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Biochemistry and Biophysics |
Reproductive Biology Unit and Division of Gynaecologic Oncology, Departments of Obstetrics and Gynaecology and Cellular and Molecular Medicine, University of Ottawa, and Loeb Health Research Institute, The Ottawa Hospital, Civic Campus, Ottawa, Ontario, Canada K1Y 4E9 [H. S., Y. S., B. K. T]; and Department of Obstetrics and Gynaecology, Fukui Medical University, Fukui, Japan 910-1193 [F. K.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although platinum derivatives (e.g., cisplatin, carboplatin) and paclitaxel are first-line chemotherapeutic agents for the treatment of ovarian epithelial cancer, chemoresistance is a major therapeutic problem, and the molecular mechanisms involved are poorly understood. The development of resistance may be cell-type specific and related to the dosing schedule (1) . The mechanisms of chemoresistance appear to be multifactorial and are generally thought of in terms of altered pharmacodynamics and gene expression (including multidrug resistant genes), modified drug target, increased rate of DNA repair, or decreased rates of drug-induced DNA or macromolecule damage (2, 3, 4) . The effect of cisplatin appears to result from inhibition of replication by cisplatin-DNA adducts and G1 arrest with subsequent induction of apoptosis (5) . p53 accumulation plays a key role in this cell cycle arrest. It has been shown that the p53 level is regulated by MDM24 (6) and that various mutations of p53 are frequently detected in cancer cells. Although our knowledge of the events leading to chemoresistance is incomplete, failure to activate apoptosis in these cancer cells may confer resistance to these agents (4) .
An intensive search for modulators of apoptosis has revealed the presence of several potent endogenous suppressers in mammalian cells. The IAP family, originally identified in baculovirus, currently consists of five members: neuronal apoptosis inhibitory protein [Naip], X-linked inhibitor of apoptosis protein [Xiap (8, 9, 10) ], ilp (10) ], human inhibitor of apoptosis protein-1 [Hiap-1 (8 , 9 , 11) ], human inhibitor of apoptosis protein-2 [Hiap-2 (8 , 9 , 11) ], and survivin (12) . Hiap 1 and Hiap-2 have been suggested to suppress TNF-receptor signaling by binding to the TNF receptor-associated factor (11) , and Xiap suppresses apoptosis via caspase-3 and -7 inhibition (13) . It has been reported that IAPs play a central role in the regulation of a number of physiological and pathological processes. Naip is believed to be involved in the pathogenesis of spinal muscular atrophy, in which apoptosis in the spinal ventral neurones is precipitated by the loss of this inhibitor (7) . Survivin is highly expressed in various malignant tissues (12 , 14) and has been suggested to be an important etiological factor of carcinogenesis and potential new sites for apoptosis-based therapy in cancer (15) .
Recent studies from our laboratory have demonstrated that cisplatin is capable of decreasing Xiap protein content in chemosensitive, but not in resistant, hOSE cancer cells, and that over-expression of Xiap in the sensitive cells by adenoviral Xiap sense cDNA delivery prevented the cisplatin-induced apoptosis.5 These findings suggest that.cisplatin-resistance in hOSE cancer may be due to an inability of this chemotherapeutic agent to suppress Xiap protein content and to induce apoptosis.
In the present study, we have examined this possibility by studying the influence of Xiap down-regulation on apoptosis in cisplatin-resistant hOSE cancer cells. Transient infection with adenoviral antisense Xiap cDNA significantly decreased the cellular content of this protein and induced apoptotic cell death as well as enhanced cisplatin-induced cell death in p53 wild-type (C13*) but not in mutated (A2780-cp) resistant cells. Mechanistic investigations indicate that antisense Xiap-induced apoptosis in the chemoresistant hOSE cancer cells was associated with caspase-3-mediated MDM2 cleavage and increased p53 content. These findings suggested that Xiap is not only regulating down-stream events of caspase-3, but also is triggering p53-dependent pathway when down-regulated by antisense and/or cisplatin treatment.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1,500 bp). Adenoviral
expression system was generated with an Ad E1 insertion vector. Virus
titer was determined by the plaque assay.
Cell Culture.
Cisplatin-sensitive (A2780-s), -resistant (C13*, A2780-cp, and SKOV3)
human ovarian epithelial cancer cell lines were gifts from Drs. R. Goal
and B. Vanderhyden (Ottawa Regional Cancer Center, Ottawa, Ontario,
Canada) and were maintained at 37°C and 5%
CO2, 95% atmosphere in RPMI 1640 medium (C13*),
DMEM/F12 (A2780-s and A2780-cp), or McCoy 5A (SKOV3; Life Technologies,
Inc./BRL, Burlington, Ontario, Canada) supplemented with fetal bovine
serum (10%), streptomycin (50 µg/ml), penicillin (50 units/ml), and
Fungizone (0.625 µg/ml; Life Technologies, Inc./BRL). These cell
lines were established from a patient with serous cystoadenocarcinoma
and have been well-characterized in terms of their cisplatin
sensitivity (17
, 18)
. C13* and A2780-cp cells carry wild
type p53 and mutant p53, respectively (19
, 20) . SKOV3 is a
null p53 cell line (21)
. Cells, plated in 5 x 104
cells/cm2, were
treated with adenoviral expression vectors (see below) or freshly
prepared cis-Platinum (II) Diammine Dichloride [cisplatin
0–100 µM (Sigma)] under serum-free
conditions. Serum withdrawal had no significant effect on cell
viability during the experimental period, as confirmed by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.
Hoechst 33248 Staining.
At the end of the culture period, cells attached to the growth surface
were removed by trypsin treatment [trypsin (0.05%), EDTA (0.53
mM); 37°C, 1 min]. Attached and detached cells were
pooled, pelleted, and resuspended in neutral-buffered formalin (10%)
containing Hoechst 33248 dye (12.5 ng/ml). Cells were spotted onto
slides and assessed for typical apoptotic nuclear morphology (nuclear
shrinkage, condensation, and fragmentation) with Hoechst dye under a
Zeiss fluorescence microscope with appropriate filter combination. At
least 200 cells/treatment group were counted and assessed randomly
selected fields and blinded slides to avoid experimental bias.
Protein Extraction and Immunodetection.
Cells were pelleted and lysed in ice-cold lysis buffer [NaCl (137
mM), Na2HPO4
(8.1 mM), KCl (2.68 mM),
KH2PO4 (1.47
mM), NP40 (1%; v/v), sodium deoxycholate (0.05%; w/v),
SDS (0.1%; w/v) containing phenylmethylsulfonyl fluoride (10
µM), aprotinin (57 µg/ml), leupeptin (5 µg/ml), and
pepstatin (5 µg/ml)]. Cell lysates were sonicated briefly (5
s/cycle, 3 cycles; 0°C), incubated on ice (1 h), and centrifuged
(15,000 x g; 20 min). The supernatant was
saved and stored at -20°C for subsequent analyses. Protein
concentration was determined using Bio-Rad DC protein assay kit. Equal
amounts of proteins (70–100 µg) were loaded and resolved on SDS-PAGE
(8–15%) and electroblotted (30V; 12 h) onto nitrocellulose
membranes (Bio-Rad Canada). After Western transfer, uniform protein
loading was confirmed by comparing the intensity of stained total
proteins between lanes of nitrocellulose membranes previously stained
with SYPRO Ruby protein stain (Molecular Probes, Eugene, OR). The
images were scanned and stored electronically, and the intensity of the
protein stain in each lane was quantified for future reference or for
normalization of Western blot results. Membranes were then blocked
(room temperature, 1 h) with 5% Blotto [Tris-HCl (10
mM; pH 8.0), NaCl (150 mM),
Tween 20 (0.05%; v/v; TBS-Tween 20) containing skim milk (5%; w/v)],
incubated with primary antibody [Xiap (1:4,000); caspase-3 (1:3000);
MDM2 (1:1000); p53 (1:2000)]; p21waf1 (1:2000);
room temperature, 1 h] and subsequently with horseradish
peroxidase-conjugated secondary antibody [1:2,000 in 5% Blotto; room
temperature, 30 min.]. Peroxidase activity was visualized with an ECL
kit (Amersham Pharmacia Biotech, Arlington Heights, IL) after a wash
with TBS-Tween 20 or TBS [Tris-HCl (10
mM; pH 8.0), NaCl (150
mM)]. Signal intensity was determined
densitometrically and normalized against those of total proteins
present in the corresponding lane on the membrane using Molecular
Analyst software, version 1.5 (Bio-Rad Canada).
Adenoviral Infection.
After 24 h of plating (106 cells/60-mm
culture dish), cells were infected with adenoviral antisense
full-length Xiap and/or sense p53 at MOIs of 5, 10, 20, or 40
for C13*, 50, 100, or 200 for A2780-cp; 200 or 400 for SKOV3 (Xiap);
and 5, 10, or 20 (p53). Infection with an equivalent concentration
(MOI) of LacZ was carried out to provide proper control. LacZ was also
used to adjust the total concentration of adenovirus when two different
viral preparations were used in the same experiment (double infection).
To test the changes on cisplatin sensitivity, infected cells were
treated with or without cisplatin (10 µM; 24 h)
after a 72-h infection period. The number of apoptotic cells was
determined by Hoechst stain. Xiap and p53 protein contents as well as
caspase-3 and MDM2 contents and cleavage were assessed by Western blot.
The infection efficiency over 48 h, as determined by X-gal assay,
was >90% in all cell lines, and the expression of Xiap was confirmed,
if necessary, by Western blot before cisplatin treatment.
In Vitro Caspase Treatment.
Whole cell lysate (50 µg of total protein) of C13* cells was
incubated with PIPES assay buffer [PIPES (20 mM; pH 7.2),
NaCl (100 mM), DTT (10 mM), EDTA (1
mM),
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
(0.1%; w/v), and sucrose (10%; w/v)] containing recombinant active
caspase-3 in the presence or absence of DEVD-CHO. The incubation was
terminated by the addition of equal volumes of 2 x sample buffer [Tris-HCl (100 mM; pH 6.8), DTT (200
mM), SDS (4%; w/v), glycerol (20%; v/v), and Bromphenol
blue (0.2%)]. MDM2 cleavage in the cell lysate was assessed by
Western blot.
Statistical Analysis.
All experiments were repeated at least three times. Data were subjected
to one- or two-way ANOVA (PRISM software, version 3.0; GraphPad, San
Diego, CA). Differences between experimental groups were determined by
the Tukey or Bonferroni post-test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A, a and b) had no apparent effects on
cell morphology, down-regulation of Xiap with adenovirus antisense
(MOI = 10; 72 h) evoked typical apoptotic features
such as membrane blebbing (arrow), cell shrinkage and
detachment (Fig. 1
A, c) and nuclear condensation
and fragmentation (Fig. 1
A, d).
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B,
a). Although antisense Xiap infection (MOI = 10) resulted in a statistically nonsignificant (10.9 ± 2.8% versus 5.9 ± 0.5%;
P > 0.05) increase in the number of
apoptotic cells at 24 h, significant and much larger increases
were noted at 48 h (21.3 ± 3.4% versus
5.2 ± 0.3%; P < 0.01),
60 h (29.3 ± 3.2% versus 7.1 ± 2.1%; P < 0.001) and 72 h
(32.6 ± 4.0% versus 10.2 ± 4.3%; P < 0.001) when compared with LacZ
controls (Fig. 1
B, b).
Xiap Down-Regulation Sensitizes Cisplatin-resistant C13* Cells to
the Cytotoxic Action of Cisplatin.
Whereas C13* cells failed to respond to cisplatin at concentrations as
high as 30 µM, a higher concentration (100
µM) was capable of inducing apoptosis in 16.4 ± 3.9% and 16.6 ± 3.0% of these cells.
Antisense Xiap treatment sensitized the resistant cells to the
proapoptotic action of cisplatin. The number of apoptotic cells in the
antisense Xiap treatment group was significantly higher than the LacZ
group at 30 (14.7 ± 3.5%; P < 0.05) and 100 (48.7 ± 4.9%;
P < 0.001) µM
cisplatin and the cisplatin concentration-response curve was shifted to
the left by antisense Xiap expression (Fig. 2A)
. Significant effects of antisense Xiap
(P < 0.001) and cisplatin concentration
(P < 0.001) as well as their interaction
(P < 0.001) were confirmed by two-way ANOVA.
The effect of antisense Xiap was concentration-dependent, as
demonstrated in studies with C13* cells infected with various
concentrations (MOI = 0–40) of LacZ or antisense Xiap
and then challenged with cisplatin (10 µM;
24 h). Antisense Xiap-treated cells showed increased sensitivity
to cisplatin at MOIs of 20 (P < 0.05) and 40
(P < 0.001) compared with LacZ-treated
cells. Two-way ANOVA indicates significant antisense
(P < 0.001) and concentration
(P < 0.001) effects and interaction
(P < 0.001; Fig. 2
B).
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A, a and b). Xiap down-regulation was
associated with cleavage of procaspase-3, a 32-kDa member of the
cysteine protease family involved in the execution of apoptosis and
activated by proteolytic cleavage to form 20- and 18-kDa fragments
(prodomain + large subunit and large subunit alone,
respectively) by the other upstream caspases (22)
. The
cleavage of procaspase-3 was dependent on the antisense concentration,
and significant increases of p20 and p17 protein contents were
observable at MOIs of 10 (P < 0.001) and 20
(P < 0.001; Fig. 3
A). Significant
antisense (P < 0.001) and concentration
(P < 0.001) effects as well as interaction
between these factors (P < 0.001) were
noted. Consistent with the observed onset of apoptosis, p20
(P < 0.01) and p17 (P < 0.05) levels were significantly increased (compared with LacZ)
as early as 48 h after adenoviral Xiap antisense infection (Fig. 3
B, a and b). A significant
interaction (P < 0.05) existed between
antisense Xiap and the duration of infection, which was brought about
by the significantly higher levels of these active cleavage fragments
after 24 h of infection with the antisense. Interestingly,
procaspase-3 levels were decreased, though not consistently, by
antisense Xiap treatment.
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. Furthermore, p53 accumulation following
Xiap down-regulation at a MOI of 10 (P < 0.05) or 20 (P < 0.001) was associated with
MDM2 cleavage when compared with respective LacZ controls (Fig. 4
A, b). The addition of recombinant active
caspase-3, but not of caspase-7, to C13* whole cell lysate elicited a
concentration- and time-dependent MDM2 cleavage pattern identical to
that observed in extracts from cells following Xiap down-regulation
(Fig. 4
B, a). MDM2 cleavage by caspase-3 was
blocked by the presence of the caspase-3 inhibitor, DEVD-CHO (Fig. 4
B, b). Because C13* cells possess wild type p53
(27)
, these findings suggest that Xiap down-regulation not
only induces caspase-3 cleavage and activation, but possibly also
activates a p53-dependent apoptotic pathway.
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A, a and b), whereas it was ineffective
in its p53-mutated resistant variant, A2780-cp (Ref. 19
;
P > 0.05; Fig. 5
A, c
and d). Contrary to the wild type p53 C13* cells, Xiap
down-regulation in A2780-cp alone failed to significantly induce
apoptosis (P > 0.05) even at a concentration
of the adenoviral Xiap antisense as high as a MOI of 200, which
effectively decreased Xiap content after 72 h (Fig. 6
A, a and b). In contrast,
overexpression of wild type p53 alone in these p53 mutants by
adenoviral sense cDNA resulted in a concentration (MOI)-dependent
increase in apoptosis (P < 0.001; Fig. 6
A, c and d). Whereas expression of
wild type p53 with low concentration of sense p53 (MOI = 10) had a slight, but nonsignificant, effect on apoptosis
(P > 0.05), a higher concentration
(MOI = 40) markedly increased the population of
apoptotic cells (25.0 ± 6.0% versus
0.8 ± 0.3%; P < 0.001)
compared with corresponding LacZ-treated cells.
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B). Moreover, the proapoptotic effects of Xiap
down-regulation (antisense Xiap; MOI = 100) were
dependent on the concentration of sense p53 (Fig. 6
C).
Expression of antisense Xiap significantly enhanced the sensitivity of
the cells to wild type p53 resulting in a shift of the p53 sense
concentration-apoptosis response curve to the left. Two-way ANOVA
indicates significant antisense Xiap and p53 sense concentration
effects, and interaction between the two factors
(P < 0.01).
Upon wild type p53 restoration (MOI = 10), the
proapoptotic effect of antisense Xiap treatment was
concentration-dependent (P < 0.01), and
significant increases in the number of apoptotic cells were evident
with adenoviral Xiap antisense at MOIs of 50 (37.8 ± 5.3%) and 100 (51.4 ± 0.4%) compared with LacZ
(7.7 ± 3.6% and 11.1 ± 7.0%,
respectively; P < 0.01; Fig. 7A, a
). Adenoviral Xiap antisense expression
resulted in a concentration-dependent Xiap down-regulation, however a
greater decrease in Xiap level was observed when the A2780-cp cells
were coinfected with Xiap antisense and wild type p53 sense (Fig. 7
A, b and c). Expression of
p21waf1 and 90-kDa MDM2 protein were found
predominantly in cells in which wild type p53 was reconstituted. Xiap
down-regulation significantly increased p53 content (about three-fold)
in p53 mutant reconstituted with wild type p53 and marked by decreased
p53-induced p90 and p60 MDM2 levels (approximately 50% and 30%,
respectively; Fig. 7
A, d). These results
suggested that caspase-3 activation is necessary, but not sufficient,
for the induction of apoptosis in p53-deficient cells, and that a
functional p53 may be involved in the completion of the apoptotic
process.
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B). Induction of intact MDM2 protein was
observed in cells with sense p53 expression, irrespective of antisense
Xiap delivery, and a caspase-mediated MDM2 fragment was evident
in the cells treated with antisense Xiap and sense p53 constructs.
Although the proportions of apoptotic cells induced by adenoviral gene
delivery were smaller than in C13* and A2780-cp cell lines,
adenoviral infection of cells with antisense Xiap and sense p53
significantly induced apoptosis (3.4 ± 0.39%)
compared with viral LacZ (0.6 ± 0.3%;
P < 0.001), antisense Xiap (1.7 ± 0.1%; P < 0.05), and sense p53
(0.47 ± 0.27%; P < 0.001)
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DISCUSSION |
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We have also shown in the present studies that antisense Xiap expression alone in cisplatin-resistant wild type p53 hOSE cancer cells (C13*) results in a concentration- and time-dependent decrease in Xiap content and an increase in apoptosis. These findings not only demonstrate a key regulatory role of Xiap in apoptosis in these cells, but also suggest that, despite being resistant to cisplatin, they are capable of and committed to apoptosis but are prevented from undergoing the death process due to the presence of Xiap. Xiap has a strong affinity for caspase-3, a cysteine protease involved in the execution of apoptosis (13 , 30) and is an inhibitor of caspase-3 activation (cleavage by upstream caspases) and activity. The physiological role of Xiap has been considered as a final guardian for preventing and regulating this cell-death process. Thus, removal of the antiapoptotic factor would be expected to have resulted in unleashing of the apoptotic process, probably mediated by the release of caspase-3 from its inhibition. However, our present observations are that Xiap down-regulation alone was only effective in p53 wild type cells, and failed to induce apoptosis in p53 mutated and null cell lines.
The mechanism(s) by which caspase-3 elicits its proapoptotic action in hOSE cancer cells is unclear. It has been suggested that proteins involved in DNA breakdown [e.g., inhibitor of caspase activated DNase (31) ] and signal transduction [e.g., focal adhesion kinase, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (32) ] and cytoskeletal proteins [e.g., actin (33) ] are possible physiological substrates for caspase-3, and that this death protease mediates the apoptotic signal by disrupting survival signals and/or cellular integrity. In the present studies, we have observed that pro-caspase-3 cleavage following Xiap down-regulation in wild type p53 hOSE cancer cells was temporally associated with MDM2 cleavage. In both C13* and A2780-cp cells, the 60-kDa MDM2 was the dominant isoform. MDM2 were undetectable in SKOV3 cells, although a 90-kDa native protein became detectable after wild type p53 over-expression. Whereas the 60-kDa isoform is believed to be a caspase cleavage product of the 90-kDa protein and is frequently found in cancer cells (34) , our in vitro studies with C13* whole-cell lysate and human recombinant active caspase-3 and -7 indicate that MDM2 may be a physiological substrate for caspase-3. We demonstrated the caspase-3-mediated cleavage of the 60-kDa MDM2 to a smaller fragment (30 kDa), as well as the 90- to 60-kDa cleavage when the 90-kDa protein was up-regulated. Although how the conversion of the 60-kDa protein to the 30-kDa form (but not of the 90- to 60-kDa form) is suppressed in the nonapoptotic cells is unclear, the possible involvement of Xiap-independent caspases in the initial processing of the 90-kDa protein cannot be excluded. These findings are, however, consistent with the fact that MDM2 has caspase-sensitive sites (23) , and are in good agreement with previously published observations (24 , 35) . The physiological significance of the observed caspase-3 specificity is not known.
MDM2 is an oncoprotein that binds p53 and facilitates ubiquitin-mediated degradation of the tumor suppresser protein (6) . It has been reported that a decrease in MDM2 content stabilizes p53, whereas the opposite is true when the oncoprotein is over-expressed (36) . Wild type p53 accumulation is known to induce cell cycle arrest and apoptosis, and p53 mutation is frequently identified in various cancer cells (37) . In the present investigations, we have demonstrated Xiap down-regulation is associated with significant cleavage of caspase-3 and MDM2, p53 accumulation, and increased apoptosis in wild type p53-expressing C13* cells. When wild type p53 was supplemented, accumulation of p53 was also evident in p53 mutated (A2780-cp) and null (SKOV3) cells treated with antisense Xiap. Xiap down-regulation together with wild type p53 reconstitution (by sense p53 expression), however, not only further increased p53 content, but significantly increased apoptosis in these otherwise p53-deficient cells. These studies provide the first evidence for a role of Xiap in the regulation of cellular p53 level and suggest that caspase-3-mediated MDM2 processing may be an additional regulatory point for the antiapoptotic protein in promoting survival of wild type p53-expressing hOSE cancer cells.
The regulation of Xiap levels in hOSE cancer cells remains to be investigated. In the present studies, over-expression of wild type p53 decreased basal Xiap content and enhanced the effect of antisense Xiap expression, suggesting a possible involvement of p53 in the transcriptional control of Xiap expression. On the other hand, posttranscriptional processing of Xiap may also be an important determinant of the steady-state levels of Xiap and, thus, of apoptosis. Our present studies indicate that at least 60–72 h are required to sufficiently decrease Xiap content following antisense expression, suggesting that the antiapoptotic protein may have a relatively long half-life. This is consistent with the observation that, although adenoviral infection efficiency was close to 100%, only about 60% of the cells were apoptotic 72 h after infection (MOI = 20).
In summary, we have shown that Xiap plays an important role of regulation of apoptosis in hOSE cancer cells and is an important determinant in cisplatin resistance. In addition to its well-established inhibitory action on caspase-3 activity, this antiapoptotic protein appears to be central in the control of p53 accumulation and p53-mediated apoptosis, possibly via the MDM2-p53-ubiquitin degradation pathway. Xiap down-regulation not only induces apoptosis but also sensitizes chemoresistant wild type p53 hOSE cancer cells to the proapoptotic action of cisplatin. In p53 mutant cancer cells, coexpression of Xiap antisense and wild type p53 sense is more effective than wild type p53 restoration alone. These findings provide a new concept for the development of novel therapeutic approaches in the treatment of chemoresistant hOSE cancer. Studies with xenograft models are required to establish whether these strategies are indeed applicable in vivo.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the Canadian Institutes
of Health Research (MT-15691, to B. K. T), Grant-in-Aid for
International Scientific Research from the Ministry of Education,
Science, Sports and Culture of Japan (Joint Research 10044255, to F. K
and B. K. T.), and the Ottawa Hospital Foundation. 
2 These authors contributed equally in the present
investigations.
3 To whom requests for reprints should be
addressed, at Loeb Health Research Institute, 725 Parkdale Avenue,
Ottawa, Ontario, K1Y 4E9, Canada. Phone: (613) 761-4081; Fax:
(613) 761-5365; E-mail: btsang{at}lri.ca
4 The abbreviations used are: MDM2, murine double
minute 2; IAP, inhibitor of apoptosis proteins; Xiap, X-linked
inhibitor of apoptosis protein; hOSE, human ovarian surface epithelial;
DEVD-CHO, N-acetyl-Asp-Glu-Val-Asp-CHO (aldehyde); TBS,
Tris-buffered saline; MOI, multiplicity of infection.
5 J-L. Li, Q. Feng, J-M. Kim, D. Schneiderman, P.
Liston, M. Li, B. Vanderhyden, W. Faught, M. F. K. Fung, M.
Senterman, R. G. Korneluk and B. K. Tsang. Human ovarian cancer and
cisplatin resistance: possible role of inhibitor of apoptosis proteins,
Endocrinology, in press.
Received 2/18/00. Accepted 8/16/00.
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 H. Wang, J.-Y. Jiang, C. Zhu, C. Peng, and B. K. Tsang Role and Regulation of Nodal/Activin Receptor-Like Kinase 7 Signaling Pathway in the Control of Ovarian Follicular Atresia Mol. Endocrinol., October 1, 2006; 20(10): 2469 - 2482. [Abstract] [Full Text] [PDF]
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 X. Yang, M. Fraser, U. M. Moll, A. Basak, and B. K. Tsang Akt-Mediated Cisplatin Resistance in Ovarian Cancer: Modulation of p53 Action on Caspase-Dependent Mitochondrial Death Pathway. Cancer Res., March 15, 2006; 66(6): 3126 - 3136. [Abstract] [Full Text] [PDF]
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D. Lane, M. Cote, R. Grondin, M.-C. Couture, and A. Piche Acquired resistance to TRAIL-induced apoptosis in human ovarian cancer cells is conferred by increased turnover of mature caspase-3. Mol. Cancer Ther., March 1, 2006; 5(3): 509 - 521. [Abstract] [Full Text] [PDF]
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 A. B. Alvero, W. Chen, A. C. Sartorelli, P. Schwartz, T. Rutherford, and G. Mor Triapine (3-aninopyridine-2-carboxaldehyde thiosemicarbazone) Induces Apoptosis in Ovarian Cancer Cells Reproductive Sciences, February 1, 2006; 13(2): 145 - 152. [Abstract] [PDF]
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 A. P. Kater, F. Dicker, M. Mangiola, K. Welsh, R. Houghten, J. Ostresh, A. Nefzi, J. C. Reed, C. Pinilla, and T. J. Kipps Inhibitors of XIAP sensitize CD40-activated chronic lymphocytic leukemia cells to CD95-mediated apoptosis Blood, September 1, 2005; 106(5): 1742 - 1748. [Abstract] [Full Text] [PDF]
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J. C. Reed and M. Pellecchia Apoptosis-based therapies for hematologic malignancies Blood, July 15, 2005; 106(2): 408 - 418. [Abstract] [Full Text] [PDF]
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 U. Fischer and K. Schulze-Osthoff New Approaches and Therapeutics Targeting Apoptosis in Disease Pharmacol. Rev., June 1, 2005; 57(2): 187 - 215. [Abstract] [Full Text] [PDF]
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B. Z. Carter, M. Gronda, Z. Wang, K. Welsh, C. Pinilla, M. Andreeff, W. D. Schober, A. Nefzi, G. R. Pond, I. A. Mawji, et al. Small-molecule XIAP inhibitors derepress downstream effector caspases and induce apoptosis of acute myeloid leukemia cells Blood, May 15, 2005; 105(10): 4043 - 4050. [Abstract] [Full Text] [PDF]
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 Y. Mizutani, H. Nakanishi, K. Yamamoto, Y. N. Li, H. Matsubara, K. Mikami, K. Okihara, A. Kawauchi, B. Bonavida, and T. Miki Downregulation of Smac/DIABLO Expression in Renal Cell Carcinoma and Its Prognostic Significance J. Clin. Oncol., January 20, 2005; 23(3): 448 - 454. [Abstract] [Full Text] [PDF]
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F. A. Sinicrope, R. C. Penington, and X. M. Tang Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis Is Inhibited by Bcl-2 but Restored by the Small Molecule Bcl-2 Inhibitor, HA 14-1, in Human Colon Cancer Cells Clin. Cancer Res., December 15, 2004; 10(24): 8284 - 8292. [Abstract] [Full Text] [PDF]
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S. Mabuchi, M. Ohmichi, Y. Nishio, T. Hayasaka, A. Kimura, T. Ohta, J. Kawagoe, K. Takahashi, N. Yada-Hashimoto, H. Seino-Noda, et al. Inhibition of Inhibitor of Nuclear Factor-{kappa}B Phosphorylation Increases the Efficacy of Paclitaxel in in Vitro and in Vivo Ovarian Cancer Models Clin. Cancer Res., November 15, 2004; 10(22): 7645 - 7654. [Abstract] [Full Text] [PDF]
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 Z. Wang, M. Cuddy, T. Samuel, K. Welsh, A. Schimmer, F. Hanaii, R. Houghten, C. Pinilla, and J. C. Reed Cellular, Biochemical, and Genetic Analysis of Mechanism of Small Molecule IAP Inhibitors J. Biol. Chem., November 12, 2004; 279(46): 48168 - 48176. [Abstract] [Full Text] [PDF]
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 G. Xu, Y. Zhong, S. Munir, B. B. Yang, B. K. Tsang, and C. Peng Nodal Induces Apoptosis and Inhibits Proliferation in Human Epithelial Ovarian Cancer Cells via Activin Receptor-Like Kinase 7 J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5523 - 5534. [Abstract] [Full Text] [PDF]
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A. D. Schimmer Inhibitor of Apoptosis Proteins: Translating Basic Knowledge into Clinical Practice Cancer Res., October 15, 2004; 64(20): 7183 - 7190. [Abstract] [Full Text] [PDF]
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 N. PURI, M. S. ELLER, H. R. BYERS, S. DYKSTRA, J. KUBERA, and B. A. GILCHREST Telomere-based DNA damage responses: a new approach to melanoma FASEB J, September 1, 2004; 18(12): 1373 - 1381. [Abstract] [Full Text] [PDF]
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S. Mabuchi, M. Ohmichi, Y. Nishio, T. Hayasaka, A. Kimura, T. Ohta, M. Saito, J. Kawagoe, K. Takahashi, N. Yada-Hashimoto, et al. Inhibition of NF{kappa}B Increases the Efficacy of Cisplatin in in Vitro and in Vivo Ovarian Cancer Models J. Biol. Chem., May 28, 2004; 279(22): 23477 - 23485. [Abstract] [Full Text] [PDF]
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C.-Q. Li, A. I. Robles, C. L. Hanigan, L. J. Hofseth, L. J. Trudel, C. C. Harris, and G. N. Wogan Apoptotic Signaling Pathways Induced by Nitric Oxide in Human Lymphoblastoid Cells Expressing Wild-Type or Mutant p53 Cancer Res., May 1, 2004; 64(9): 3022 - 3029. [Abstract] [Full Text] [PDF]
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H. C. Dan, M. Sun, S. Kaneko, R. I. Feldman, S. V. Nicosia, H.-G. Wang, B. K. Tsang, and J. Q. Cheng Akt Phosphorylation and Stabilization of X-linked Inhibitor of Apoptosis Protein (XIAP) J. Biol. Chem., February 13, 2004; 279(7): 5405 - 5412. [Abstract] [Full Text] [PDF]
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B. Z. Carter, S. M. Kornblau, T. Tsao, R.-Y. Wang, W. D. Schober, M. Milella, H.-G. Sung, J. C. Reed, and M. Andreeff Caspase-independent cell death in AML: caspase inhibition in vitro with pan-caspase inhibitors or in vivo by XIAP or Survivin does not affect cell survival or prognosis Blood, December 1, 2003; 102(12): 4179 - 4186. [Abstract] [Full Text] [PDF]
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D.-S. Byun, K. Cho, B.-K. Ryu, M.-G. Lee, M.-J. Kang, H.-R. Kim, and S.-G. Chi Hypermethylation of XIAP-associated Factor 1, a Putative Tumor Suppressor Gene from the 17p13.2 Locus, in Human Gastric Adenocarcinomas Cancer Res., November 1, 2003; 63(21): 7068 - 7075. [Abstract] [Full Text] [PDF]
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M. Fraser, B. M. Leung, X. Yan, H. C. Dan, J. Q. Cheng, and B. K. Tsang p53 Is a Determinant of X-Linked Inhibitor of Apoptosis Protein/Akt-Mediated Chemoresistance in Human Ovarian Cancer Cells Cancer Res., November 1, 2003; 63(21): 7081 - 7088. [Abstract] [Full Text] [PDF]
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L. Yang, Z. Cao, H. Yan, and W. C. Wood Coexistence of High Levels of Apoptotic Signaling and Inhibitor of Apoptosis Proteins in Human Tumor Cells: Implication for Cancer Specific Therapy Cancer Res., October 15, 2003; 63(20): 6815 - 6824. [Abstract] [Full Text] [PDF]
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M. Krajewska, S. Krajewski, S. Banares, X. Huang, B. Turner, L. Bubendorf, O.-P. Kallioniemi, A. Shabaik, A. Vitiello, D. Peehl, et al. Elevated Expression of Inhibitor of Apoptosis Proteins in Prostate Cancer Clin. Cancer Res., October 15, 2003; 9(13): 4914 - 4925. [Abstract] [Full Text] [PDF]
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J. A. Shabbits, Y. Hu, and L. D. Mayer Tumor Chemosensitization Strategies Based on Apoptosis Manipulations Mol. Cancer Ther., August 1, 2003; 2(8): 805 - 813. [Full Text] [PDF]
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Y. Hu, M. Bally, W. H. Dragowska, and L. Mayer Inhibition of Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase Enhances Chemotherapeutic Effects on H460 Human Non-Small Cell Lung Cancer Cells through Activation of Apoptosis Mol. Cancer Ther., July 1, 2003; 2(7): 641 - 649. [Abstract] [Full Text] [PDF]
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Y. Hu, G. Cherton-Horvat, V. Dragowska, S. Baird, R. G. Korneluk, J. P. Durkin, L. D. Mayer, and E. C. LaCasse Antisense Oligonucleotides Targeting XIAP Induce Apoptosis and Enhance Chemotherapeutic Activity against Human Lung Cancer Cells in Vitro and in Vivo Clin. Cancer Res., July 1, 2003; 9(7): 2826 - 2836. [Abstract] [Full Text] [PDF]
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 X. Hong, L. Lei, and R. Glas Tumors Acquire Inhibitor of Apoptosis Protein (IAP)-mediated Apoptosis Resistance through Altered Specificity of Cytosolic Proteolysis J. Exp. Med., June 16, 2003; 197(12): 1731 - 1743. [Abstract] [Full Text] [PDF]
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 Y. Wang, P. U. Rippstein, and B. K. Tsang Role and Gonadotrophic Regulation of X-Linked Inhibitor of Apoptosis Protein Expression During Rat Ovarian Follicular Development In Vitro Biol Reprod, February 1, 2003; 68(2): 610 - 619. [Abstract] [Full Text] [PDF]
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S. Wittmann, P. Bali, S. Donapaty, R. Nimmanapalli, F. Guo, H. Yamaguchi, M. Huang, R. Jove, H. G. Wang, and K. Bhalla Flavopiridol Down-Regulates Antiapoptotic Proteins and Sensitizes Human Breast Cancer Cells to Epothilone B-induced Apoptosis Cancer Res., January 1, 2003; 63(1): 93 - 99. [Abstract] [Full Text] [PDF]
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C.-P. Ng and B. Bonavida X-linked Inhibitor of Apoptosis (XIAP) Blocks Apo2 Ligand/Tumor Necrosis Factor-related Apoptosis-inducing Ligand-mediated Apoptosis of Prostate Cancer Cells in the Presence of Mitochondrial Activation: Sensitization by Overexpression of Second Mitochondria-derived Activator of Caspase/Direct IAP-binding Protein with Low pI (Smac/DIABLO) Mol. Cancer Ther., October 1, 2002; 1(12): 1051 - 1058. [Abstract] [Full Text] [PDF]
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 C.-Q. Li, L. J. Trudel, and G. N. Wogan Nitric oxide-induced genotoxicity, mitochondrial damage, and apoptosis in human lymphoblastoid cells expressing wild-type and mutant p53 PNAS, August 6, 2002; 99(16): 10364 - 10369. [Abstract] [Full Text] [PDF]
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K.-C. Huang, P. H. Rao, C. C. Lau, E. Heard, S.-K. Ng, C. Brown, S. C. Mok, R. S. Berkowitz, and S.-W. Ng Relationship of XIST Expression and Responses of Ovarian Cancer to Chemotherapy Mol. Cancer Ther., August 1, 2002; 1(10): 769 - 776. [Abstract] [Full Text] [PDF]
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C. G. Ferreira, M. Epping, F. A. E. Kruyt, and G. Giaccone Apoptosis: Target of Cancer Therapy Clin. Cancer Res., July 1, 2002; 8(7): 2024 - 2034. [Abstract] [Full Text] [PDF]
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F. Guo, R. Nimmanapalli, S. Paranawithana, S. Wittman, D. Griffin, P. Bali, E. O'Bryan, C. Fumero, H. G. Wang, and K. Bhalla Ectopic overexpression of second mitochondria-derived activator of caspases (Smac/DIABLO) or cotreatment with N-terminus of Smac/DIABLO peptide potentiates epothilone B derivative-(BMS 247550) and Apo-2L/TRAIL-induced apoptosis Blood, May 1, 2002; 99(9): 3419 - 3426. [Abstract] [Full Text] [PDF]
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S. Alas, C.-P. Ng, and B. Bonavida Rituximab Modifies the Cisplatin-mitochondrial Signaling Pathway, Resulting in Apoptosis in Cisplatin-resistant Non-Hodgkin's Lymphoma Clin. Cancer Res., March 1, 2002; 8(3): 836 - 845. [Abstract] [Full Text] [PDF]
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 Y. Deng, Y. Lin, and X. Wu TRAIL-induced apoptosis requires Bax-dependent mitochondrial release of Smac/DIABLO Genes & Dev., January 1, 2002; 16(1): 33 - 45. [Abstract] [Full Text] [PDF]
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A. Gruslin, Q. Qiu, and B. K. Tsang Influence of Maternal Smoking on Trophoblast Apoptosis Throughout Development: Possible Involvement of Xiap Regulation Biol Reprod, October 1, 2001; 65(4): 1164 - 1169. [Abstract] [Full Text] [PDF]
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G. K. Yiu, W. Y. Chan, S.-W. Ng, P. S. Chan, K. K. Cheung, R. S. Berkowitz, and S. C. Mok SPARC (Secreted Protein Acidic and Rich in Cysteine) Induces Apoptosis in Ovarian Cancer Cells Am. J. Pathol., August 1, 2001; 159(2): 609 - 622. [Abstract] [Full Text]
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C. G. Ferreira, P. van der Valk, S. W. Span, I. Ludwig, E. F. Smit, F. A. E. Kruyt, H. M. Pinedo, H. van Tinteren, and G. Giaccone Expression of X-linked Inhibitor of Apoptosis as a Novel Prognostic Marker in Radically Resected Non-Small Cell Lung Cancer Patients Clin. Cancer Res., August 1, 2001; 7(8): 2468 - 2474. [Abstract] [Full Text] [PDF]
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E. Asselin, G. B. Mills, and B. K. Tsang XIAP Regulates Akt Activity and Caspase-3-dependent Cleavage during Cisplatin-induced Apoptosis in Human Ovarian Epithelial Cancer Cells Cancer Res., March 1, 2001; 61(5): 1862 - 1868. [Abstract] [Full Text]
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C. W. Xiao, K. Ash, and B. K. Tsang Nuclear Factor-{{kappa}}B-Mediated X-Linked Inhibitor of Apoptosis Protein Expression Prevents Rat Granulosa Cells from Tumor Necrosis Factor {{alpha}}-Induced Apoptosis Endocrinology, February 1, 2001; 142(2): 557 - 563. [Abstract] [Full Text] [PDF]
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J. Li, Q. Feng, J.-M. Kim, D. Schneiderman, P. Liston, M. Li, B. Vanderhyden, W. Faught, M. F. K. Fung, M. Senterman, et al. Human Ovarian Cancer and Cisplatin Resistance: Possible Role of Inhibitor of Apoptosis Proteins Endocrinology, January 1, 2001; 142(1): 370 - 380. [Abstract] [Full Text] [PDF]
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) or antisense Xiap (
) (MOI = 0, 5, 10, or 20; 72 h; panel a), or for various
durations (24, 48, 60, or 72 h; MOI = 20,
panel b).
) (MOI = 20; 72 h)
or without adenovirus (control, 
) were treated with various
concentrations of cisplatin (0, 10, 30, and 100 µM;
24 h). B, C13* cells infected with various
concentrations of adenoviral antisense Xiap (•) or LacZ (
) and sense p53 (