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Pretreatment Sensitizes Human Melanoma Cells to TRAIL/Apo2 Ligand-Induced Apoptosis1
Center for Drug Discovery and Development, Taussig Cancer Center, Cleveland Clinic Foundation, Cleveland, OH 44195
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and had been correlated with
apoptosis induction. Because IFN- induced other gene products that
have been associated with apoptosis, it was postulated that one or more
IFN-stimulated genes might sensitize cells to TRAIL/Apo2L. Melanoma
cell lines were treated with IFN- for 16–24 h before treatment with
TRAIL/Apo2L. Regardless of their sensitivity to either cytokine alone,
>30% of cells underwent apoptosis in response to the combined
treatment. Induction of apoptosis by IFN- and TRAIL/Apo2L in
combination correlated with synergistic activation of caspase-9, a
decrease in mitochondrial potential, and cleavage of poly(ADP-ribose)
polymerase. Cleavage of X-linked inhibitor of apoptosis following
IFN- and TRAIL/Apo2L treatment was observed in sensitive WM9, A375,
or WM3211 cells but not in resistant WM35 or WM164 cells. Thus, in
vitro IFN- and TRAIL/Apo2L combination treatment had more potent
apoptotic and anti-growth effects when compared with either
cytokine alone in melanoma cells lines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, Fas ligand, death receptor
(DR)43, and
TRAIL/Apo2L initiates cellular death cascades. Both in vitro and in
vivo studies have demonstrated tumoricidal activity without significant
toxicity toward normal cells or tissues (3). TRAIL/Apo2L
binds to TRAIL-R1 (DR4) and TRAIL-R2 (DR5) (4, 5) and
mediates its effects by activating both NF-
B (antiapoptotic) and
caspase-8 (proapoptotic) pathways. Once activated, DRs bind the
cytoplasmic adapter molecule Fas-associated death domain protein, which
in turn recruits either an initiator cysteine protease (caspase-8) or
the receptor-interacting protein and TNFR-associated factor 2.
Recruitment of caspase-8 initiates the apoptotic cascade, whereas
receptor-interacting protein and TNFR-associated factor 2 activate
NF-B (6). In contrast, two other TRAIL receptors,
TRAIL-R3 and TRAIL-R4 (DcR1, DcR2), lack a functional death domain and
cannot transduce apoptotic signals (4, 5).
IFNs transcriptionally regulate >100 genes (7, 8). Some
of these IFN-stimulated genes have been associated with induction of
apoptosis5
(9), including TRAIL/Apo2L (10, 11, 12). IFN-
preferentially induced TRAIL/Apo2L and had greater antiproliferative
(13) and apoptotic effects in vitro in melanoma cells when
compared with IFN-2 (11). A rough correlation was
observed in induction of TRAIL/Apo2L by IFN- and apoptosis in
melanoma cell lines. IFN- induced apoptosis by activating the
caspase cascade, releasing cytochrome c from mitochondria,
and promoting DNA fragmentation. However, this activation occurred late
(>72 h), implicating an intermediate cellular effector(s).
Neutralizing experiments using Ab to TRAIL or dominant negative mutant
of TRAIL-R2 (DR5) confirmed a functional role of TRAIL/Apo2L in
IFN--mediated apoptosis in melanoma cells (11) and in
multiple myeloma cells (12). Furthermore, all cell lines
that underwent apoptosis in response to IFN- exhibited TRAIL/Apo2L
induction. However, a subset of melanoma cell lines, including WM35,
exhibited TRAIL/Apo2L induction but did not apoptose in response to
IFN-. These data suggested that TRAIL/Apo2L was necessary but not
sufficient to mediate IFN--induced apoptosis.
Enhanced antitumor activity of TRAIL/Apo2L in combination with
chemotherapeutic agents that disrupt cellular metabolism and mitotic
activity has been reported (14, 15, 16, 17, 18). We postulated that
IFNs might sensitize melanoma cells to TRAIL/Apo2L, because IFN-
induced other genes associated with apoptosis. In this work, we report
that IFN- and TRAIL/Apo2L in combination synergistically induced
apoptosis and caspase activation in melanoma cell lines. This occurred
at least in part by cleavage of the X-linked inhibitor of apoptosis
(XIAP).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human melanoma cell lines WM9, WM35, WM3211, WM793, WM164 (19), A375, FEMX, Guilliams, and Minors (American Type Culture Collection, Manassas, VA) were grown in DMEM (Life Technologies, Rockville, MD) supplemented with heat-inactivated 10% FCS (HyClone Laboratories, Logan, UT) in a humidified chamber of 95% air/5% CO2 at 37°C. Collection of biopsies and preparation of primary melanoma cell culture was conducted following Institutional Review Board guidelines and approval. Low passage, melanoma cells (CCFMel-1H, CCFMel-2B), primary astrocytes (CCF-TEN, CCF-Bon) (20), human foreskin fibroblasts (HFF) (Cleveland Clinic Foundation, Cleveland, OH), HUVECs, and human fibroblast cell line WI-38 (American Type Culture Collection) were cultured in DMEM-F12 medium supplemented with 10% FCS. Cells were periodically confirmed as mycoplasma free.
IFN-2b (intron A; Schering-Plough, Kenilworth, NJ) or IFN-
(Rebif; Ares-Serono, Geneva, Switzerland) used in the study were of
equivalent specific activity (2 x 108 U/mg
protein). All experiments were done using different preparations of
recombinant human TRAIL/Apo2L; that from Genentech (San
Francisco, CA) is denoted by (G) and that from PeproTech (Rocky Hill,
NJ) is denoted by (P) in the text. TRAIL/Apo2L (G) consisted of >99%
trimeric protein with Zn2+ (21).
Presence of Zn2+ has been reported as necessary
for its optimal activity (4).
Cells were treated with IFN- and/or TRAIL/Apo2L for different time
periods based on the experiment. For analysis of early apoptosis events
such as annexin V positivity, cells were treated with TRAIL/Apo2L for
16–24 h and for late apoptosis TUNEL or antiproliferative effects
(24–36 h). To analyze enzymes or proteins involved in initiation and
execution of apoptosis pathway, cells were treated with
TRAIL/Apo2L for <16 h (2, 6, or 12 h) to avoid too many dead
cells. Doses and time for IFN (100 and 500 U/ml) used in this study
were based on a previous report (12). All treatments were
performed at 37°C in a humidified chamber of 95% air/5%
CO2.
Antiproliferative assays
Cells were plated at a cell density of 10,000 cells/well in
96-well plates and IFN-2 or IFN- were added in different
dilutions (100 and 500 U/ml) to the assay plate. Quadruplicates of each
treatment were performed. After 24 h, recombinant human
TRAIL/Apo2L (P) was added at different concentrations. After 36 h,
plates were fixed with 10% TCA (4°C) for 1 h, rinsed with
water, and allowed to air dry. Cell numbers were estimated by staining
with 0.4% sulforodamine B (w/v) (Sigma-Aldrich, St. Louis, MO) and
measuring the absorbance at 570 nm (22). Results were
calculated as follows: % growth = (ODexp -
ODini)/(ODfin -
ODini) x 100, where
ODfin corresponds to
A570 of wells with no treatment,
ODini corresponds to 0% growth, and
ODexp corresponds to wells treated with different
concentrations of IFN. The multiple drug-effect analysis method of Chou
and Talalay (23) was used to measure interaction between
IFN- and TRAIL/Apo2L.
Gel electrophoresis and immunoblot analyses
Whole cell lysates were prepared in 1x lysis buffer (50 mm Tris-Cl (pH 8), 1% Triton X-100, 10% glycerol, 1 mM EDTA, 250 mM NaCl, 1 mM DTT, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin) for subsequent immunoblotting studies (11). SDS-PAGE was conducted by using Laemmli buffer system on 12% polyacrylamide gels, and proteins separated on gels were transferred onto a polyvinylidene difluoride membrane by the semidry method (Trans Blot SD; Bio-Rad, Hercules, CA). Binding of the primary and secondary Abs was performed according to standard protocols (11). Membranes were immunoblotted with the mAb to apoptotic protease-associated factor-1 (Apaf-1), Bcl2, Bax (Santa Cruz Biotechnology, Santa Cruz, CA), or with the polyclonal Ab to caspase-3 (BD PharMingen, San Diego, CA), cellular inhibitor of apoptosis (cIAP)1, cIAP2 (Santa Cruz Biotechnology), and XIAP (BD PharMingen), followed by incubation with HRP-conjugated secondary Abs (Pierce, Rockford, IL). Immunoreactive bands were visualized by using ECL (PerkinElmer, Boston, MA). Equal protein loading was confirmed by reprobing with actin mAb (Sigma-Aldrich). All the immunoblots in this study were repeated two to three times with reproducible results.
EMSA
A375 cells were treated with IFN-2 (24 h), IFN- (24 h), or
TRAIL/Apo2L (2 h), or were treated with IFN (24 h) followed by
TRAIL/Apo2L (2 h), and cytoplasmic extracts were prepared. NF-B
binding consensus (5'-AGTTGAGGGGACTTTCCCAGGC-3') sequence from the
IFN- gene promoter was end labeled with
[
32-P]dATP (3000 Ci/mol) using T4
polynucleotide kinase. DNA binding reactions were performed in a
20-µl volume containing 10 µg nuclear protein, 20 mM HEPES, 10 mM
KCl, 0.1% Nonidet P-40, 0.5 mM DTT, and 10% glycerol. The binding
reaction was performed for 20 min at room temperature. Complexes were
separated from the free probe on a 6% nondenaturing polyacrylamide gel
in 0.5x Tris-borate EDTA buffer at 200 V for 2 h. Gels were dried
and exposed to film.
Caspase activity assay
Caspase-3, caspase-8, and caspase-9 activities were measured
using a commercially available ApoAlert assay kit (Clontech
Laboratories, Palo Alto, CA). Briefly, cells treated with IFN- (40
h), TRAIL/Apo2L (2, 6, or 16 h), or IFN- (24 h), followed by
TRAIL/Apo2L for 2, 6, or 16 h, were washed twice with cold PBS and
lysed on ice in 50 µl of cold lysis buffer. Cell lysates were
centrifuged at 10,000 x g for 10 min to precipitate
cellular debris. Assay was performed in triplicate on a 96-well plate
based on the manufacturer’s protocol.
TUNEL assay
DNA fragmentation was detected in IFN-- and
TRAIL/Apo2L-treated cells by TUNEL staining using the APO-BRDU
kit (BD PharMingen) as per the manufacturer’s protocol. The percentage
of FITC-positive cells was analyzed by FACS (FACSVantage; BD
Biosciences, San Diego, CA).
Annexin V/PI assay
Annexin V staining of exposed membrane phospholipid phosphatidylserine was done using the annexin V assay kit (BD PharMingen) following the manufacturer’s protocol. The percentages of annexin V- and propidium iodide (PI)-positive cells were analyzed by FACS (FACSVantage).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Defects in TRAIL/Apo2L induction by IFNs (11) were
postulated to be a factor mediating resistance to apoptosis by IFNs in
melanoma cell lines. Based on the hypothesis that exogenous TRAIL/Apo2L
might induce apoptosis in resistant melanoma cells, functional in vitro
studies were performed with recombinant TRAIL/Apo2L protein. Based on
previous studies (24, 25), increasing doses of TRAIL/Apo2L
were tested in A375 melanoma cells. No significant (<10%) apoptosis
(assessed by annexin V/PI staining) was observed at concentrations
ranging from 25 to 200 ng/ml (24–48 h). To analyze sensitivity of
other melanoma cell lines, cells were treated with TRAIL/Apo2L (100
ng/ml) for 24 h and IFN- (500 U/ml) for 40 h
(11). No significant cytotoxic effects were observed in
response to TRAIL/Apo2L in WM9, WM3211, A375, WM35, and WM164 melanoma
cells (Fig. 1
). At 40 h, IFN-
alone induced 13–16% apoptosis in WM9 and WM3211 cells. To confirm
that cells responded to TRAIL/Apo2L, they were cotreated with the
metabolic inhibitor actinomycin D (10 ng/ml) and TRAIL/Apo2L.
TRAIL/Apo2L induced apoptosis in most melanoma cells in the presence of
actinomycin D (Fig. 1), confirming that they expressed functional TRAIL
receptors and downstream apoptotic components. All the apoptosis assays
in this study were repeated at least three times and a variation of
±5% between individual experiments was considered acceptable.
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-induced apoptosis was
chosen. Cells were either left untreated or treated in triplicate with
IFN- (500 U/ml for 40 h) or TRAIL/Apo2L (100 ng/ml for 16
h). In parallel, cells were cotreated with IFN- and TRAIL/Apo2L (16
h) or pretreated with IFN- for 8 or 24 h followed by
TRAIL/Apo2L (16 h). Apoptotic cell death was measured by annexin
V/PIstaining followed by bivariate FACS analysis. No
significant apoptosis was observed in cells treated with either
cytokine as a single agent or together. However, cells pretreated with
IFN- for 8 h followed by TRAIL/Apo2L had an 8–10% increase in
annexin V/PI positivity. This increased to >30% in cells pretreated
with IFN- for 24 h (Fig. 2A). Similar results were
obtained with TUNEL analyses (Fig. 2B).
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-induced apoptosis) and A375 (resistant to IFN--induced
apoptosis) were treated in parallel with recombinant TRAIL/Apo2L (P) or
with Zn2+ (G). Cells were treated with similar
doses as described previously and assessed by annexin V/PI staining.
Again, both cell lines showed synergistic increase in apoptosis
following IFN- pretreatment. Neither of the TRAIL/Apo2L preparations
alone induced apoptosis in either of the cell lines (Fig. 3).
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sensitized other cells to
TRAIL/Apo2L-induced apoptosis, annexin V/PI and TUNEL staining were
performed. TRAIL/Apo2L (16–24 h) alone had no significant effect on
apoptosis (3–9%) in most melanoma cells. However, IFN- (40 h)
induced partial apoptosis (12–15%) in a subset of melanoma cell
lines. IFN- pretreatment sensitized seven of nine melanoma cell
lines (WM3211, FeMX, WM793, CCFMel2H, CCFMelB) to TRAIL/Apo2L-induced
cytotoxicity. However, a subset (two of nine) of cell lines (WM35,
WM164) that were resistant to IFN--induced apoptosis was also
resistant to proapoptotic effects of the IFN- and TRAIL/Apo2L
combination (Table I). Primary
nonmalignant human cells, HUVECs, HFF, WI-38, CCF-TEN, and CCF-BON,
were resistant to apoptotic effects of TRAIL/Apo2L alone as well as in
combination with IFNs (Table I).
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is more potent then IFN-2 in sensitizing cells to
TRAIL/Apo2L-induced antiproliferative effects
Short-term antiproliferative assays (60 h) were performed with
IFN-2 and IFN- with TRAIL/Apo2L in combination to assess growth
inhibition. A375 cells were pretreated with either IFN-2 or
IFN- (100 and 500 U/ml) for 24 h followed by increasing doses
(0–200 ng/ml) of TRAIL/Apo2L for 36 h. Cells were fixed and
relative cell numbers were assessed by sulforhodamine B
staining. Unlike apoptotic effects, TRAIL/Apo2L had growth inhibitory
effects on A375 melanoma cells (ID50 of 
200
ng/ml). Compared with IFN-2, IFN- was more potent in sensitizing
cells to TRAIL/Apo2L. The IFN- and TRAIL/Apo2L combination effect
was synergistic (23). At an IFN concentration of 100 U/ml,
the ID50 for TRAIL/Apo2L was 25 ng/ml with
IFN- but was 200 ng/ml for IFN-2-treated cells (data not shown).
With an increased concentration of IFNs (500 U/ml), the
ID50 for TRAIL/Apo2L was 12.5 ng/ml and 100 ng/ml
with IFN- and IFN-2, respectively (Fig. 4A). The differences in
potency of IFN-2 and IFN- alone (p 
0.05) or in combination with TRAIL/Apo2L were statistically significant
(p 0.01).
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2 and IFN- pretreatment on
TRAIL-induced apoptosis, A375 cells were treated with either IFN-2
and IFN- (100 and 500 U/ml) or TRAIL/Apo2L (100 ng/ml) alone
or were pretreated with IFN followed by TRAIL/Apo2L. Apoptosis was
assessed by annexin V/PI staining. As observed in antiproliferative
assays, IFN- pretreatment was more potent in inducing apoptosis
(24–34%) when compared with IFN-2 (7–12%) at equivalent
TRAIL/Apo2L concentrations (Fig. 4
B).
IFN- did not modulate activation of NF-B or expression of
proteins involved in DR-mediated apoptotic signaling
An IFN--dependent inhibition of NF-B activation might
sensitize cells to TRAIL-induced apoptosis. To test this possibility,
A375 cells treated with IFN-2, IFN-, or TRAIL/Apo2L alone or with
the IFN and TRAIL/Apo2L in combination were assessed for NF-B
activation by EMSA. IFN alone did not induce NF-B activation.
TRAIL/Apo2L induced equivalent amounts of NF-B in untreated or
IFN-pretreated A375 cells (Fig. 5A).
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and TRAIL/Apo2L. IFN-
treatment did not induce or inhibit expression of any of these proteins
(Fig. 5B).
IFN- and TRAIL/Apo2L in combination synergistically activated
caspase-9
To identify the mechanism of synergism of IFN and TRAIL/Apo2L, we
assessed cleavage of key death substrates that indicate activation of
the cell death machinery. To test caspase activation, time course study
(2–16 h) was done following TRAIL/Apo2L treatment. A375 cells were
treated with either IFN- (500 U/ml for 40 h) or TRAIL/Apo2L
(100 ng/ml) alone or pretreated with IFN- (24 h) followed by
TRAIL/Apo2L (2, 6, and 16 h). Cell extracts were analyzed for
caspase-3, caspase-8, and caspase-9 activity using specific fluorogenic
caspase tetrapeptide substrates. IFN- alone had no effect on any of
the caspases in A375 cells. Consistent with a previous report
(24), at 6- and 16-h time points, TRAIL/Apo2L alone
resulted in 10- to 30-fold increased caspase-8 and caspase-3 activity.
However, IFN- treatment further augmented caspase-3 and caspase-8
activity by 2-fold (Fig. 6, A
and B). Furthermore, despite increased caspase-3 activity,
poly(ADP-ribose) polymerase, an apoptotic protease downstream of
caspase-3, was not cleaved in cells treated with TRAIL/Apo2L alone
(data not shown).
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(24 h)
followed by TRAIL/Apo2L (2 h), a marked increase (>20-fold) in
caspase-9 activity resulted. This was further enhanced with increasing
time (Fig. 6C). In IFN--sensitive WM9 cells, enhanced
caspase-9 activity was observed in cells treated with IFN- or
IFN- plus TRAIL/Apo2L. TRAIL/Apo2L alone had no significant effect
on caspase-9 activity (Fig. 6D). These data suggested that
failure to activate caspase-9 resulted in the lack of
TRAIL/Apo2L-induced apoptosis.
To ascertain the role of caspase-9 in mediating IFN- pretreatment
effects, A375 cells were treated either with the caspase-9 inhibitor
(LEHD-fmk) alone or with the combination of IFN- and TRAIL/Apo2L.
The caspase-3 inhibitor (DEVD-fmk) was also used with IFN- and
TRAIL/Apo2L in combination as a positive control. Caspase inhibitors
alone had no significant cytotoxic effects. IFN- followed by
TRAIL/Apo2L resulted in >30% apoptotic cells, but in the presence of
the caspase-9 inhibitor (LEHD-fmk) only 7–8% cells were annexin V/PI
positive. As expected, the caspase-3 inhibitor (DEVD-fmk) blocked
IFN-- and TRAIL/Apo2L-mediated apoptosis completely (Fig. 6E).
Caspase-3 cleavage was confirmed in resistant and sensitive
melanoma cells by immunoblot analyses. Consistent with the fluorogenic
caspase assays, the 32-kDa procaspase-3 was partially cleaved to a
20-kDa active form after TRAIL/Apo2L (P) or (G) treatment of
cells both sensitive (A375, WM9) and resistant (WM35) to the
combination treatment. However, the p17 and p11 subunits
resulting from the autocatalytic activity of caspase-3 were observed
only in sensitive (A375, WM9, WM3211) cell lines following treatment
with IFN- and TRAIL/Apo2L in combination (Fig. 7).
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and TRAIL/Apo2L in combination synergistically
induced cleavage of XIAP in apoptosis-sensitive melanoma cell lines
Because TRAIL/Apo2L resulted in cleavage of caspase-3, a
block in apoptosis downstream of caspase-3 was postulated. XIAP is a
potent inhibitor of apoptosis (IAP) that binds to and inhibits
caspase-9 and caspase-3 activity (26) and has been
associated with TRAIL/Apo2L resistance in melanoma cells
(27). Thus, the effect of IFN and TRAIL/Apo2L in
combination on XIAP was examined. A375, WM9, WM3211, WM35, and WM 164
cells were treated with IFN- (40 h), TRAIL/Apo2L (P) or (G) (12 h),
and the combination IFN- and TRAIL/Apo2L (P) or (G). Immunoblot
analysis with XIAP Ab revealed synergistic cleavage of XIAP to an
inactive 29-kDa fragment in WM9, WM3211, and A375 cells following
combination treatment with IFN- and TRAIL/Apo2L (Fig. 8). No cleavage was observed in resistant
WM35 or WM164 cells. Both preparations of TRAIL/Apo2L (P) and (G) had
comparable activity. IFN--sensitive cells WM9 and WM3211 had slight
cleavage of XIAP with IFN- alone. TRAIL/Apo2L alone had no
significant effect on XIAP expression in melanoma cell lines.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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100–200 ng/ml). However, no apoptotic effects of recombinant
TRAIL/Apo2L protein produced in Escherichia coli by two
independent sources (Genentech and PeproTech) were observed in any of
the melanoma cell lines.
In contrast to results obtained with TRAIL/Apo2L as a single agent,
combination of TRAIL/Apo2L with the metabolic inhibitor actinomycin D
induced apoptosis, suggesting that TRAIL receptors, apoptotic
initiators, and caspases were functional in these melanoma cells.
Other tumor cell types resistant to TRAIL/Apo2L have been rendered
sensitive by cotreatment with chemotherapeutic agents such as
actinomycin D, CPT11, or 5-fluorouracil (14, 15, 16, 17, 18). IFN-2
also has been suggested to sensitize cells to TNF--mediated
apoptosis (28). Thus, it was postulated that IFNs might
sensitize melanoma cells to TRAIL/Apo2L-mediated apoptosis.
Cell lines sensitive (WM9) and resistant (A375) to
IFN--induced apoptosis were treated concomitantly with IFN-
and TRAIL/Apo2L. Unlike metabolic inhibitors such as
actinomycin D, concomitant treatment with IFN- and TRAIL/Apo2L had
no greater effects compared with either single agent. Irrespective of
sensitivity to either cytokine alone, when cells were pretreated with
IFN- for 12–48 h followed by TRAIL/Apo2L apoptosis was augmented in
most of the melanoma cell lines. In vitro, IFN-2 was weak
compared with IFN- in inducing apoptosis alone (11) or
in sensitizing cells to TRAIL/Apo2L in melanomas. However, IFN-2
induced apoptosis by activating the DR-mediated caspase pathway in
multiple myeloma cells (12). Similarly, the IFN- and
TRAIL/Apo2L combination was 2-fold less potent (15–20% apoptosis)
compared with IFN- (30–40%) in melanoma cells (data not shown).
Unlike IFN-2 or IFN-, IFN- may sensitize cells to DR-mediated
apoptosis by up-regulation of DR5 (29). Similar
observations regarding augmentation of TRAIL/Apo2L-induced apoptosis by
IFN- was reported in breast carcinoma cells (30). Very
little cytotoxicity was observed with IFN- or TRAIL/Apo2L alone or
with the IFN-/TRAIL combination on primary nonmalignant human cells
such as HUVECs, fibroblasts (HFF, WI-38), and astrocytes (CCF-TEN,
CCF-BON).
TRAIL/Apo2L activates the NF-B-mediated prosurvival signaling
pathway. Inhibition of NF-B activation by IFN-2 has been reported
as the mechanism for IFN-2-dependent sensitivity to TNF- in Daudi
cells (28). However, IFN- neither activated nor
inhibited TRAIL-induced NF-B, suggesting no direct role of NF-B
in mediating the effects of IFN- on TRAIL/Apo2L sensitivity.
Resistance to TRAIL/Apo2L has been attributed to differential
expression of DRs (31), defects in caspase-8
(32), higher expression of FLIP (33) or XIAP
(27), or defects in Apaf-1 (34) expression.
Because treatment of melanoma cell lines with actinomycin D rendered
them TRAIL sensitive, a possible role of downstream inhibitory proteins
like FLIP and IAPs that bind to Fas-associated death domain
protein or other proteins in the caspase pathway was postulated.
Expression of these apoptotic regulators was assessed in various
melanoma cell lines. Although constitutive expression of these proteins
varied from cell to cell, there was no correlation between levels of
expression and sensitivity to apoptosis induced by IFN- alone or the
combination. Furthermore, IFN- treatment (24 h) did not alter
expression of FLIP, Apaf-1, caspase-9, caspase-8, caspase-3, cIAP-1, or
cIAP-2 in melanoma cells. No correlation with apoptotic sensitivity has
been observed in expression of DRs (TRAIL-R1, TRAIL-R2) and decoy
receptors and in resistance to TRAIL/Apo2L in melanoma cells including
WM9, WM35, WM3211, WM793, and WM-981 (25). IFN-
treatment had no effect on transcript levels of TRAIL-R1 and TRAIL-R2
in sensitive and resistant melanoma cells (11).
Activities of the initiator (caspase-8) and the executioner (caspase-3)
caspases were analyzed in A375 cells following TRAIL/Apo2L, IFN-,
and the combination treatment. IFN- failed to induce TRAIL/Apo2L in
A375 cells (11) and had no significant effect on caspase
activity. Both caspase-8 and caspase-3 resulted in cleavage of their
specific fluorogenic substrates in response to TRAIL/Apo2L
(35). However, TRAIL/Apo2L alone couldnot activate
caspase-9 without prior IFN- treatment. Activation of caspase-9 may
have provided additional apoptotic signals to enhance
disruption of mitochondrial functions. Synergistic cytochrome
c release from mitochondria followed disruption of
mitochondrial potential (
m) with the
combination treatment with IFN- and TRAIL/Apo2L (data not
shown).
IAP proteins are defined by a novel conserved motif termed the
baculoviral IAP repeat (36). XIAP (also known as human
IAP-likeprotein/minor histocompatibility Ag), the most potent
caspase inhibitor, directly binds and inhibits caspase-3,
caspase-9, and caspase-7 but not caspases-1, caspase-6, caspase-8, or
caspase-10 (26, 37). TRAIL/Apo2L alone has been implicated
in cleavage of XIAP in TRAIL-sensitive melanoma cell lines
(27). No significant effects on expression or cleavage of
XIAP were observed following treatment with TRAIL/Apo2L alone in this
study. However,when cells were pretreated with IFN- followed by
TRAIL/Apo2L treatment, XIAP, but not cIAP-1 or cIAP-2, was cleaved to
its inactive 29-kDa form. Cleavage of XIAP could contribute to
activation of caspase-9 and cleavage of poly(ADP-ribose) polymerase and
Bid following the combination treatment.
TRAIL/Apo2L binds to its DRs, activates caspase-8, and results in
cleavage of procaspase-3 to active caspase-3 (p20). However, the p17
subunit resulting from autocatalytic activity of active caspase-3 was
detectable only in cells pretreated with IFN-. It seems probable
that active XIAP bound to the p20 subunit of caspase-3 prevents the
second catalytic cut that is necessary for downstream events (27, 38). These results implicated XIAP as a significant inhibitor of
TRAIL/Apo2L-induced apoptosis.
IFN- may modulate TRAIL/Apo2L-mediated cleavage of XIAP through
induction of XIAP-associated factor-1 (XAF1)
(41), a negative regulator of
XIAP (39, 40). XAF1 protein was strongly
up-regulated in WM-9, A375, and WM3211 cells but not in resistant WM35
and WM164 cells (41). IFN- alone had no effect on XIAP
expression. The mechanism involved in cleavage of XIAP may not be
induction of XAF1 by IFN but rather caspase-3 and caspase-9, freed of
inhibitory effects of XIAP. It is conceivable that potentiation of
activities of caspase-3 and caspase-9 may render resistant
cells sensitive to TRAIL/Apo2L. Thus, one or more novel IFN-stimulated
genes, such as XAF1, may have an important role in IFN-mediated
sensitization to TRAIL/Apo2L. These studies provide a novel role of
IFNs in mediating sensitivity to DR-mediated apoptosis in vitro by
modulating IAPs. Further in vivo studies with IFN and TRAIL/Apo2L in
combination are needed to ascertain its antitumor effects.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Biological Sciences, University of Toledo, Toledo, OH 43606.
3 Address correspondence and reprint requests to Dr. Ernest C. Borden, Center for Drug Discovery and Development, Taussig Cancer Center, Cleveland Clinic Foundation, 9500 Euclid Avenue T-40, Cleveland, OH 44195. E-mail address: bordene{at}cc.ccf.org
4 Abbreviations used in this paper: DR, death receptor; IAP, inhibitor of apoptosis; XIAP, X-linked IAP; cIAP, cellular IAP; Apaf-1, apoptotic protease-associated factor-1; XAF1, XIAP-associated factor-1; PI, propidium iodide; HFF, human foreskin fibroblast.
5 D. W. Leaman, M. Chawla-Sarkar, K. Vyas, A. Ozdemir, and E. C. Borden. Greater potency of IFN- compared with IFN-2 in inducing IFN stimulated genes in melanoma: identification of new ISGs by oligonucleotide microarray. Submitted for publication.
Received for publication February 20, 2002. Accepted for publication May 9, 2002.
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compared with IFN-2: correlation with TRAIL/Apo2L induction in melanoma cell lines. Clin. Cancer Res. 7:1821. and for diploid and transformed human cells. Cancer Res. 42:4948. suppresses activation of nuclear transcription factors NF-B and activator protein 1 and potentiates TNF-induced apoptosis. J. Immunol. 165:4927.. Exp. Cell Res. 262:154.[Medline]
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