Jun 3;139(2):271-9
1710634
Nature. 2000 Aug 24;406(6798):855-62
10972280
Nature. 1996 Jan 25;379(6563):349-53
8552191
Nature. 2000 Dec 21-28;408(6815):1008-12
11140638
J Immunol. 1999 Feb 15;162(4):2399-405
9973521
Nat Med. 2002 Aug;8(8):808-15
12118245
Cell Tissue Res. 2000 Jul;301(1):143-52
10928287
Annu Rev Biochem. 1999;68:383-424
10872455
Nature. 1980 Apr 10;284(5756):555-6
6245367
J Clin Oncol. 2005 Jul 20;23(21):4602-8
16034041
Proc Natl Acad Sci U S A. 2003 Jun 24;100(13):7533-8
12808146
J Biol Chem. 1999 Oct 22;274(43):30651-6
10521451
Science. 1995 Mar 10;267(5203):1456-62
7878464
J Urol. 2001 Oct;166(4):1300-4, discussion 1304-5
11547062
Science. 2004 Sep 3;305(5689):1471-4
15353805
J Pathol. 1971 Sep;105(1):13-20
4108566
Mol Cancer Ther. 2007 Mar;6(3):1022-30
17363495
Br J Cancer. 1972 Aug;26(4):239-57
4561027
Clin Biochem. 2004 Aug;37(8):673-8
15302609
J Biol Chem. 2002 Nov 15;277(46):44236-43
12218061
Endocrinology. 2001 Jan;142(1):370-80
11145600
Cancer Res. 2004 May 15;64(10):3386-90
15150089
Cancer Res. 2004 Dec 15;64(24):8973-9
15604261
Blood. 2005 Nov 15;106(10):3474-82
16037389