Content uploaded by Hong Xin
Author content
All content in this area was uploaded by Hong Xin on Apr 08, 2021
Content may be subject to copyright.
Enhanced Killing of Melanoma Cells by Simultaneously
Targeting Mcl-1 and NOXA
Jian-Zhong Qin, Hong Xin, Leonid A. Sitailo, Mitchell F. Denning, and Brian J. Nickoloff
Department of Pathology, Loyola University Medical Center, Maywood, Illinois
Abstract
By deciphering the dysregulation of apoptosis in melanoma
cells, new treatment approaches exploiting aberrant control
mechanisms regulating cell death can be envisioned. Among
the Bcl-2 family, a BH3-only member, NOXA, functions in a
specific mitochondrial-based cell death pathway when mela-
noma cells are exposed to a proteasome inhibitor (e.g.,
bortezomib). Some therapeutic agents, such as bortezomib,
not only induce proapoptotic Bcl-2 family members and active
conformational changes in Bak and Bax but also are
associated with undesirable effects, including accumulation
of antiapoptotic proteins, such as Mcl-1. To enhance the
bortezomib-mediated killing of melanoma cells, the apoptotic
pathway involving NOXA was further investigated, leading to
identification of an important target (i.e., the labile Bcl-2
homologue Mcl-1 but not other survival proteins). To reduce
Mcl-1 levels, melanoma cells were pretreated with several
different agents, including Mcl-1 small interfering RNA
(siRNA), UV light, or the purine nucleoside analogue fludar-
abine. By simultaneously triggering production of NOXA
(using bortezomib) as well as reducing Mcl-1 levels (using
siRNA, UV light, or fludarabine), significantly enhanced kill-
ing of melanoma cells was achieved. These results show
binding interactions between distinct Bcl-2 family members,
such as NOXA and Mcl-1, in melanoma cells, paving the way
for novel and rational therapeutic combination strategies,
which target guardians of the proapoptotic Bak- and Bax-
mediated pathways, against this highly aggressive and often
fatal malignancy. (Cancer Res 2006; 66(19): 9636-45)
Introduction
Not only is the lifetime risk of developing melanoma increasing
steadily (f1 in 75), but also in metastatic melanoma patients the
average survival rate is only 6 to 10 months (1, 2). The treatment of
metastatic melanoma is frequently futile because of the aggressive
growth and apoptotic resistance of the tumor to various
therapeutic regimens (3). The resistance of melanoma to existing
treatments is linked to the large number of survival pathways and
inactivation of death pathways (2). The treatment approaches that
have been investigated in numerous phase III clinical trials include
immunotherapy with or without dendritic cells, cytokine-based
therapy, radiation therapy, and chemotherapy (4–6). Even the use of
combinations of chemotherapeutic agents has not significantly
changed the overall survival benefit in any randomized clinical
trials for the past 20 years. One of the primary obstacles to progress
has been the lack of precise mechanistic insights by which
conventional therapeutic agents either succeed or fail in killing
various tumor cell types. Added to this issue has been the growing
number of survival factors defined in melanoma cells that seem to
present a daunting challenge for investigators attempting to
overcome the apoptotic resistance of melanoma cells (7). These
survival factors include members of the Bcl-2 family, such as Bcl-2
and Bcl-x
L
, as well as elevated levels of survivin and Mcl-1 (8–10).
Recently, we and others have successfully overcome the apoptotic
resistance of melanoma cells using proteasome inhibitors (11–13),
and this study extends our efforts to optimize the effectiveness of
this class of antineoplastic reagents (14).
To probe into the molecular black box of melanoma cells
regarding apoptosis and drug resistance (7, 8, 15), we sought to
define the molecular mechanism by which a proteasome inhibitor
[e.g., bortezomib (PS-341; Velcade)] can overcome the well-known
apoptotic resistance of melanoma cells. Defining this pathway led
to an important role for the BH3-only protein, NOXA, in the
successful apoptotic response both in vitro and in animal-based
xenograft tumor models (11–13). The induction of NOXA was found
to be independent of the p53 status of the treated cells and
included a transcriptional component (11–13). Because a recent
phase II clinical trial using bortezomib as a single agent revealed
minimal efficacy, we decided to identify molecular targets for
NOXA with the perspective of using this new insight for designing
a rational approach for combination therapy rather than relying
on purely empirical approaches for future clinical trials (16–18).
A growing body of evidence highlights the molecular machinery
involving the intrinsic or mitochondrial-based apoptotic pathway
(19, 20). The overall integrity of mitochondrial function is
controlled by the Bcl-2 family of proteins that include both
antiapoptotic and proapoptotic members (21). These family
members belong to three different classes of proteins based on
their Bcl-2 homology domains, which include multidomain
antiapoptotic proteins (Bcl-2, Bcl-x
L
, Mcl-1, Bcl-w, and A1),
multidomain proapoptotic proteins (Bak and Bax), and BH3-only
proapoptotic proteins (Bid, Bad, Bim, PUMA, NOXA, and Bik). The
proapoptotic BH3-only proteins, such as NOXA, are the most apical
regulators of death signaling cascades and hence have become
intensely studied in a variety of normal and malignant cell types
(22). A recent elegant model has been proposed in which the
overall apoptotic response of cells can be defined around the
proapoptotic Bak and Bax family members (23–25).
According to this model, the activation of multidomain
proapoptotic proteins, such as Bak and Bax, mediating toxicity is
required for cell death and is normally blocked in healthy cells by
the copresence of Mcl-1 and Bcl-x
L
. Thus, Mcl-1 and Bcl-x
L
serve as
complimentary guardians to protect against cell death by binding
to Bak and Bax and hence preventing their conformational state to
be converted from an inactive to active or toxic status. However,
Note: J-Z. Qin and H. Xin contributed equally to this work.
Requests for reprints: Brian J. Nickoloff, Oncology Institute, Cardinal Bernardin
Cancer Center, Loyola University Medical Center, Building 112, Room 301, 2160 South
First Avenue, Maywood, IL 60153. Phone: 708-327-3241; Fax: 708-327-3239; E-mail:
bnickol@lumc.edu.
I2006 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-06-0747
Cancer Res 2006; 66: (19). October 1, 2006 9636 www.aacrjournals.org
Research Article
Research.
on April 8, 2021. © 2006 American Association for Cancercancerres.aacrjournals.org Downloaded from
when NOXA is induced, it can displace Mcl-1 leading to the ubi-
quitination and proteasome-mediated degradation of Mcl-1; when
Bad is present, it can displace Bcl-x
L
, thereby allowing Bak and Bax
to oligomerize, and in this activated conformation, Bak and Bax
can mediate toxic reactions in the mitochondria culminating in cell
death. Thus, a combination of both NOXA and Bad are postulated
to be required to trigger cell death following a cytotoxic stimulus,
although the relative contribution of the complementary guardians
of Bak and Bax (e.g., Mcl-1 and Bcl-x
L
) has not been defined in
melanoma cells. In addition, the relative roles for activated Bak and
Bax in the bortezomib-mediated cell death of melanoma cells has
not been delineated. In other cell types, the relative roles for
activated Bak and/or Bax have been found to be dependent on the
stimulus (25–28).
Because we observed previously that inhibition of proteasome
function served as a cytotoxic stimulus in melanoma cells but not
in normal melanocytes, which resulted from differential induction of
NOXA, the binding partner for NOXA was sought in melanoma cells
and identified as Mcl-1. Once we established NOXA targeted coun-
teracting Mcl-1 in bortezomib-treated melanoma cells, coupled with
accumulation of Mcl-1 by proteasome inhibitors, agents that could
be used to pretreat melanoma cells were identified to reduce Mcl-1
levels, thereby enhancing the effectiveness of bortezomib to
augment the extent of NOXA-mediated cell death. Agents selected
to reduce Mcl-1 levels included a genetic approach [e.g., small
interfering RNA (siRNA) knockdown], a physical agent (e.g., low-dose
UV light; ref. 29), and a pharmacologic agent (e.g., purine nucleoside
analogue, fludarabine). Previous studies have revealed the transcrip-
tional and translational control of Mcl-1 is characterized by a short
half-life at the mRNA and protein levels (29, 30). Thus, the agents
selected share the ability to disrupt both transcriptional and/or
translational events and hence can reduce Mcl-1 levels (31). We were
particularly interested in a purine nucleoside analogue (e.g.,
fludarabine) because it can inhibit both DNA and RNA synthesis
and has been successfully combined in a clinical setting with other
promising agents, including bortezomib, to achieve enhanced results
(31–33). Although activated conformation of both Bak and Bax
could be detected, the relative kinetics and extent of activation was
different with higher levels of activated Bax compared with Bak
in bortezomib-treated melanoma cells.
As all melanoma cell lines examined constitutively expressed
Bad as well as Bak and Bax, the current results support future
clinical trials for melanoma patients in which bortezomib is used to
induce NOXA, combined together with an agent, such as
fludarabine, which can reduce Mcl-1 levels. Such new strategies
targeting mitochondria (34) exploit the specificity of BH3-only
proteins and requirement for coordinated inactivation of distinct
survival factors that culminate in selective killing of melanoma cells
while sparing normal melanocytes. It is essential for rational design
of drug combinations to better understand the genetic and mole-
cular basis for aberrant apoptosis control in melanoma cells (16, 22),
and the current results provide insight into specific therapeutic
opportunities requiring bench to bedside translational studies.
Materials and Methods
Cell culture. A late-passage (>60 passages) human cutaneous melanoma
cell line (C8161; mutant p53 allele) and low-passage (<20 passages)
pulmonary metastatic melanoma cells (RJ002L; wild-type p53 allele) were
maintained in RPMI 1640 supplemented with 10% fetal bovine serum as
described previously (12). Removal of the metastatic melanoma lesion was
done after patient informed consent as part of a phase I Food and Drug
Administration–approved clinical trial and approval of the Loyola
Institutional Review Board.
Chemical reagents and antibodies. Bortezomib (pyrazylcarbonyl-
Phe-Leu-boronate), manufactured by Millenium Pharmaceuticals (Cam-
bridge, MA), was obtained from the Loyola University Medical Center
pharmacy. Fludarabine was purchased from Sigma Chemical Co. (St. Louis,
MO), and a 10 mmol/L stock solution was prepared in DMSO. Antibodies
used in this study were obtained as follows: NOXA and activated Bak (35)
were from Oncogene Research Products (La Jolla, CA) and Mcl-1, Bcl-2,
Bcl-x
L
, Bad, and poly(ADP-ribose) polymerase (PARP) were from Santa
Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody to detect activated Bax
from BD PharMingen, Inc. (San Diego, CA; ref. 36). Antibody against h-actin
(ICN, Irvine, CA) served as loading control.
Cell death detection. Cell viability was assessed using Apo Target
Annexin V-FITC staining kits (Biosource, Camarillo, CA) according to the
manufacturer’s instructions. The relative percentage of cells undergoing
apoptosis was quantified by flow cytometric analysis using FACSCalibur
(Becton Dickinson, Palo Alto, CA) as described previously (13).
Mcl-1 retroviral constructs and infection. The Mcl-1 cDNA (kindly
provided by Dr. W. Douglas Cress, University of South Florida College
of Medicine) was subcloned into the BamHI and Not I of LZRS-based
retroviral expression vector. The Phoenix-Ampo retroviral packaging cells
were transfected with calcium precipitation method and viral supernatants
were prepared as described previously (30). RJ002L melanoma cells were
seeded in six-well plates and infected with viral supernatants containing
either control vector (Linker) or Mcl-1 cDNA and then were subjected to
1Amol/L bortezomib treatment for 24 hours.
Mcl-1 siRNA transfection. Smart pools of Mcl-1 siRNA duplexes and
scrambled control duplexes were purchased from Dharmacon Research,
Inc. (Lafayette, CO). C8161 melanoma cells were plated in six-well plates at a
density of 15 10
4
per well, and transfection was accomplished using
Oligofectamine (Invitrogen, Carlsbad, CA) in Opti-MEM following the
manufacturer’s protocol. After 48 hours, transfected cells were treated with
bortezomib for another 24 hours.
UV light exposure. To reduce Mcl-1 levels using UV light, melanoma
cells were grown in 10-cm tissue culture plates and the lid and medium
were removed followed by UV irradiation using a Panelite unit (Ultralite
Enterprise, Inc., Lawrenceville, GA). This light source consists of four UVB
bulbs (FS36T12/UVB-VHO) and the output wavelengths of the bulbs are
65% UVB, 34% UVA, and 1% UVC as described previously (37). The UV dose
was monitored with an International Light, Inc. (Newburyport, CT)
radiometer fitted with a UVB detector. After irradiation, the medium was
readded and the lid was replaced onto the Petri dish.
Western blot analysis. Whole-cell extracts were prepared as described
previously (13). Briefly, cells were harvested by scraping monolayers and
washed with PBS. Cell pellets were resuspended in CHAPS containing a
protease inhibitor cocktail. Extracts were vigorously shaken at 4jCfor
15 minutes followed by centrifugation. Supernatants were collected and
protein concentration determined using Bradford reagent (Bio-Rad
Laboratories, Hercules, CA). Protein samples (30 Ag) were resolved by
SDS-PAGE and transferred to polyvinylidene difluoride membrane by
electroblotting. Membranes were probed with various primary antibodies
overnight at 4jC and washed, and fluorescence-labeled secondary antibody
was added. After 1 hour, membranes were washed and detected with
LI-COR image analysis system.
Detection of intracellular levels of activated multidomain proa-
poptotic proteins. Melanoma cells were fixed with 2% paraformaldehyde
(10 minutes, room temperature), washed, and incubated with primary
antibodies that detect the activated configuration of Bak and activated Bax
diluted in fluorescence-activated cell sorting (FACS) buffer supplemented
with 0.3% saponin as described previously (35, 36). Cells were then washed
and incubated with FITC-labeled secondary antibody to detect levels of Bak
and Bax in their activated configuration using FACS analysis. The per-
centage of melanoma cells containing activated Bak or Bax was assessed
based on fluorescence intensity beyond the control antibody baseline levels
(expressed in each figure as a relative percentage under a horizontal
bracket, indicating the fluorescence intensity set points).
NOXA Targets and Counteracts Mcl-1 in Melanoma
www.aacrjournals.org 9637 Cancer Res 2006; 66: (19). October 1, 2006
Research.
on April 8, 2021. © 2006 American Association for Cancercancerres.aacrjournals.org Downloaded from
Immunoprecipitation. Cells treated with or without bortezomib
were lysed in >10 volume of lysis buffer [50 mmol/L Tris-HCl (pH 7.4),
1% NP40, 0.25% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA,
freshly added 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L
Na
3
VO
4
, 1 mmol/L NaF, protease inhibitor cocktail]. Lysate (500 Ag)
from each sample was precleaned with protein A/G-plus agarose (Santa
Cruz Biotechnology) and incubated with 2 Ag of either Mcl-1 or NOXA
(Zymed, San Francisco, CA) antibodies or control rabbit IgG (Santa Cruz
Biotechnology) for 4 hours followed by rotating with agarose beads
overnight at 4jC. After extensive washes with lysis buffer and PBS, bound
proteins were eluted with sample buffer and processed for Western blotting.
Immunofluorescence staining. C8161 melanoma cells were seeded on
coverslips in six-well plates, transfected with 100 nmol/L Mcl-1 siRNA or
control RNA for 48 hours, and then treated with the 1 Amol/L bortezomib
for 6 hours. Before finishing the treatment, the cells were labeled with
50 nmol/L MitoTracker Red CMXRos (Molecular Probes, Eugene, OR) for
30 minutes. The cells were washed with PBS and fixed with 2%
paraformaldehyde (20 minutes at room temperature), permeabilized with
0.2% Triton X-100 for 10 minutes, blocked with 2% bovine serum albumin
(BSA) for 30 minutes, and then stained with 1 Ag/mL antibody against
activated Bax (BD Transduction Laboratories, San Diego, CA) in 2% BSA for
1 hour at room temperature. After an extensive wash, cells were further
incubated with goat anti-mouse IgG (Alexa Fluor 488, Molecular Probes) at
1:400 dilution for 1 hour. Before mounting, cells were stained with Hoechst
33342 at 10 Ag/mL for 5 minutes. Stained cells were visualized by Zeiss
microscope, Oberkochen, Germany and the images were captured with
Sensys camera (run by Macprobe version 4.1 software).
Bax cross-link assay. C8161 melanoma cells were treated with either
5 mJ/cm
2
UV light, 1 Amol/L bortezomib alone, or 6-hour UV light
pretreatment plus bortezomib. The cells were collected by brief trypsini-
zation, washed with PBS, and suspended in 250 AL PBS. Proteins were
cross-linked with 0.5% formaldehyde with shaking at room temperature for
1 hour as described (38). Cross-linking was quenched by adding 2 volumes
of 2% glycine. The cells were spun, washed with PBS, and suspended in
isotonic sucrose buffer. Whole-cell lysates were prepared and subjected to
Western blot analysis with anti-Bax NT (Upstate, Lake Placid, NY).
Statistical analysis. The mean and SE were derived from at least three
independent experiments and assessed by Student’s ttest. Results were
considered significant when P< 0.05.
Results
Association between NOXA and Mcl-1 in bortezomib-treated
melanoma cells. To determine if there were any molecular
interactions between NOXA and Mcl-1, immunoprecipitations
followed by Western blot analyses were done using melanoma
cells before and after bortezomib exposure (1 Amol/L; 24 hours).
When whole-cell lysates were immunoprecipitated for Mcl-1, the
immunoblots of C8161 melanoma cells after bortezomib exposure
revealed an association between Mcl-1 and NOXA (Fig. 1, left).
Conversely, when bortezomib-treated melanoma cells were immu-
noprecipitated for NOXA, the immunoblot confirmed an associa-
tion between NOXA and Mcl-1 (Fig. 1, right). Based on these
coimmunoprecipitation and Western blot studies, a physical
interaction between Mcl-1 and NOXA was identified in bortezo-
mib-treated melanoma cells.
Overexpression of Mcl-1 reduces bortezomib-induced mel-
anoma cell apoptosis. To determine if a binding partner of NOXA,
which was determined to be Mcl-1 (Fig. 1), was influencing
apoptotic susceptibility, retroviral-mediated infection to overex-
press Mcl-1 in melanoma cells was followed by bortezomib
exposure (1 Amol/L; 24 hours). RJ002L melanoma cells are more
sensitive than C8161 melanoma cells to bortezomib-induced
apoptosis (13); therefore, the ability of overexpression of Mcl-1
to protect against bortezomib-induced apoptosis was studied
using RJ002L cells. After confirming an f2-fold level of over-
expression of Mcl-1 (Fig. 2A), RJ002L melanoma cells were
examined before and after bortezomib treatment and analyzed
for extent of cell death. In control (Linker) infected melanoma
cells, exposure to bortezomib increased cell death from 12% to
>50% (Fig. 2B). Compared with control (Linker) infected mela-
noma cells, the melanoma cells overexpressing Mcl-1 were
significantly less susceptible to bortezomib-induced apoptosis as
the Mcl-1-overexpressing melanoma cell death response follow-
ing bortezomib was 30% (Fig. 2B). In the next series of experi-
ments, the effect of knocking down Mcl-1 levels was determined
following bortezomib exposure accompanied by a more detailed
biochemical dissection of molecular pathways involved in the
apoptotic response.
Knockdown of Mcl-1 by siRNA enhances bortezomib-
induced cell death of melanoma cells, including activation of
Bak and Bax. By using a specific siRNA to knockdown Mcl-1 levels
but not other Bcl-2 family members, such as Bcl-2 or Bcl-x
L
,the
apoptotic dependence on Mcl-1 levels could be assessed in C8161
Figure 1. Binding between Mcl-1 and NOXA in bortezomib-treated C8161 melanoma cells (1 Amol/L; 24 hours). Immunoprecipitation followed by Western blot
analysis reveals a binding partner for Mcl-1 is NOXA and vice versa depending on the antibody used for either immunoprecipitation or Western blotting. Note
when whole-cell lysates were immunoprecipitated with an anti-Mcl-1 antibody but not rabbit IgG, the bortezomib-treated melanoma cell immunoprecipitate for Mcl-1
also contained NOXA as detected by immunoblot analysis (left ). Using a complementary approach in which anti-NOXA antibody, but not rabbit IgG, led to an
immunoprecipitate that also contained Mcl-1 as detected by subsequent immunoblotting (right ). For both approaches, the input analysis revealed that NOXA and
Mcl-1 levels were increased in melanoma cells after bortezomib exposure. Actin levels confirm equivalent protein loading. Taken together, these results show binding
between Mcl-1 and NOXA in bortezomib-treated melanoma cells.
Cancer Research
Cancer Res 2006; 66: (19). October 1, 2006 9638 www.aacrjournals.org
Research.
on April 8, 2021. © 2006 American Association for Cancercancerres.aacrjournals.org Downloaded from
melanoma cells. Assessment of cell death using Annexin V/FACS
analysis revealed no significant differences in the spontaneous
apoptotic response to Mcl-1 siRNA exposure (10-11%), but the
bortezomib-induced cell death (1 Amol/L; 24 hours) significantly
increased from 25% in control siRNA-treated melanoma cells to
45% in Mcl-1 siRNA-treated melanoma cells (Fig. 3A). The Mcl-1
siRNA not only reduced constitutive Mcl-1 levels compared with
control siRNA-treated cells, but the Mcl-1 siRNA also reduced
bortezomib-induced accumulation of Mcl-1 (Fig. 3B). There was
reduction in low constitutive NOXA levels by Mcl-1 siRNA and only
a slight reduction in bortezomib-induced NOXA levels when
comparing control siRNA with Mcl-1 siRNA-treated melanoma
cells. The Mcl-1 siRNA treatment had no effects on constitutive or
bortezomib-treated levels of either Bcl-2 or Bcl-x
L
. Protein levels of
Bad, which were relatively high before bortezomib treatment, were
reduced (but still detectable) in bortezomib-treated melanoma
cells and not influenced by the Mcl-1 siRNA. Bortezomib treatment
produced increased total cellular levels of Bak and decreased total
Bax levels. Greater induction of cleaved PARP was apparent in
bortezomib-exposed melanoma cells in the Mcl-1 siRNA-pretreated
cells compared with control siRNA-treated melanoma cells. Taken
together, the forced overexpression of Mcl-1, or the knockdown of
Mcl-1, significantly influenced the bortezomib-induced death
response of melanoma cells.
To further delineate the death machinery in melanoma cells,
activated forms for both Bak and Bax were detected by FACS
Figure 2. Overexpression of Mcl-1 by retroviral infection
reduces bortezomib-induced apoptosis (1 Amol/L; 24 hours)
in RJ002L melanoma cells. A, Westernblot analysis of Mcl-1
levels before and 24 hours after bortezomib exposure
(1 Amol/L) in control (Linker) and Mcl-1-overexpressing
melanoma cells. These results indicate a 2-fold increase in
Mcl-1 levels by retroviral infection. Actin levels confirm
equivalent protein loading. Whole-cell extracts and
immunoblotting was done as described in Materials and
Methods. B, quantitative assessment of apoptosis in
RJ002L melanoma cells reveals no difference in
spontaneous apoptosis but reduced apoptosis in Mcl-1-
overexpressing melanoma cells. *, P< 0.005, significantly
reduced killing of melanoma cells when Mcl-1 levels are
increased and bortezomib is added to melanoma cells
compared with control (Linker) infected cells. Cell death
assessment was done by Annexin V/FACS analysis as
described in Materials and Methods. Columns, mean of
three independent experiments; bars, SE.
Figure 3. Knockdown of Mcl-1 levels using siRNA enhances bortezomib-induced cell death (1 Amol/L; 24 hours) of C8161 melanoma cells. A, quantitative assessment
of cell death in C8161 melanoma cells reveals no difference in spontaneous levels between control siRNA and Mcl-1 siRNA treatment. However, Mcl-1 siRNA
treatment followed by bortezomib exposure enhanced cell death compared with control siRNA-treated cells. *, P< 0.005, significantly enhanced killing of melanoma
cells by bortezomib in Mcl-1 siRNA-treated cells compared with control siRNA-treated cells. Cell death assessment was done by Annexin V/FACS analysis as
described in Materials and Methods. Columns, mean of three independent experiments; bars, SE. B, Western blot analysis of Mcl-1 levels before and after siRNA
treatment reveals knockdown of constitutive levels as well as reduction in accumulation of Mcl-1 24 hours after bortezomib exposure (1 Amol/L). Mcl-1 siRNA
treatment also slightly reduced constitutive NOXA levels and slightly increased Bak levels but did not significantly influence Bcl-2, Bcl-x
L
, Bad, or PARP levels. In
bortezomib-treated melanoma cells, the Bad levels were reduced in both control siRNA and Mcl-1 siRNA-treated cultures, with enhanced levels of cleaved PARP
present in Mcl-1 siRNA-treated cells following bortezomib exposure. Actin levels confirm equivalent protein loading. Whole-cell extracts and immunoblotting was done
as described in Materials and Methods.
NOXA Targets and Counteracts Mcl-1 in Melanoma
www.aacrjournals.org 9639 Cancer Res 2006; 66: (19). October 1, 2006
Research.
on April 8, 2021. © 2006 American Association for Cancercancerres.aacrjournals.org Downloaded from
analysis in both control siRNA and Mcl-1 siRNA-pretreated cells
following bortezomib exposure (Fig. 4A). Although Bak and Bax in
several experimental cell systems seem to be functionally
equivalent in mediating cell death, and complete resistance is
dependent on inactivation of both Bak and Bax (39), their
activation by exposure of ordered NH
2
-terminal sequences
following bortezomib treatment of melanoma cells was examined.
The antibodies used are well characterized and only recognize an
epitope exposed when either Bak or Bax becomes activated (35, 36).
In control siRNA-treated C8161 melanoma cells, no constitutive
activated Bak or Bax levels are detected (Fig. 4A), and addition of
bortezomib (1 Amol/L; 24 hours) only increased high levels of
activated Bax (30.5%), with a less dramatic increase in activated
Bak (5-7%). However, when Mcl-1 siRNA-treated melanoma cells
were examined, addition of bortezomib (1 Amol/L; 24 hours)
triggered prominent increase in both activated Bak (16.3%) and
activated Bax (46.6%).
To confirm and extend these findings, subcellular localization
profiles were done using MitoTracker Red CMXRos to identify
intracellular mitochondria together with immunofluorescence
staining to detect and localize activated Bax (Fig. 4Band C).
When control siRNA-treated C8161 melanoma cells were examined
in the absence of bortezomib, there was no detection of activated
Bax in the cells in either the mitochondrial compartment or else-
where in the cytosol. Addition of bortezomib (1 Amol/L; 6 hours)
revealed occasional melanoma cells with detectable activated Bax
(green), which colocalized in the merged views with the red-labeled
mitochondrial compartment (yellow; white arrows depicting
double staining). When Mcl-1 siRNA-treated C8161 melanoma
cells were examined, no detection of activated Bax was identified in
the absence of bortezomib (Fig. 4C). However, with the addition of
bortezomib (1 Amol/L; 6 hours), numerous melanoma cells
expressing activated Bax (green), and the colocalization to the
mitochondrial compartment (red) resulted in yellow stained cells
when the images were merged (Fig. 4C, white arrows). Taken
together, these results indicate the increased number of C8161
melanoma cells expressing activated Bak and Bax can be detected
when bortezomib is added to the Mcl-1 siRNA-treated cells
compared with control siRNA-treated cells. In the following
sections, other approaches to reducing Mcl-1 levels were used
(e.g., UV light or fludarabine), and the subsequent molecular and
cellular efforts were defined following bortezomib exposure.
Modulation of Mcl-1 levels in melanoma cells following UV
light exposure and bortezomib treatment. Because Mcl-1 is a
Figure 4. Knockdown of Mcl-1 levels with siRNA promotes bortezomib-induced Bak and Bax activation in C8161 melanoma cells. A, C8161 melanoma cells were
transfected with control siRNA or Mcl-1 siRNA for 48 hours and then treated with 1 Amol/L bortezomib for 24 hours. Intracellular staining for activated Bak and Bax
was done as described in Materials and Methods. Representative flow cytometric profiles from three independent experiments reveal increased levels of Bak (top )
and Bax (bottom) with activated conformation in the cells treated with Mcl-1 siRNA and bortezomib compared with the control siRNA and bortezomib-treated
cells. The brackets and underlying percentages indicate the relative increase in cells expressing either activated Bak or Bax. Band C, C8161 melanoma cells
transfected with control siRNA (B) and Mcl-1 siRNA (C) were treated with 1 Amol/L bortezomib for 6 hours and labeled with MitoTracker Red CMXRos followed by
immunofluorescence staining with anti-active form of Bax as described in Materials and Methods. The corresponding images of MitoTracker Red CMXRos (red ) and
activated Bax (green) were merged to show the colocalization of mitochondria and activated Bax (white arrows ). In the untreated melanoma cells (no bortezomib),
no activated Bax is identified within the mitochondrial compartment. Note the greater number of melanoma cells expressing activated Bax in the bortezomib-treated
Mcl-1 siRNA cells compared with control siRNA cells.
Cancer Research
Cancer Res 2006; 66: (19). October 1, 2006 9640 www.aacrjournals.org
Research.
on April 8, 2021. © 2006 American Association for Cancercancerres.aacrjournals.org Downloaded from
short-lived protein normally degraded via the ubiquitin-proteasome
pathway (29), addition of a proteasome inhibitor (e.g., bortezomib)
leads to accumulation of Mcl-1 (11–13). In other systems, reduction
or loss of Mcl-1 is required for optimal apoptotic responses, and UV
irradiation has been described previously as potent method to
modulate Mcl-1 levels (30, 40). To determine the effect of UV
light alone and in combination with bortezomib, C8161 melanoma
cells were examined before and after treatment and analyzed for
relative levels of important proteins regulating apoptosis as well
as the extent of cell death under each experimental condition.
C8161 melanoma cells are characterized by high constitutive levels
of several prosurvival proteins, including Mcl-1, Bcl-2, and Bcl-x
L
,
as well as proapoptotic Bad and Bax, but only relatively low Bak
and even lower NOXA levels (Fig. 5A). A single low dose of UV
light (5 mJ/cm
2
) reduces Mcl-1 levels as early as 1 hour after
irradiation and was 4-fold lower after 6 hours of irradiation. The
reduced Mcl-1 levels were sustained 30 hours after UV light
exposure with no comparable effects on the other protein levels
(compare lanes 2 and 5with lane 1 ). Exposure to bortezomib alone
leads to accumulation of Mcl-1 levels (lane 3 ), and the pretreatment
with UV light for 6 hours reduces the bortezomib-induced Mcl-1
levels (compare lane 4 with lane 3 ) after a subsequent 24-hour
exposure interval to bortezomib (1 Amol/L) without affecting other
protein levels, except for Bad, which was decreased 30 hours after a
single dose of UV light and also reduced by bortezomib alone or
UV light plus bortezomib. UV light irradiation alone did not induce
NOXA after 6 hours and only slightly increased NOXA levels after
30 hours. When melanoma cells were pretreated with UV light
for 6 hours and then exposed to bortezomib for 24 hours (lane 4),
the high NOXA levels and lowered Mcl-1 levels were associated
with higher levels of cleaved PARP.
Quantitative analysis of cell death was accomplished by Annexin
V/FACS analysis using C8161 melanoma cells before and after the
aforementioned single and combination treatments (Fig. 5B). A
single exposure to UV light (5 mJ/cm
2
) produced a 22% cell death
response after 30 hours compared with untreated cells (7%).
Bortezomib alone (1 Amol/L) triggered a cell death response of
29% after 24 hours. Preirradiating C8161 melanoma cells for
6 hours followed by an additional 24 hours of bortezomib exposure
triggered significant enhancement of cell death reaching 65% of
the total cell population. Following up on the previous results using
FACS and immunofluorescence-based cell staining to detect
activated Bax, an additional biochemical approach was used that
involved detection of Bax dimers by Western blot analysis.
Activation of Bax can be detected by cross-linking intracellular
proteins as described previously (38), and Fig. 5Crevealed the
presence of Bax dimers in C8161 melanoma cells after UV light or
bortezomib treatment. Furthermore, when both UV light and
bortezomib are combined, an increased level of Bax dimmer can be
detected compared with the individual treatments.
Based on these results, it seemed that the apoptotic response of
melanoma cells to bortezomib included a pathway involving Mcl-1;
therefore, additional experiments were designed to exploit this
observation from a therapeutic perspective by combining borte-
zomib with a pretreatment protocol taking advantage of the ability
of fludarabine to reduce Mcl-1 levels.
Combination of fludarabine and bortezomib enhances
apoptotic response of melanoma cell killing. To further define
Figure 5. Pretreating C8161 melanoma cells with low-dose UV light reduces Mcl-1 levels and enhances bortezomib-induced cell death and Bax activation. A, Western
blot analysis of whole-cell extracts before and after low-dose UV light (5 mJ/cm
2
) and/or bortezomib treatment reveals high constitutive levels of Mcl-1, Bcl-2,
Bcl-x
L
, Bad, Bak, and Bax with barely detectable NOXA and intact (noncleaved) PARP (lane 1 ). After 6 hours of UV light exposure, there is a reduction in Mcl-1 levels
with no other significant changes in the other protein levels (lane 2). Twenty-four hours after bortezomib exposure (1 Amol/L), there is accumulation of Mcl-1 and
induction of high NOXA levels with PARP cleavage (lane 3 ). When melanoma cells are pretreated with low-dose UV light and 6 hours later bortezomib is added, the
following 24-hour incubation period is characterized by reduced accumulation of Mcl-1 and slightly lower Bad levels but high NOXA and cleaved PARP levels (lane 4 ).
The relative protein levels 30 hours following low-dose UV light exposure revealed lower Mcl-1, Bcl-2, Bcl-x
L
, and Bax levels compared with untreated cells with
slight increase in NOXA levels and cleaved PARP (lane 5 ). Actin levels confirm equivalent protein loading. Whole-cell extracts and immunoblotting was done as
described in Materials and Methods. B, quantitative assessment of cell death in C8161 melanoma cells reveals a low spontaneous level (7%), which is increased to
22% after 30 hours following a single low dose (5 mJ/cm
2
) of UV light. Bortezomib treatment alone (1 Amol/L; 24 hours) triggered 29% cell death response. A single
low dose (5 mJ/cm
2
) of UV light followed 6 hours later by addition of bortezomib for an additional 24 hours increased the cell death response to 65%. *, P< 0.05,
significantly enhanced killing of melanoma cells by a combination of single low dose (5 mJ/cm
2
) of UV light pretreatment followed by bortezomib compared with either
agent used alone. Cell death assessment was done by Annexin V/FACS analysis as described in Materials and Methods. Columns, mean; bars, SE. C, C8161
melanoma cells were pretreated with UV light (5 mJ/cm
2
) for 6 hours and then treated with or without bortezomib for 24 hours followed by cross-linking with
formaldehyde as described in Materials and Methods. The Western blot analysis with Bax antibody shows Bax monomer (1) and Bax dimer (2). Note the Bax dimer
level significantly increased in cells treated with UV light + bortezomib.
NOXA Targets and Counteracts Mcl-1 in Melanoma
www.aacrjournals.org 9641 Cancer Res 2006; 66: (19). October 1, 2006
Research.
on April 8, 2021. © 2006 American Association for Cancercancerres.aacrjournals.org Downloaded from
the relative role for Mcl-1 levels in the bortezomib-mediated
apoptotic response of melanoma cells, a therapeutically relevant
strategy was employed using fludarabine. Fludarabine was selected
for this drug combination approach because of its ability to reduce
Mcl-1 levels (31), and other groups have reported enhanced kill-
ing of tumor cells by targeting Mcl-1 using other agents (41, 42).
A dose-dependent (1, 5, and 10 Amol/L) reduction in high
constitutive Mcl-1 levels was observed at 24 hours in C8161
melanoma cells following exposure to fludarabine alone, with no
significant effect on levels of other Bcl-2 family members, such
as Bcl-2, Bcl-x
L
, and Bad (Fig. 6A). Fludarabine treatment alone
over the dose range used in this experimental setting for 24 hours
had minimal effect on NOXA levels and no effect on total cell-
ular levels of Bak, Bax, or PARP levels. C8161 melanoma cells
pretreated with increasing concentrations of fludarabine for
24 hours followed by washing and then exposed to bortezomib
produced a dose-dependent reduction in the accumulation of
Mcl-1 with minimal effects on Bcl-2, Bcl-x
L
, Bak, and Bax (Fig. 6A).
Whereas the pretreatment with fludarabine did not influence
bortezomib-induced NOXA levels or Bcl-2 and Bcl-x
L
levels, there
was an overall reduction in Bad levels in cells exposed to bor-
tezomib. The pretreatment with fludarabine followed by bor-
tezomib was also associated with a dose-dependent increase in
cleaved PARP levels, being greatest at the two highest fludarabine
doses.
Compared with spontaneous levels of C8161 melanoma cell
death (untreated; 8% by Annexin V/FACS analysis), there was no
significant induction in cell death after 24 hours by fludarabine
Cancer Research
Cancer Res 2006; 66: (19). October 1, 2006 9642 www.aacrjournals.org
Research.
on April 8, 2021. © 2006 American Association for Cancercancerres.aacrjournals.org Downloaded from
when used alone (1, 5, and 10 Amol/L; 9-11% by Annexin V/FACS
analysis; Fig. 6B). The cell death response in C8161 melanoma
cells exposed to increasing concentrations of fludarabine alone for
24 hours followed by washing and an additional 24 hours revealed
increased cell death at the 5 and 10 Amol/L doses (21% and 39%,
respectively). When bortezomib (1 Amol/L) was added as a single
agent, the 24-hour cell death response was 24%. Although
pretreating C8161 melanoma cells with 1 Amol/L fludarabine for
24 hours and then washing and adding bortezomib for an addi-
tional 24 hours did not increase the cell death response (23 %),
when the fludarabine pretreatment concentration was increased
to 5 and 10 Amol/L, addition of bortezomib increased the cell
death response to 43% and 57%, respectively. These increased
death responses were consistent with the increased PARP levels as
depicted in Fig. 6A.
To further characterize the response of melanoma cells to the
combination of fludarabine pretreatment followed by bortezomib
exposure, the relative levels of activated Bak and Bax were
examined using FACS analysis. Exposure to fludarabine alone
(24 hours; 1, 5, and 10 Amol/L) triggered no increase in activated
Bak levels but increased the activated Bax levels at the 5 and
10 Amol/L concentrations from 6% in untreated cells to 12% and
17%, respectively (Fig. 6C). When C8161 melanoma cells were
treated with fludarabine at the indicated concentrations for
24 hours, washed, and then exposed to bortezomib (1 Amol/L)
for an additional 24 hours, activated Bak levels increased from 2%
to 8%, 10%, and 12% at the 1, 5, and 10 Amol/L fludarabine
concentrations, respectively (Fig. 6C, top). When C8161 melanoma
cells were treated with fludarabine at the indicated concentrations
for 24 hours, washed, and then exposed to bortezomib (1 Amol/L)
for an additional 24 hours, activated Bax levels increased from
18% to 29%, 44%, and 46% at the 1, 5, and 10 Amol/L fludara-
bine concentrations, respectively (Fig. 6C, bottom). Taken together,
these results indicate that pretreatment of melanoma cells with
fludarabine followed by bortezomib exposure enhances intracellu-
lar levels of activated conformations of both Bak and Bax.
A summary of the working model by which a combination of
fludarabine and bortezomib produce enhanced melanoma cell
killing, in which NOXA, Mcl-1, and activated Bak and Bax are
highlighted, is provided in Fig. 7.
Discussion
Targeting the proteasome function has turned out to be an excel-
lent antireplicative strategy for oncologists in a variety of clinical
settings (43). The 26S proteasome is composed of a multicatalytic
protease functioning to regulate a larger number of intracellular
protein levels. Various inhibitors can reversibly block the chymo-
tryptic-like proteolytic activity of the proteasome complex (14).
Of particular note is the relative sparing by proteasome inhibitors
of normal, nonmalignant cells from apoptosis but also the ability to
bypass classic multidrug resistance mechanisms (44).
Precise molecular details controlling cell death decisions are
rapidly emerging from several laboratories (45–47). As regards the
multidomain-mediated apoptotic pathway, a distinctive mode of
regulation has been proposed by which proapoptotic proteins,
such as Bak and Bax, are normally prevented from activation by
direct sequestration due to two different prosurvival proteins,
Mcl-1 and Bcl-x
L
(24, 25). The conformational change associated
with Bak and Bax toxicity when Mcl-1 and Bcl-x
L
are displaced by
NOXA and Bad, respectively, can be quantitated using antibodies
directed against a NH
2
-terminal epitope in Bak and Bax (35). To
determine if this model could be exploited therapeutically, we
defined the molecular machinery engaged by a proteasome inhib-
itor in melanoma cells. Moreover, the relative roles for NOXA/
Mcl-1 versus Bad/Bcl-x
L
could be assessed in melanoma cells.
Indeed, this model was validated and successfully exploited
because we showed that NOXA bound to Mcl-1 and by simul-
taneously inducing NOXA and reducing Mcl-1 levels, leading to
enhanced levels of activated Bak and Bax accompanied by killing
of melanoma cells. A summary of our current working model for
these molecular mechanistic pathway by which a combination of
bortezomib plus fludarabine produced enhanced killing of mela-
noma cells is presented in Fig. 7.
The accumulation of the prosurvival protein Mcl-1 is not sur-
prising following bortezomib exposure, given that the proteasome
Figure 6. Enhanced C8161 melanoma cell killing by pretreating with fludarabine followed by bortezomib. A, Western blot analysis of whole-cell extracts before and
after increasing concentrations of fludarabine and/or bortezomib treatment reveals high constitutive levels of Mcl-1, Bcl-2, Bcl-x
L
, Bad, Bak, and Bax with barely
detectable NOXA and intact (noncleaved) PARP (lane 1 ). After 24 hours of fludarabine exposure, there is a reduction in Mcl-1 levels with no other significant changes
in the other protein levels, except for slight induction of NOXA levels (lanes 2-4 ). Twenty-four hours after bortezomib exposure (1 Amol/L), there is accumulation of Mcl-1
and induction of high NOXA levels with ubiquitinated forms of Bcl-2, with lower Bad and higher Bak levels compared with untreated cells, accompanied by PARP
cleavage (lane 5). When melanoma cells are pretreated with increasing doses of fludarabine for 24 hours and washed and then bortezomib is added, the following
24-hour incubation period is characterized by reduced accumulation of Mcl-1 but high NOXA and particularly high levels of cleaved PARP detected at the 5 and
10 Amol/L doses of fludarabine (lanes 6-8). Actin levels confirm equivalent protein loading. Whole-cell extracts and immunoblotting was done as described in Materials
and Methods. B, quantitative assessment of cell death in C8161 melanoma cells reveals a low spontaneous level (8%), which is not significantly increased by
24-hour exposure to fludarabine alone (1, 5, and 10 Amol/L). When fludarabine is used alone for 24 hours, and the cells are washed and examined after an
additional 24 hours, there is a dose-dependent increase in cell death at the 5 Amol/L (24%) and 10 Amol/L (39%) concentrations. Bortezomib treatment alone (1 Amol/L;
24 hours) triggered 24% cell death response. When fludarabine was added for 24 hours, and the cells were washed and then exposed to bortezomib for an additional
24 hours, although there was no increased cell death at the concentration of 1 Amol/L fludarabine, the use of fludarabine at 5 and 10 Amol/L did increase the cell
death response (43% and 57%, respectively). *, P< 0.01, significantly enhanced killing of melanoma cells by a combination of fludarabine pretreatment at 5 and
10 Amol/L doses followed by bortezomib compared with either agent used alone. Cell death assessment was done by Annexin V/FACS analysis as described in
Materials and Methods. Columns, mean; bars, SE. C, response of C8161 melanoma cells to fludarabine (1, 5, and 10 Amol/L) and/or bortezomib (1 Amol/L) reveals
altered intracellular levels of activated Bak and activated Bax. Compared with low constitutive activated Bak or Bax levels (2% and 6%, respectively), the cell
death response to fludarabine alone at all concentrations after 24 hours is characterized by no to minimal increases in intracellular levels of activated Bak, whereas
activated Bax is increased at 5 and 10 Amol/L to 12% and 17%, respectively. Pretreatment of C8161 melanoma cells with fludarabine (24 hours) followed by
washing and exposure to bortezomib (24 hours) triggered enhanced activated Bak levels at 1, 5, and 10 Amol/L concentrations of fludarabine of 8%, 10%, and 12%,
respectively. Pretreatment of C8161 melanoma cells with fludarabine (24 hours) followed by washing and exposure to bortezomib (24 hours) triggered enhanced
activated Bax levels at 1, 5, and 10 Amol/L concentrations of fludarabine of 29%, 44%, and 46%, respectively. Taken together, these results show that pretreatment with
fludarabine followed by bortezomib triggers conformational changes leading to activation of both Bak and Bax in melanoma cells. Quantitation of melanoma cells
expressing activated Bak or Bax was done using FACS and setting the gates based on isotype control antibody staining profiles versus staining intensity detected by
using antibodies specific for activated conformations of Bak and Bax as described in Materials and Methods. The bars and the underlying percentages indicate the
relative increase in cells expressing either activated Bak or Bax.
NOXA Targets and Counteracts Mcl-1 in Melanoma
www.aacrjournals.org 9643 Cancer Res 2006; 66: (19). October 1, 2006
Research.
on April 8, 2021. © 2006 American Association for Cancercancerres.aacrjournals.org Downloaded from
mediates the degradation of many proteins involved in cellular
proliferation and apoptosis. Thus, to increase the effectiveness of
killing by bortezomib, it is important to consider adding a second
agent that can interfere with the Mcl-1 accumulation. By
pretreating melanoma cells with either Mcl-1 siRNA, low-dose UV
light, or fludarabine, Mcl-1 levels were reduced sufficiently to
enhance the effectiveness of NOXA accompanied by increased
melanoma cell death. Another set of important observations was
the relatively low levels of Bad present in bortezomib-treated
melanoma cells (Figs. 5 and 6), suggesting a greater role for
NOXA in displacing Mcl-1, compared with Bad displacing Bcl-x
L
,
in this neoplastic system using a proteasome inhibitor. These
results provide a molecular basis for rational combination
therapies operating through the mitochondrial apoptotic pathway
in which bortezomib is used together with other agents targeting
Mcl-1 (48).
Using proteasome inhibitors with melanoma cells has provided
new insights not only into the apoptotic resistance of these
aggressive tumor cells but also into new pathways that can be
designed to overcome the apoptotic resistance of melanoma while
sparing normal melanocytes (43). Exposing melanoma cells to
conventional chemotherapeutic agents have yielded only modest
proapoptotic responses, and several antiapoptotic factors have
been invoked to explain this relative refractoriness. The best single
agent activating against melanoma is dacarbazine or its derivative
temozolomide, but only a 10% to 15% response rate is observed
with a 4-month median response duration (49). Specific mecha-
nisms by which melanoma cells acquire their notorious resistance
to either extrinsic or intrinsic pathway-mediated killing include
activation of Ras with increased levels of Bcl-2 (50, 51), increased
survivin levels (52), loss of death receptors (53, 54), and reduction
in Apaf-1 expression (55–57).
To overcome this apoptotic resistance, we and others have
discovered a key role for the proapoptotic BH3-only protein NOXA,
which is selectively induced by proteasome inhibitors (in a p53-
independent fashion) but not conventional chemotherapeutic
agents, such as Adriamycin or etoposide (11–13, 58). Of perhaps
equal importance is the sparing of NOXA induction or killing of
normal melanocytes by bortezomib (11–13, 58). Given the
disappointing phase II clinical trial results using bortezomib as a
single agent (18), we decided to further interrogate the apoptotic
machinery engaged within melanoma cells that were exposed to
bortezomib to determine a rational basis for selecting an
appropriate agent to combine with bortezomib and hence
potentially improve the killing of melanoma cells. The clear-cut
binding partnership established between NOXA and Mcl-1 paved
the pathway to the studies herein focused on Mcl-1. As revealed in
this report, the use of several different mediators to modulate
Mcl-1 influenced the apoptotic susceptibility in melanoma cells
exposed to bortezomib, validating the approach for a rational drug
combination therapy.
For practical considerations, we selected fludarabine as an
appropriate pharmacologic agent to combine with bortezomib
given the ability of fludarabine to reduce Mcl-1 levels that became
elevated and thereby counteract the proapoptotic effects of NOXA
induction by bortezomib. Another group observed synergistic
killing of leukemia cells when either fludarabine or another purine
nucleoside analogue, cladribine, was combined with bortezomib
(33). By combining bortezomib with fludarabine, not only is it likely
that a greater and more durable clinical effect as regards killing
of melanoma cells can be achieved, but also it may also be possible
to use lower drug concentrations and avoid or minimize toxicities
that can limit the usefulness of either drug administered
individually in patients with melanoma. In conclusion, the current
results support further studies, including a clinical trial in which
patients with metastatic melanoma are treated with a combination
of bortezomib plus fludarabine, and tissue samples were examined
to assess the relative levels of NOXA and Mcl-1 within tumor cells
before and after this combination therapy in responders and
nonresponders.
Acknowledgments
Received 2/27/2006; revised 5/26/2006; accepted 7/19/2006.
Grant support: NIH grants CA 59327, CA 27502 (B.J. Nickoloff ), and CA083784
(M.F. Denning).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Lynn Walter for expert figure and article preparation and Jeffrey Ziffra,
Barbara Bodner, and Larry Stennett for expert technical assistance.
Figure 7. Model for apoptotic pathway
by which a combination of bortezomib +
fludarabine produces enhanced conversion
of inactive to active conformation of Bak and
Bax accompanied by increased PARP
cleavage and killing of melanoma cells.
The key elements of this model include a
strategy by which inhibition of proteasome
activity (via bortezomib) not only triggers
proapoptotic NOXA induction but also
counteracts the undesirable accumulation
of antiapoptotic Mcl-1 levels (using siRNA
against Mcl-1 or using fludarabine
pretreatment).
Cancer Research
Cancer Res 2006; 66: (19). October 1, 2006 9644 www.aacrjournals.org
Research.
on April 8, 2021. © 2006 American Association for Cancercancerres.aacrjournals.org Downloaded from
References
1. Jemal A, Thomas A, Murray T, Thun M. Cancer
statistics, 2002. CA Cancer J Clin 2002;52:23–47.
2. Soengas MS, Lowe SW. Apoptosis and melanoma
chemoresistance. Oncogene 2003;22:3138–51.
3. Chang AE, Karnell LH, Menck HR. The National
Cancer Data Base report on cutaneous and noncuta-
neous melanoma: a summary of 84,836 cases from the
past decade. The American College of Surgeons
Commission on Cancer and the American Cancer
Society. Cancer 1998;83:1664–78.
4. Grossman D, Kim PJ, Sche chner JS, Altieri D C.
Inhibition of melanoma tumor growth in vivo by
survivin targeting. Proc Natl Acad Sci U S A 2001;98:
635–40.
5. Helmbach H, Rossmann E, Kern MA, Schadendorf D.
Drug-resistance in human melanoma. Int J Cancer 2001;
93:617–22.
6. Mandara M, Nortilli R, Sava T, Cetto GL. Chemother-
apy for metastatic melanoma. Expert Rev Anticancer
Ther 2006;6:121–30.
7. Chin L, Merlino G, DePinho RA. Malignant melanoma:
modern black plague and genetic black box. Genes Dev
1998;12:3467–81.
8. Grossman D, Altieri DC. Drug resistance in melanoma:
mechanisms, apoptosis, and new potential therapeutic
targets. Cancer Metastasis Rev 2001;20:3–11.
9. Hussein MR, Haemel AK, Wood GS. Apoptosis and
melanoma: molecular mechanisms. J Pathol 2003;199:
275–88.
10. Tron VA, Krajewski S, Klein-Parker H, et al. Immu-
nohistochemical analysis of Bcl-2 protein regulation in
cutaneous melanoma. Am J Pathol 1995;146:643–50.
11. Fernandez Y, Verhaegen M, Miller TP, et al.
Differential regulation of noxa in normal melanocytes
and melanoma cells by proteasome inhibition: thera-
peutic implications. Cancer Res 2005;65:6294–304.
12. Qin JZ, Stennett L, Bacon P, et al. p53-independent
NOXA induction overcomes apoptotic resistance of
malignant melanomas. Mol Cancer Ther 2004;3:895–902.
13. Qin JZ, Ziffra J, Stennett L, et al. Proteasome
inhibitors trigger NOXA-mediated apoptosis in melano-
ma and myeloma cells. Cancer Res 2005;65:6282–93.
14. Dalton WS. The proteasome. Semin Oncol 2004;
31:3–9.
15. Satyamoorthy K, Bogenrieder T, Herlyn M. No longer
a molecular black box—new clues to apoptosis and drug
resistance in melanoma. Trends Mol Med 2001;7:191–4.
16. Chudnovsky Y, Khavari PA, Adams AE. Melanoma
genetics and the development of rational therapeutics. J
Clin Invest 2005;115:813–24.
17. Amiri KI, Horton LW, LaFleur BJ, Sosman JA,
Richmond A. Augmenting chemosensitivity of malig-
nant melanoma tumors via proteasome inhibition:
implication for bortezomib (Velcade, PS-341) as a
therapeutic agent for malignant melanoma. Cancer
Res 2004;64:4912–8.
18. Markovic SN, Geyer SM, Dawkins F, et al. A phase II
study of bortezomib in the treatment of metastatic
malignant melanoma. Cancer 2005;103:2584–9.
19. Green DR, Kroem er G. The pathophysiology of
mitochondrial cell death. Science 2004;305:626–9.
20. Huang DC, Strasser A. BH3-only proteins—essential
initiators of apoptotic cell death. Cell 2000;103:839–42.
21. Strasser A. The role of BH3-only proteins in the
immune system. Nat Rev Immunol 2005;5:189–200.
22. Gelinas C, White E. BH3-only proteins in control:
specificity regulates MCL-1 and BAK-mediated apopto-
sis. Genes Dev 2005;19:1263–8.
23. Lindsten T, Ross AJ, King A, et al. The combined
functions of proapoptotic Bcl-2 family members bak
and bax are essenti al for normal development of
multiple tissues. Mol Cell 2000;6:1389–99.
24. Chen L, Willis SN, Wei A, et al. Differential targeting
of prosurvival Bcl-2 proteins by their BH3-only ligands
allows complementary apoptotic function. Mol Cell
2005;17:393–403.
25. Willis SN, Chen L, Dewson G, et al. Proapoptotic Bak
is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until
displaced by BH3-only proteins. Genes Dev 2005;19:
1294–305.
26. Lang-Rollin I, Maniati M, Jabado O, et al. Apoptosis
and the conformational change of Bax induced by
proteasomal inhibition of PC12 cells are inhibited by
bcl-xL and bcl-2. Apoptosis 2005;10:809–20.
27. Panaretakis T, Pokrovskaja K, Shoshan MC, Grander
D. Activation of Bak, Bax, and BH3-only proteins in the
apoptotic response to doxorubicin. J Biol Chem 2002;
277:44317–26.
28. von Haefen C, Gillissen B, Hemmati PG, et al.
Multidomain Bcl-2 homolog Bax but not Bak mediates
synergistic induction of apoptosis by TRAIL and 5-FU
through the mitochondrial apoptosis pathway. Onco-
gene 2004;23:8320–32.
29. Iglesias-Serret D, Pique M, Gil J, Pons G, Lopez JM.
Transcriptional and translational control of Mcl-1
during apoptosis. Arch Biochem Biophys 2003;417:
141–52.
30. Chaturvedi V, Sitailo LA, Qin JZ, et al. Knockdown of
p53 levels in human keratinocytes accelerates Mcl-1 and
Bcl-x(L) reduction thereby enhancing UV-light induced
apoptosis. Oncogene 2005;24:5299–312.
31. Maggio SC, Rosato RR, Kramer LB, et al. The histone
deacetylase inhibitor MS-275 interacts synergistically
with fludarabine to induce apoptosis in human
leukemia cells. Cancer Res 2004;64:2590–600.
32. Robak T, Korycka A, Kasznicki M, Wrzesien-Kus A,
Smolewski P. Purine nucleoside analogues for the
treatment of hematological malignancies: pharmacology
and clinical applications. Curr Cancer Drug Targets
2005;5:421–44.
33. Duechler M, Linke A, Cebula B, et al. In vitro
cytotoxic effect of proteasome inhibitor bortezomib in
combination with purine nucleoside analogues on
chronic lymphocytic leukaemia cells. Eur J Haematol
2005;74:407–17.
34. Armstrong JS. Mitochondria: a target for cancer
therapy. Br J Pharmacol 2005;147:239–48.
35. Griffiths GJ, Dubrez L, Morgan CP, et al. Cell damage-
induced conformational changes of the pro-apoptotic
protein Bak in vivo precede the onset of apoptosis. J Cell
Biol 1999;144:903–14.
36. Nechushtan A, Smith CL, Hsu YT, Youle RJ.
Conformation of the Bax C-terminus regulates subcel-
lular location and cell death. EMBO J 1999;18:2330–41.
37. Qin JZ, Bacon P, Panella J, et al. Low-dose UV-
radiation sensitizes keratinocytes to TRAIL-induced
apoptosis. J Cell Physiol 2004;200:155–66.
38. Sitailo LA, Tibudan SS, Denning MF. Bax activation
and induction of apoptosis in human keratinocytes by
the protein kinase Cycatalytic domain. J Invest
Dermatol 2004;123:434–43.
39. Wei MC, Zong WX, Cheng EH, et al. Proapoptotic
BAX and BAK: a requisite gateway to mitochondrial
dysfunction and death. Science 2001;292:727–30.
40. Nijhawan D, Fang M, Traer E, et al. Elimination
of Mcl-1 is required for the initiation of apoptosis
following ultraviolet irradiation. Genes Dev 2003;17:
1475–86.
41. Battle TE, Arbiser J, Frank DA. The natural product
honokiol induces caspase-dependent apoptosis in B-cell
chronic lymphocytic leukemia (B-CLL) cells. Blood 2005;
106:690–7.
42. Ma Y, Cress WD, Haura EB. Flavopiridol-induced
apoptosis is mediated through up-regulation of E2F1
and repression of Mcl-1. Mol Cancer Ther 2003;2:
73–81.
43. Adams J. The proteasome: a suitable antineoplastic
target. Nat Rev Cancer 2004;4:349–60.
44. Dalton W. Drug resistance in hematologic malignan-
cies. Clin Adv Hematol Oncol 2005;3:267–8.
45. Danial NN, Korsmeyer SJ. Cell death: critical control
points. Cell 2004;116:205–19.
46. Reed JC. Apoptosis-targeted therapies for cancer.
Cancer Cell 2003;3:17–22.
47. Strasser A, O’Connor L, Dixit VM. Apoptosis
signaling. Annu Rev Biochem 2000;69:217–45.
48. Shore GC, Viallet J. Modulating the bcl-2 family of
apoptosis suppressors for potential therapeutic benefit
in cancer. Am Soc Hematol Educ Program 2005;226–30.
49. Sun W, Schuchter LM. Metastatic melanoma. Curr
Treat Options Oncol 2001;2:193–202.
50. Borner C, Schlagbauer Wadl H, Fellay I, et al. Mutated
N-ras upregulates Bcl-2 in human melanoma in vitro
and in SCID mice. Melanoma Res 1999;9:347–50.
51. Davies H, Bignell GR, Cox C, et al. Mutations of the
BRAF gene in human cancer. Nature 2002;417:949–54.
52. Grossman D, McNiff JM, Li F, Altieri DC. Expression
and targeting of the apoptosis inhibitor, survivin, in
human melanoma. J Invest Dermatol 1999;113:1076–81.
53. Hersey P, Zhang XD. How melanoma cells evade trail-
induced apoptosis. Nat Rev Cancer 2001;1:142–50.
54. Ivanov VN, Bhoumik A, Ronai Z. Death receptors and
melanoma resistance to apoptosis. Oncogene 2003;22:
3152–61.
55. Allen JD, Zhang XD, Scott CL, et al. Is Apaf-1
expression frequently abrogated in melanoma? Cell
Death Differ 2005;12:680–1.
56. Peltenburg LT, de Bruin EC, Meersma D, et al.
Expression and function of the apoptosis effector Apaf-1
in melanoma. Cell Death Differ 2005;12:678–9.
57. Soengas MS, Capodieci P, Polsky D, et al. Inactivation
of the apoptosis effector Apaf-1 in malignant melanoma.
Nature 2001;409:207–11.
58. Fernandez Y, Miller TP, Denoyelle C, et al. Chemical
blockage of the proteasome inhibitory function of
bortezomib: impact on tumor cell death. J Biol Chem
2006;281:1107–18.
NOXA Targets and Counteracts Mcl-1 in Melanoma
www.aacrjournals.org 9645 Cancer Res 2006; 66: (19). October 1, 2006
Research.
on April 8, 2021. © 2006 American Association for Cancercancerres.aacrjournals.org Downloaded from
2006;66:9636-9645. Cancer Res
Jian-Zhong Qin, Hong Xin, Leonid A. Sitailo, et al.
Targeting Mcl-1 and NOXA
Enhanced Killing of Melanoma Cells by Simultaneously
Updated version
http://cancerres.aacrjournals.org/content/66/19/9636
Access the most recent version of this article at:
Cited articles
http://cancerres.aacrjournals.org/content/66/19/9636.full#ref-list-1
This article cites 57 articles, 18 of which you can access for free at:
Citing articles
http://cancerres.aacrjournals.org/content/66/19/9636.full#related-urls
This article has been cited by 21 HighWire-hosted articles. Access the articles at:
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
.pubs@aacr.orgDepartment at
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site.
(CCC)
Click on "Request Permissions" which will take you to the Copyright Clearance Center's
.http://cancerres.aacrjournals.org/content/66/19/9636
To request permission to re-use all or part of this article, use this link
Research.
on April 8, 2021. © 2006 American Association for Cancercancerres.aacrjournals.org Downloaded from
