Oncogene (2006) 25, 47984811
& 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00
www.nature.com/onc
REVIEW
Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy
S Fulda and K-M Debatin
University Childrens Hospital, Ulm, Germany
Apoptosis or programmed cell death is a key regulator
of physiological growth control and regulation of tissue
homeostasis. One of the most important advances in
cancer research in recent years is the recognition that
cell death mostly by apoptosis is crucially involved in the
regulation of tumor formation and also critically deter-
mines treatment response. Killing of tumor cells by most
anticancer strategies currently used in clinical oncology,
for example, chemotherapy, c-irradiation, suicide gene
therapy or immunotherapy, has been linked to activation of
apoptosis signal transduction pathways in cancer cells such
as the intrinsic and/or extrinsic pathway. Thus, failure to
undergo apoptosis may result in treatment resistance.
Understanding the molecular events that regulate apop-
tosis in response to anticancer chemotherapy, and how
cancer cells evade apoptotic death, provides novel oppor-
tunities for a more rational approach to develop molecular-
targeted therapies for combating cancer.
Oncogene (2006) 25, 47984811. doi:10.1038/sj.onc.1209608
Keywords: apoptosis; death receptor; mitochondria;
cancer; chemotherapy
Introduction
Current cancer therapies, for example, chemotherapy,
g-irradiation, immunotherapy or suicide gene therapy,
primarily exert their antitumor effect by triggering
apoptosis in cancer cells (Makin and Dive, 2001; Fulda
and Debatin, 2004). Apoptosis is an evolutionary
conserved, intrinsic program of cell death that occurs
in various physiological and pathological situations
(Hengartner, 2000). Apoptosis is characterized by
typical morphological and biochemical hallmarks,
including cell shrinkage, nuclear DNA fragmentation
and membrane blebbing (Hengartner, 2000). The under-
lying mechanisms for initiation of an apoptosis response
upon cytotoxic therapy may depend on the individual
stimulus and have often not exactly been identied.
However, damage to DNA or to other critical molecules
is considered to be a common initial event which is then
propagated by the cellular stress response (Rich et al.,
Correspondence: Dr S Fulda, University Childrens Hospital, Eythstr.
24, Ulm D-89075, Germany.
E-mail: simone.fulda@uniklinik-ulm.de
2000). Multiple stress-inducible molecules, for example,
c-Jun N-terminal kinase (JNK), mitogen-activated
protein kinase (MAPK)/extracellular signal-regulated
protein kinase (ERK), nuclear factor kappa B (NF-kB)
or ceramide, have been implied in transmitting the
apoptotic signal (Davis, 2000; Karin et al., 2002).
Proteolytic enzymes such as caspases are important
effector molecules in apoptosis (Degterev et al., 2003).
Activation of caspases in response to anticancer
chemotherapy can be initiated through activation of
the extrinsic (receptor) pathway or at the mitochondria
by stimulating the intrinsic pathway (Fulda and
Debatin, 2004). Because of the potential detrimental
effects on cell survival in case of inappropriate activa-
tion of apoptosis programs, apoptosis pathways have to
be tightly controlled. The antiapoptotic mechanisms
regulating cell death have also been implicated in
conferring drug resistance to tumor cells (Fulda and
Debatin, 2004). However, the concept that apoptosis
represents the major mechanism by which cancer cells
are eliminated may not universally apply and caspase-
independent apoptosis or other modes of cell death have
also to be considered as cellular response to anticancer
therapy (Brown and Wilson, 2003; Brown and Attardi,
2005). Thus, a better understanding of these diverse
modes of cell death in cancer therapy will provide a
molecular basis for new strategies targeting cell death
pathways in resistant forms of cancer.
Apoptosis signaling pathways
In most cases, anticancer therapies eventually result in
activation of caspases, a family of cysteine proteases
that act as common death effector molecules in various
forms of cell death (Degterev et al., 2003). Caspases are
synthesized as inactive proforms and upon activation,
they cleave next to aspartate residues (Degterev et al.,
2003). The fact that caspases can activate each other by
cleavage at identical sequences results in amplication
of caspase activity through a protease cascade (Degterev
et al., 2003). Caspases cleave a number of different
substrates in the cytoplasm or nucleus leading to many
of the morphologic features of apoptotic cell death
(Degterev et al., 2003). For example, polynucleosomal
DNA fragmentation is mediated by cleavage of ICAD
(inhibitor of caspase-activated DNase), the inhibitor of
the endonuclease CAD (caspase-activated DNase) that
cleaves DNA into the characteristic oligomeric frag-

ments (Nagata, 2000). Likewise, proteolysis of several
cytoskeletal proteins such as actin or fodrin leads to loss
of overall cell shape, whereas degradation of lamin
results in nuclear shrinking (Degterev et al., 2003).
Activation of caspases can be initiated from different
entry points, for example, at the plasma membrane
upon ligation of death receptor (receptor pathway) or at
the mitochondria (mitochondrial pathway) (Hengartner,
2000) (Figure 1). Stimulation of death receptors of the
tumor necrosis factor (TNF) receptor superfamily such
as CD95 (APO-1/Fas) or TNF-related apoptosis-indu-
Figure 1 Apoptosis signaling pathways. Apoptosis pathways can
be initiated through different entry sites, for example, at the plasma
membrane by death receptor ligation (receptor pathway) or at the
mitochondria (mitochondrial pathway). Stimulation of death
receptors of the tumor necrosis factor (TNF) receptor superfamily
such as CD95 (APO-1/Fas) or TNF-related apoptosis-inducing
ligand (TRAIL) receptors by CD95 ligand (CD95-L) or TRAIL
results in receptor aggregation and recruitment of the adaptor
molecule Fas-associated death domain (FADD) and caspase-8.
Upon recruitment, caspase-8 becomes activated and initiates
apoptosis by direct cleavage of downstream effector caspases.
The mitochondrial pathway is initiated by stress signals through
the release of apoptogenic factors such as cytochrome c, apoptosis
inducing factor (AIF), or Smac/DIABLO from the mitochondrial
intermembrane space. The release of cytochrome c into the cytosol
triggers caspase-3 activation through formation of the cytochrome
c/Apaf-1/caspase-9-containing apoptosome complex. The Smac/
DIABLO promotes caspase activation through neutralizing the
inhibitory effects to IAPs, whereas AIF causes DNA condensation.
The receptor and the mitochondrial pathway can be interconnected
at different levels, for example, by Bid, a BH3 domain-containing
protein of the Bcl-2 family which assumes cytochrome-c-releasing
activity upon cleavage by caspase-8. Activation of caspases is
negatively regulated at the receptor level by FLIP, which block
caspase-8 activation, at the mitochondria by Bcl-2 family proteins
and by inhibitor of apoptosis proteins (IAPs). See text for more
details.
Apoptosis pathways in anticancer chemotherapy
S Fulda and K-M Debatin
4799
cing ligand (TRAIL) receptors results in activation of the
initiator caspase-8 which can propagate the apoptosis
signal by direct cleavage of downstream effector caspases
such as caspase-3 (Walczak and Krammer, 2000). The
mitochondrial pathway is initiated by the release of
apoptogenic factors such as cytochrome c, apoptosis-
inducing factor (AIF), Smac (second mitochondria-
derived activator of caspase)/DIABLO (direct inhibitor
of apoptosis protein (IAP)-binding protein with low PI),
Omi/HtrA2 or endonuclease G from the mitochondrial
intermembrane space (Cande et al., 2002; Saelens et al.,
2004). The release of cytochrome c into the cytosol
triggers caspase-3 activation through formation of the
cytochrome c/Apaf-1/caspase-9-containing apoptosome
complex, whereas Smac/DIABLO and Omi/HtrA2
promote caspase activation through neutralizing the
inhibitory effects to the IAPs (Saelens et al., 2004).
Links between the receptor and the mitochondrial
pathway exist at different levels. Upon death receptor
triggering, activation of caspase-8 may result in cleavage
of Bid, a Bcl-2 family protein with a BH3 domain only,
which in turn translocates to mitochondria to release
cytochrome c thereby initiating a mitochondrial ampli-
cation loop (Cory and Adams, 2002). In addition,
cleavage of caspase-6 downstream of mitochondria may
feed back to the receptor pathway by cleaving caspase-8
(Cowling and Downward, 2002).
Nonapoptotic forms of cell death
Although apoptosis pathways and signal-transducing
molecules have been shown to play a crucial role for
killing of tumor cells in response to cytotoxic agents used
in the treatment of cancer patients, the signicance of
apoptosis as the main mechanism of cancer cell death
in response to anticancer chemotherapy has also been
challenged. So far, no clear pattern has emerged between
the level of apoptosis or proteins that regulate apoptosis
and treatment response in most solid tumors. In addition
to apoposis, cancer cells have also been shown to be
effectively eliminated after DNA damage by necrosis,
mitotic catastrophe and autophagy (Brown and Wilson,
2003; Brown and Attardi, 2005). Thus, nonapoptotic
modes of cell death have also been taken into consi-
deration as response to cytotoxic therapy. Although the
signaling pathways and molecules involved in these
alternative forms of cell death have not yet exactly been
dened, non-caspase proteases such as calpains or
cathepsins, lysosomal enzymes or PARP-1 may be
involved (Okada and Mak, 2004). The form of cell
death may depend on the context, including the cell type,
the genotype of the cell, the type of DNA damage and
the dose of the agent used. Thus, the relative contribu-
tion of these different modes of cell death for chemo-
responses in vitro and in vivo remains to be dened.
Extrinsic pathway
Death receptors are members of the tumor necrosis
factor (TNF) receptor gene superfamily that consists of
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 Apoptosis pathways in anticancer chemotherapy
S Fulda and K-M Debatin
4800
more than 20 proteins with a broad range of biological
functions, including regulation of cell death and
survival, differentiation or immune regulation (Walczak
and Krammer, 2000; Ashkenazi, 2002). Members of
the TNF receptor family share similar, cysteine-rich
extracellular domains. In addition, death receptors are
dened by a cytoplasmic domain of about 80 amino
acids called death domain, which plays a crucial role in
transmitting the death signal from the cells surface to
intracellular signaling pathways. The best-characterized
death receptors include CD95 (APO-1/Fas), TNF
receptor 1 (TNFRI), TNF-related apoptosis-inducing
ligand-receptor 1 (TRAIL-R1) and TRAIL-R2, whereas
the role of DR3 (TRAMP/Apo-3/WSL-1/LARD) or
DR6 has not exactly been dened (Walczak and
Krammer, 2000). The corresponding ligands of the
TNF superfamily comprise death receptor ligands such
as CD95 ligand (CD95L), TNFa, lymphotoxin-a (the
later two bind to TNFRI), TRAIL and TWEAK, a
ligand for DR3 (Walczak and Krammer, 2000).
CD95
The CD95 receptor/CD95L system is a key signal
pathway involved in the regulation of apoptosis in
several different cell types, for example, in the immune
system (Krammer, 2000). CD95, a 48 kDa type I trans-
membrane receptor, is expressed on activated lympho-
cytes, on a variety of tissues of lymphoid or non-
lymphoid origin, as well as on tumor cells (Krammer,
2000). CD95 ligand is produced by activated T cells
and plays a crucial role in the regulation of the immune
system by triggering autocrine suicide or paracrine
death in lymphocytes or other target cells (Krammer,
2000). Furthermore, CD95L expression on cancer
cells has been implicated in immune escape of tumors
(Krammer, 2000). By constitutive expression of death
receptor ligands such as CD95L, tumors may adopt
a killing mechanism from cytotoxic lymphocytes to
delete the attacking antitumor T cells through induction
of apoptosis via CD95/CD95L interaction. However,
this model of tumor counterattack has also been
challenged, since no study has so far conclusively
demonstrated that tumor counterattack is a relevant
immune escape mechanism in vivo (Igney and Krammer,
2002).
TNF-related apoptosis-inducing ligand and its receptors
TNF-related apoptosis-inducing ligand/Apo-2L was
identied in 1995 based on its sequence homology to
other members of the TNF superfamily and is consti-
tutively expressed in a wide range of tissues (LeBlanc
and Ashkenazi, 2003). The two agonistic TRAIL recep-
tors, TRAIL-R1 and TRAIL-R2, contain a conserved
cytoplasmic death domain motif, which enables them to
engage the cells apoptotic machinery upon ligand
binding, whereas TRAIL-R3 to R5 are antagonistic
decoy receptors, which bind TRAIL, but do not
transmit a death signal (LeBlanc and Ashkenazi, 2003).
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Signaling through CD95 or TRAIL receptors
Ligation of death receptors such as CD95 or the
agonistic TRAIL receptors TRAIL-R1 and TRAIL-
R2 by their cognate ligands or agonistic antibodies
results in receptor trimerization, clustering of the
receptors death domains and recruitment of adaptor
molecules such as Fas-associated death domain
(FADD) through homophilic interaction mediated by
the death domain (Walczak and Krammer, 2000). Fas-
associated death domain in turn recruits caspase-8 to the
activated CD95 receptor to form the CD95 death-
inducing signaling complex (DISC) (Kischkel et al.,
1995). Oligomerization of caspase-8 upon DISC forma-
tion drives its activation through self-cleavage. Caspase-
8 then activates downstream effector caspases such as
caspase-3. For the CD95 signaling pathway, two distinct
prototypic cell types have been identied (Scafdi et al.,
1998). In type I cells, caspase-8 is activated upon death
receptor ligation at the DISC in quantities sufcient to
directly activate downstream effector caspases such as
caspase-3 (Scafdi et al., 1998). In type II cells, however,
the amount of active caspase-8 generated at the DISC is
insufcient to activate caspase-3 and a mitochondrial
amplication loop is required for full activation of
caspases (Scafdi et al., 1998). Also, a similar cell type-
dependent organization (type I and type II) of the
TRAIL signaling pathway has been described (Fulda
et al., 2002a).
Disruption of the extrinsic pathway in cancers
Cancer cells have evolved numerous strategies to resist
cell death induction via the extrinsic pathway. Signaling
to cell death in response to death receptor ligation can
principally be inhibited by an increase in antiapoptotic
molecules or by a decrease or defective function in
proapoptotic proteins.
For example, surface expression of death receptors
may vary between different cell types and can be
downregulated or absent in resistant forms of tumors.
To this end, drug-resistant leukemia or neuroblastoma
cells showed strong downregulation of CD95 expression
suggesting that critical levels of CD95 expression may
have an impact on drug sensitivity (Friesen et al., 1997;
Fulda et al., 1998b). Also, decient transport of the
agonistic TRAIL receptors TRAIL-R1 and TRAIL-R2
from intracellular stores, for example, endoplasmatic
reticulum, to the cell surface was identied as a
mechanism conferring TRAIL resistance in colon cancer
cells (Jin et al., 2004). Mutations of the CD95 gene have
been identied in a variety of hematological malignan-
cies and solid tumors (Debatin et al., 2003). The
incidence of CD95 mutations found in human tumors
has been taken as evidence that the CD95 system exerts
a tumor suppressor function. Moreover, decoy receptor
3 (DcR3), which act as decoy receptors by competitively
binding CD95L, can interfere with CD95-triggered
apoptosis and was found to be genetically amplied or
overexpressed in lung carcinoma, colon carcinoma or
glioblastoma (Pitti et al., 1998; Roth et al., 2001). A
decoy receptor for TRAIL, TRAIL-R3, was found to be

overexpressed in gastric carcinoma (Sheikh et al., 1999).
Moreover, loss of expression of the agonistic TRAIL
receptors TRAIL-R1 and -R2 may account for TRAIL
resistance. Both receptors are located on chromosome
8p, a region of frequent loss of heterozygosity (LOH) in
tumors (LeBlanc and Ashkenazi, 2003). In a small
percentage of cancers, for example, non-Hodgkins
lymphoma, colorectal, breast, head and neck cancer,
osteosarcoma or lung carcinoma, deletions or muta-
tions were found, which resulted in loss of both copies
of TRAIL-R1 or R2 (Pai et al., 1998; Dechant et al.,
2004).
Impaired surface expression of CD95 or TRAIL
receptors may also be caused by epigenetic changes such
as CpG-island hypermethylation of gene promoters, for
example, in neuroblastoma or colon carcinoma cells
(van Noesel et al., 2002; Petak et al., 2003). Also,
alterations in chromatin structure, for example, chro-
matin condensation because of histone deacetylation,
may block transcription by preventing the access of
transcription factors to the DNA (Marks et al., 2001).
Interestingly, epigenetic changes in CD95 expression
have been reported to determine immune escape and
response to therapy (Maecker et al., 2002). In tumors
with epigentically silenced CD95, restoration of CD95
expression by histone deacetylase inhibitors resulted in
suppression of tumor growth and restoration of
chemosensitivity in an natural killer (NK) cell-depen-
dent manner (Maecker et al., 2002).
Signaling by death receptors can also be negatively
regulated by proteins that associate with their cytoplas-
matic domains, for example, FLIP or phosphoprotein
enriched in diabetes/phosphoprotein enriched in astro-
cytes-15 kDa (PED/PEA-15) (Hao et al., 2001; Krueger
et al., 2001). Two splice variants of FLIP, a long form
(FLIPL) and a short form (FLIPS), have been identied
in human cells, which have sequence homology to
caspase-8 and caspase-10, but lack its catalytic site
(Krueger et al., 2001). Consequently, the recruitment of
FLIP to the DISC instead of procaspase-8 or -10 can
block caspase activation (Krueger et al., 2001). High
FLIP expression has been found in many tumor cells
and has been correlated with resistance to CD95- or
TRAIL- and also to chemotherapy-induced apoptosis
(Fulda et al., 2000; Longley et al., 2006). However, the
impact of FLIP on apoptosis sensitivity towards
cytotoxic drugs may vary between cell types, since
overexpression of FLIP did not confer protection
against cytotoxic drugs in T-cell leukemia cells (Kataoka
et al., 1998), whereas FLIP antisense oligonucleotides or
short interference RNA sensitized osteosarcoma or
colorectal cancer cells for chemotherapeutic drugs
(Kinoshita et al., 2000; Longley et al., 2006). The
PEA-15 is another death effector domain (DED)-
containing protein, which blocks CD95-, TRAIL- or
TNFa-triggered apoptosis in a receptor-proximal man-
ner by disrupting FADD and caspase-8 inter-
actions (Hao et al., 2001). Recently, PEA-15 has also
been implicated in mediating AKT-dependent chemo-
resistance in human breast cancer cells (Stassi et al.,
2005).
Apoptosis pathways in anticancer chemotherapy
S Fulda and K-M Debatin
4801
Signaling through the extrinsic pathway in cancer therapy
The CD95 system has been implicated in chemotherapy-
induced tumor cell death in a number of studies. To t
his end, treatment with anticancer drugs triggered an
increase in CD95L expression, which stimulated the
receptor pathway in an autocrine or paracrine manner
by binding to its receptor CD95 (Friesen et al., 1996;
Fulda et al., 1997, 1998a; Houghton et al., 1997; Muller
et al., 1997). Various anticancer agents with different
primary intracellular targets have been used in these
studies including DNA-damaging agents such as dox-
orubicin, etoposide, cisplatin or bleomycin (Friesen
et al., 1996; Fulda et al., 1997, 1998a; Muller et al.,
1997). The CD95 receptor/ligand system has also been
implicated in thymine-less death in colon carcinoma
cells following treatment with 5-uorouracil (Houghton
et al., 1997). Activation of the transcription factors
activator protein-1 (AP-1) and NF-kB was shown to
mediate the increase in CD95L transcription and
mRNA levels in response to chemotherapy and AP-1-
and NF-kB-binding sites were identied in the human
CD95L promotor (Kasibhatla et al., 1998). In addition
to CD95L, CD95 expression on the cells surface
increased upon drug treatment, in particular in cells
harboring wild-type p53 (Muller et al., 1997). p53-
responsive elements have been identied in the rst
intron of the CD95 gene, as well as three putative p53-
binding sites within the CD95 promotor, which showed
limited homology with the p53 consensus-binding site
(Muller et al., 1998). Moreover, antagonistic CD95L
antibodies, soluble antagonistic CD95 receptors or DN-
FADD were found to reduce drug-induced apoptosis
under certain circumstances. In addition to upregulation
of CD95L and CD95, anticancer agents have been
reported to activate the CD95 pathway by modulating
expression and recruitment of pro- or antiapoptotic
components of the CD95 DISC to activated receptors.
Upregulation of FADD and procaspase-8 was found
upon treatment with doxorubicin, cisplatin or mitomy-
cin C in colon carcinoma cells (Micheau et al., 1999).
Also, increased recruitment of FADD and procaspase-8
to the CD95 receptor to form the CD95 DISC was
observed in certain tumor cells upon drug treatment
in a CD95L-dependent or CD95L-independent manner
(Fulda et al., 2001). These ndings indicate that in cells
with an inducible CD95 receptor/ligand system, drug-
induced apoptosis may involve CD95L-initiated DISC
formation and activation of downstream effector
programs similar to activation-induced cell death
(AICD) in T cells.
Despite the reproducibility of these ndings in
different model systems, other reports challenged the
model that death receptor signaling is involved in drug-
mediated cell death (Eischen et al., 1997; Villunger et al.,
1997). To that end, antagonistic antibodies against
CD95L or CD95 did not confer protection against
apoptosis induced by cytotoxic drugs in other cell lines.
Enforced expression of FLIP, DN-FADD or the serpin
crmA did not inhibit drug-induced apoptosis, although
it inhibited caspase-8 activation (Kataoka et al., 1998).
Also, targeted disruption of genes involved in death
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 Apoptosis pathways in anticancer chemotherapy
S Fulda and K-M Debatin
4802
receptor signaling conferred no protection against
cytotoxic drug treatment, at least in nontransformed
cells. The FADD / and caspase-8 / broblasts were
sensitive to cytotoxic drugs, whereas they remained
resistant to death receptor stimulation (Varfolomeev
et al., 1998; Yeh et al., 1998). In contrast, caspase-9 /
embryonic stem cells and Apaf-1 / thymocytes remain
sensitive to death receptor triggering, however they are
resistant to cytotoxic drugs (Hakem et al., 1998;
Yoshida et al., 1998)
The discrepancies in data may be explained by the
relative contribution of the extrinsic versus the intrinsic
pathway depending on the cytotoxic drug, dose or
kinetics or on the differences between certain cell types.
The discrepancies in data may also be explained by
differences in the inhibitory reagents used to block
CD95/CD95L interaction. The quality of CD95/
CD95L-blocking agents including anti-CD95 antibody,
anti-CD95L antibody or soluble decoy CD95-Fc re-
ceptor constructs may vary depending on their origin
and preparation. Also, the lack of efcacy of these
CD95/CD95L-neutralizing agents may be owing to
inaccessibility of their proposed targets. Experiments
with adenoviral delivery of a CD95L-green uorescent
protein (GFP) construct showed that CD95 and CD95L
are stored intracellularly and colocalize to the same
intracellular compartments, for example, the Golgi and/
or endoplasmatic reticulum (Hyer et al., 2000). An anti-
CD95-blocking antibody did not inhibit CD95L-in-
duced cell death suggesting that CD95L may trigger
CD95 within the same intracellular compartment and
that these two molecules may already interact before
surface presentation (Hyer et al., 2000). Thus, CD95/
CD95L-neutralizing agents may under certain circum-
stances not even gain access to their targets prior to
triggering of the CD95 pathway. Moreover, some
studies that challenge an involvement of the CD95
system in chemotherapy of tumor cells are based on
experiments performed in nontransformed cells, for
example, embryonic broblasts, but not in cancer cells.
However, the mechanisms regulating apoptosis in non-
malignant cells may vary considerably from those in
malignant tumor cells, which is highlighted by the
differential sensitivity of these cell types to various death
stimuli.
Exploiting death receptor signaling pathways for tumor
therapy
Death receptor as targets. The idea to specically
target the extrinsic pathway to trigger apoptosis in
tumor cells, for example by ligation of death receptors,
is attractive for cancer therapy since death receptors
have a direct link to the cells death machinery
(Ashkenazi, 2002). In addition, apoptosis upon death
receptor triggering has been reported to occur indepen-
dent of the p53 tumor suppressor gene, which is deleted
or inactivated in more than half of human tumors
(El-Deiry, 2001). However, the clinical application of
CD95Lor TNFa is hampered by severe toxic side effects
(Walczak and Krammer, 2000). In contrast, TRAIL
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appears to be a relatively safe and promising candidate
for clinical application, particularly in its non-tagged,
zinc-bound homotrimeric form (LeBlanc and Ashkenazi,
2003). Studies in non-human primates such as chim-
panzees and cynomolgus monkeys showed no toxicity
upon intravenous infusion, even at high doses (Ashke-
nazi et al., 1999). In addition, no cytotoxic activity of
TRAIL was reported on a variety of normal human cells
of different lineages including broblasts, endothelial
cells, smooth muscle cells, epithelial cells or astro-
cytes (Ashkenazi et al., 1999; Lawrence et al., 2001;
Pollack et al., 2001). However, some concerns about
potential toxic side effects on human hepatocytes or
brain tissue have also been raised (Jo et al., 2000; Nitsch
et al., 2000). The loss of tumor selectivity may be related
to the TRAIL preparations used in these studies, since
TRAIL preparations, which are antibody-crosslinked or
not optimized for Zn content, have been reported to
form multimeric aggregates thereby overpassing the
threshold of sensitivity of normal cells (Lawrence et al.,
2001).
Antitumor activity of TNF-related apoptosis-inducing
ligand. Recombinant soluble TRAIL induced apopto-
sis in a broad spectrum of cancer cell lines, including
colon carcinoma, breast carcinoma, lung carcinoma,
pancreas carcinoma, prostate carcinoma, renal carcino-
ma, thyroid carcinoma, malignant brain tumors, Ewing
tumor, osteasarcoma, neuroblastoma, leukemia and
lymphoma and also exhibited potent tumoricidal
activity in vivo in several xenograft models of colon
carcinoma, breast carcinoma, malignant glioma or
multiple myeloma (Chinnaiyan et al., 2000; Nagane
et al., 2000; Walczak et al., 2000; Pollack et al., 2001;
Rohn et al., 2001). Furthermore, monoclonal antibodies
that engage the TRAIL receptors DR4 or DR5 also
demonstrated potent antitumor activity against tumor
cell lines and in preclinical cancer models (Chuntharapai
et al., 2001; Ichikawa et al., 2001). Currently, recombi-
nant soluble TRAIL or agonistic TRAIL receptors are
being evaluated in phase I clinical trials.
Combination therapy with TNF-related apoptosis-indu-
cing ligand. Although these studies provided ample
evidence of the potential of TRAIL for cancer therapy,
many tumors remain refractory towards treatment
with TRAIL, which has been related to the dominance
of antiapoptotic signals. Importantly, numerous studies
have shown that TRAIL synergized together with
cytotoxic drugs or g-irradiation to achieve antitumor
activity in various cancer cell lines including malignant
glioma, melanoma, leukemia, breast, colon or prostate
carcinoma and also cooperated to suppress tumor
growth in different mouse models of human cancers
(Gliniak and Le, 1999; Chinnaiyan et al., 2000; Keane
et al., 2000; Nagane et al., 2000; Walczak et al., 2000;
Belka et al., 2001; Rohn et al., 2001). Dependent on the
therapeutic combinations and cell type used, different
mechanisms of sensitization to TRAIL-induced apop-
tosis have been identied. For example, the molecular

mechanisms, which account for the synergistic interac-
tion of TRAIL and conventional chemotherapeutics,
may include transcriptional upregulation of the agonis-
tic TRAIL receptors TRAIL-R1 and -R2, which
occurred in a p53-dependent or p53-independent man-
ner (Takimoto and El-Deiry, 2000; Meng and El-Deiry,
2001). Of note, p53 has also been shown to transcrip-
tionally activate the antagonistic TRAIL receptors
TRAIL-R3 and -R4 (Meng et al., 2000). Recent
evidence suggest that p53 is crucial for sensitization to
TRAIL by chemotherapy through transcriptional upre-
gulation of TRAIL-R2 in some tumors, for example,
mismatch repair-decient colorectal cancer cells harbor-
ing Bax mutations (Wang and El-Deiry, 2003). Intrigu-
ingly, pre-exposure to chemotherapy restored TRAIL
sensitivity through p53-mediated increase of TRAIL-R2
expression even in resistant colorectal carcinoma cells
lacking Bax expression indicating that sequential com-
bination of anticancer agents with TRAIL may over-
come some forms of resistance (Wang and El-Deiry,
2003). In addition, chemotherapy has been reported
to enhance receptor assembly of CD95 or TRAIL
receptors, for example, in colon carcinoma cells (Lacour
et al., 2004). The synergistic interaction between
cytotoxic drugs and TRAIL may also be mediated by
downregulation of antiapoptotic proteins such as Bcl-2,
Bcl-XL or FLIP upon drug treatment (Olsson et al.,
2001). In addition, anticancer agents may sensitize
tumor cells for TRAIL treatment by upregulating
proapoptotic molecules incuding caspases or FADD
(Micheau et al., 1999). The cooperative interaction of
chemotherapeutics and TRAIL has also been explained
by the simultaneous activation of the intrinsic and
extrinsic pathway leading to enhanced cell death
through functional complementation. As TRAIL de-
monstrated synergistic interaction with anticancer
agents or irradiation, TRAIL may be most effective in
combination with conventional cancer treatments.
Intrinsic pathway of apoptosis
Signaling through the intrinsic pathway of apoptosis
In the mitochondrial pathway of apoptosis, caspase
activation is closely linked to permeabilization of the
outer mitochondrial membrane by proapoptotic mem-
bers of the Bcl family (Green and Kroemer, 2004).
Numerous cytotoxic stimuli and proapoptotic signal-
transducing molecules converge on mitochondria to
induce outer mitochondrial membrane permeabilization
(Decaudin et al., 1998; Green and Kroemer, 2004). This
permeabilization is regulated by proteins from the Bcl-2
family, mitochondrial lipids, proteins that regulate
bioenergetic metabolite ux and components of the
permeability transition pore (Green and Kroemer,
2004). Upon disruption of the outer mitochondrial
membrane, a set of proteins normally found in the space
between the inner and outer mitochondrial membranes
is released, including cytochrome c, Smac/DIABLO,
Omi/HtrA2, AIF and endonuclease G (Saelens et al.,
Apoptosis pathways in anticancer chemotherapy
S Fulda and K-M Debatin
4803
2004). Once in the cytosol, these apoptogenic proteins
trigger the execution of cell death by promoting caspase
activation or by acting as caspase-independent death
effectors (Saelens et al., 2004).
Mitochondrial mediators of caspase-dependent apoptosis
Cytochrome c. The release of cytochrome c from
mitochondria directly triggers caspase-3 activation
through formation of the cytochrome c/Apaf-1/cas-
pase-9-containing apoptosome complex (Cain et al.,
2000). Once in the cytosol, cytochrome c binds to the
C-terminal region of Apaf-1, a cytosolic protein with
an N-terminal caspase-recruitment domain (CARD), a
nucleotide-binding domain with homology to Caenor-
habditis elegans CED-4 and a C-terminal domain
containing 1213 WD-40 repeats (Zou et al., 1997).
Binding of cytochrome c to Apaf-1 facilitates the
association of dATP with Apaf-1 and exposes its
N-terminal CARD, which can now oligomerize and
become a platform on which the initiator caspase-9
is recruited and activated through a CARDCARD
interaction (Adrain et al., 1999). Consecutively, the
executioner caspase-3 is recruited to the apoptosome,
where it is activated by the resident caspase-9 (Bratton
et al., 2001). Caspase-3 then cleaves key substrates in the
cell to produce many of the cellular and biochemical
events of apoptosis.
In certain cases, caspase activity instigated by
cytosolic cytochrome c contributes to the drop of matrix
metalloproteinase (MMP), as was shown by the use of
synthetic caspase inhibitors and Apaf1 / cells
(Waterhouse et al., 2001). In addition, caspase activity
further damages the function of permeabilized mito-
chondria by affecting the activity of complexes I and II,
inevitably leading to the loss of MMP and the
generation of reactive oxygen species (Ricci et al.,
2004). Thus, secondary events resulting from cytosolic
changes, caused by the release of cytochrome c and
other mitochondrial IMS proteins, can feed back on
permeabilized mitochondria and affect their function.
Importantly, this mitochondrial amplication loop of
caspase activity may critically determine the response of
cancer cells to cytotoxic treatments.
Moreover, there is recent evidence for the existence of
a cytochrome c- and apoptosome-independent, but
Apaf-1-dependent mechanism(s) for caspase activation.
A targeted knock-in of a variant of cytochrome c that
lacks apoptogenic properties, but has electron transfer
and antioxidant activity (K72A), allowed the evaluation
of the contribution of cytochrome c to apoptosis
without affecting its function in oxidative phosphoryla-
tion (Hao et al., 2005). Interestingly, thymocytes from
the KA/KA mice were markedly more sensitive to death
stimuli including etoposide and g-irradiation than Apaf-
1( / ) thymocytes (Hao et al., 2005). Upon treatment
with g-irradiation, procaspases were efciently activated
in apoptotic KA/KA thymocytes, but Apaf-1 oligomer-
ization was not observed (Hao et al., 2005) pointing to a
differential requirement for cytochrome c and Apaf-1 in
apoptosis.
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 Apoptosis pathways in anticancer chemotherapy
S Fulda and K-M Debatin
4804
Smac/DIABLO. Other proteins released from mito-
chondria, such as Smac/DIABLO and Omi/HtrA2,
facilitate caspase activation through neutralizing endo-
genous inhibitors of caspases, the inhibitor of apoptosis
proteins (IAPs). Smac and its murine homolog DIA-
BLO are nuclear encoded mitochondrial proteins, which
contain a mitochondrial localization signal, which is
proteolytically removed upon mitochondrial import
to yield the mature 23 kDa protein (Du et al., 2000;
Verhagen et al., 2000). This maturation step exposes the
IAP-binding motif (IBM) at the N-terminus of Smac/
DIABLO (Du et al., 2000) (Table 1). Second mitochon-
dria-derived activator of caspase/DIABLO was shown
to bind to XIAP, cIAP1, cIAP2, survivin and Apollon
in a BIR-dependent way (Vaux and Silke, 2003)
(Figure 2),and acts as homodimer with the IBM present
in a bivalent conguration (Chai et al., 2000). One
Smac/DIABLO dimer binds one XIAP molecule by
both IAP-binding motifs, one interacting with BIR2 and
the other one with BIR3 (Huang et al., 2003).
Intriguingly, the same BIR3 groove binds the IBM
exposed at the N-terminus (Ala-Thr-Pro-Phe) of the
small subunit of caspase-9 following its autocatalytic
processing after Asp315, allowing Smac/DIABLO to
displace caspase-9 from XIAP (Srinivasula et al., 2001)
(Table 1).
The physiological mitochondrial function of Smac/
DIABLO is unknown, and DIABLO / mice appear
normal (Okada et al., 2002). Although the in vitro
cleavage of procaspase-3 upon addition of cytochrome c
was inhibited in lysates of Smac / cells, Smac /
mice and cells responded normally to apoptotic stimuli
such as UV irradiation, staurosporin, etoposide and
TNF/cyclohexamide (Okada et al., 2002). These ob-
servations suggest the existence of redundant factors
compensating for the loss of Smac/DIABLO, possibly
HtrA2/OMI.
Omi/HtrA2 is a nuclear-encoded, 49 kDa protein with
an N-terminal mitochondrial localization signal that
mediates its translocation into the mitochondrial inter-
membrane space (Suzuki et al., 2001; Martins et al.,
2002; van Loo et al., 2002). Omi/HtrA2 is processed in
the intermembrane space into the 37 kDa mature form,
Figure 2 Schematic diagram of domain structure of human IAP
proteins. BIR, Baculovirus IAP repeats; CARD, caspase-activating
and recruitment domain; RING, ring zinc-nger.
Oncogene
liberating an IBM at its N-terminus (Suzuki et al., 2001;
Martins et al., 2002; van Loo et al., 2002) (Table 1).
Although recombinant Omi/HtrA2 can catalyse its own
maturation in vitro, the protease responsible for its
maturation in cells remains unknown (Martins et al.,
2002). Omi/HtrA2 plays an essential role in regulating
mitochondrial homeostasis requiring its proteolytic
activity, although the molecular targets and interaction
partners of Omi/HtrA2 in the mitochondrion have not
yet been dened (Saelens et al., 2004). Once Omi/HtrA2
is released from mitochondria into the cytosol, it
promotes cell death in a caspase-dependent way by
antagonizing IAPs, and in a caspase-independent way as
a protease (Saelens et al., 2004). Similar to Smac/
DIABLO, Omi/HtrA2 blocks IAPs through its N-
terminal IAP-binding motif, presented in a trimeric
conguration (Li et al., 2002)
Although the release of cytochrome c into the cytosol
directly triggers caspase-3 activation through formation
of the cytochrome c/Apaf-1/caspase-9-containing apop-
tosome complex, Smac/DIABLO and Omi/HtrA2
indirectly promote caspase activation through antago-
nizing the inhibitory effects to IAPs (Saelens et al.,
2004). Thus, a dynamic equilibrium exists between pro-
and antiapoptotic effector molecules, which allows the
cell to cope with limited mitochondrial damage, in
which case IAPs can adequately block caspase activa-
tion initiated by a small amount of released cytochrome
c. However, under circumstances where mitochondrial
damage proceeds or simultaneously affects multiple
mitochondria, the antiapoptotic hurdle imposed by
IAPs can be overcome by the higher cytosolic concen-
tration of their antagonists Smac/DIABLO and HtrA2/
OMI, which neutralize IAPs by direct binding.
There is mounting evidence that cancer cells have an
intrinsic drive to apoptosis that is held in check by IAPs.
To this end, high basal levels of caspase-3 and caspase-8
activities and active caspase-3 fragments in the absence
of apoptosis were detected in various tumor cell lines
and cancer tissues, but not in normal cells (Yang et al.,
2003a). Tumor cells, but not normal cells also expressed
high levels of IAPs, suggesting that upregulated IAP
expression counteracted the high basal caspase activity
selectively in tumor cells (Yang et al., 2003a). Therefore,
Table 1 Alignment of the N-terminal sequences of proteins with an
IBM
Reaper MAVAF
Grim MAIAY
Hid MAVPF
Smac/DIABLO AVPI
Omi/HtrA2 AVPS
Caspase-9 ATPF
Drosophila proteins Reaper, Grim and Hid have an IAP-binding motif
(IBM) following the initiator methione, Smac/DIABLO and Omi/
HtrA2 have their IBM exposed after removal of the N-terminal
mitochondrial localization sequence, whereas the small subunit of
caspase-9 contains the IBM after autoprocessing. IBM, inhibitor of
apoptosis protein (IAP)-binding motif; Smac/DIABLO, second
mitochondria-derived activator of caspase/direct inhibitor of apoptosis
protein (IAP)-binding protein with low isoelectric point (PI).

strategies targeting IAPs are considered as a promising
approach to enhance the efcacy of cytotoxic therapies
selectively in cancer cells. To this end, transfection-
enforced expression of Smac/DIABLO lowered the
threshold for TRAIL-induced killing in different tumors
(Ng and Bonavida, 2002; Okano et al., 2003) and also
sensitized cancer cells for chemotherapy (McNeish et al.,
2003; Zhao et al., 2006). However, translocation of
endogenous Smac/DIABLO into the cytosol during
anticancer drug-induced apoptosis does not appear to
play a major role under certain conditions, for example,
in human lung carcinoma cells upon treatment with
etoposide (Bartling et al., 2004). Downregulation of
Smac/DIABLO by small interfering RNA (siRNA) did
not inuence killing by etoposide in these cells, although
an IAP-binding peptide Smac-N7 enhanced etoposide-
induced apoptosis (Bartling et al., 2004). These data
suggest that Smac/DIABLO deciency may be compen-
sated for by action of redundant determinants in certain
cancer cells.
Mitochondrial mediators of caspase-independent
apoptosis
Upon its release from the mitochondrial intermembrane
space, HtrA2/OMI contributes to cell death also in a
caspase-independent way as a protease in addition to
promoting apoptosis in a caspase-dependent way by
antagonizing IAPs (Suzuki et al., 2001; Martins et al.,
2002; van Loo et al., 2002). In vitro data demonstrated
the degradation of XIAP, cIAP1, cIAP2 and Apollon by
the protease activity of HtrA2/OMI (Suzuki et al.,
2004). In addition, Trencia et al. (2004) demonstrated
the interaction of antiapoptotic PED/PEA-15 with, and
its degradation by, cytosolic HtrA2/OMI. Decreasing
the levels of HtrA2/OMI in cells by antisense or RNA
interference lowers the sensitivity of different cancer cell
lines to cell death induced by staurosporin, Fas, UV or
cisplatin (Martins et al., 2002).
Furthermore, AIF and endonuclease G are released
from mitochondria upon outer mitochondrial mem-
brane permeabilization and translocate into the nucleus
to contribute to nuclear chromatin condensation and
large-scale DNA fragmentation (Cande et al., 2004;
Saelens et al., 2004). Whether these two proteins are
released before, together or after cytochrome c has been
controversially discussed (Arnoult et al., 2002). Also, it
is still not exactly clear how AIF contributes to nuclear
DNA fragmentation as it lacks intrinsic DNase activity.
In mammalian cells, cyclophilin A, a peptidyl-prolyl cis
trans isomerase, cooperates with AIF to induce break-
down of DNA (Cande et al., 2004).
Disruption of the intrinsic pathway in cancer
Mutations in genes involved in the regulation of the
mitochondrial pathway are very common in cancer cells.
As the majority of anticancer therapies induce apoptosis
in cancer cells by triggering the intrinsic pathway, such
mutations are usually associated with treatment resis-
tance. For example, overexpression of Bcl-2, as a result
of chromosomal translocation of the bcl-2 oncogene into
Apoptosis pathways in anticancer chemotherapy
S Fulda and K-M Debatin
4805
the immunglobulin heavy chain gene locus, is associated
with approximately 85% of human follicular lymphoma
(Tsujimoto et al., 1984). Experiments with transgenic
mice have demonstrated that Bcl-2 overexpression can
promote neoplastic transformation of B and T lympho-
cytes and myeloid cells (McDonnell and Korsmeyer,
1991; Traver et al., 1998).
Given the fact that overexpression of antiapoptotic
Bcl-2 family members promotes oncogenesis, it follows
that multi BH domain proapoptotic members of this
family act as tumor suppressors. However, since Bax
and Bak have largely overlapping roles in apoptosis, it
has been difcult to determine whether this hypothesis
holds true and indeed bax / mice are not markedly
predisposed to neoplasia (Knudson et al., 2001).
However, somatic mutations that inactivate the bax
gene have been found in certain solid tumors and
hematological malignancies. To this end, single nucleo-
tide substitution or frameshift mutations, which inacti-
vate the Bax gene in mismatch repair-decient (MMR)
colon cancer or hematopoetic malignancies, have been
described (Rampino et al., 1997; Kitada et al., 2002).
Moreover, there is mounting evidence that BH3-only
proteins may contribute to the suppression of malignant
transformation, indicating that they can function as
bona de tumor suppressors. For example, loss of a
single allele of Bim was shown to accelerate B-cell
lymphomagenesis induced by expression of a c-myc
transgene (Egle et al., 2004), in line with the key role of
Bim as a regulator of lymphoid homeostasis (Strasser,
2005). Interestingly, homozygous deletions in the
chromosomal region harboring the bim gene have
recently been identied in patients with mantle cell
lymphoma (Tagawa et al., 2005). Mice lacking bid
spontaneously develop a myeloproliferative disorder
that can progress to a malignancy resembling chronic
myelomonocytic leukemia (CMML) (Zinkel et al.,
2003). Moreover, RNAi-mediated suppression of Puma
was reported to accelerate Myc-induced lymphomagen-
esis (Hemann et al., 2004).
In addition to genetic alterations, aberrant expression
of Bcl-2 family proteins is mostly regulated at the
transcriptional or post-transcriptional level. For exam-
ple, expression of several antiapoptotic Bcl-2 family
proteins, for example, Bcl-2, Bcl-XL, Mcl-1 or B-1, is
transcriptionally regulated by NF-kB (Cory and Adams,
2002).
Besides Bcl-2 family proteins, decreased or absent
activity of Apaf-1 was found in ovarian cancer,
melanoma and leukemia. Furthermore, mutations in
the tumor suppressor gene p53, the most common
genetic defect in human cancers, impinge on the intrinsic
pathway. For example, activation of p53 upregulates a
number of genes possessing p53-responsive elements
present in their promoters, such as the proapoptotic
BH3-only proteins Puma, Noxa and Bid (Oda et al.,
2000; Yu et al., 2001; Sax et al., 2002). As a result, cells
in which p53 is stabilized are sensitized for activation of
the mitochondrial cell death pathway. In addition, p53
may directly affect mitochondrial integrity without the
need for gene activation. Indeed, it has been reported
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 Apoptosis pathways in anticancer chemotherapy
S Fulda and K-M Debatin
4806
that p53 can bind to Bcl-2 and Bcl-XL at the
mitochondria, thereby promoting mitochondrial desta-
bilization (Mihara et al., 2003).
Signaling through the intrinsic pathway in cancer therapy
Most conventional chemotherapeutic agents, for exam-
ple, etoposide, doxorubicin, cisplatin or paclitaxel, elicit
mitochondrial permeabilization in an indirect fashion by
triggering perturbations of intermediate metabolism or
by increasing the concentration of proapoptotic second
messengers, for example, by inducing p53 expression,
by inducing the ceramide/GD3 pathway, by inducing
the CD95/CD95L ligand system, affecting Bcl-2-like
proteins and/or by compromising the redox or energy
balance.
There is mounting evidence for the existence of a
nucleo-mitochondrial cross-talk following DNA da-
mage. For example, apoptotic signals arising from
DNA damage can be transmitted via the tumor
suppressor p53 to mitochondria, which in turn release
apoptogenic factors into the cytoplasm that activate
downstream destruction programs (Moll et al., 2005).
p53 can indirectly engage the mitochondrial pathway by
transcriptionally activating the expression of proapop-
totic Bcl-2 proteins, for example, Bid, Puma or Noxa
(Oda et al., 2000; Yu et al., 2001; Sax et al., 2002).
Additionally, p53 can directly trigger permeabilization
of the outer mitochondrial membrane in a transcription-
independent fashion through direct activation of proa-
poptotic Bcl-2 proteins Bax or Bak or through binding
and inactivating of antiapoptotic Bcl-2 proteins such as
Bcl-2 or Bcl-XL (Mihara et al., 2003; Chipuk et al.,
2004; Moll et al., 2005). Importantly, mitochondrially
targeted p53 has recently been shown to possess tumor
suppressor activities also in vivo (Talos et al., 2005).
Moreover, caspase-2 possesses the ability to engage
the mitochondrial apoptotic pathway in response to
DNA damage by permeabilizing the outer mitochon-
drial membrane and/or by breaching the association of
cytochrome c with the inner mitochondrial membrane.
Hence, cells stably transfected with procaspase-2 anti-
sense, or transiently expressing siRNA, were refractory
to cytochrome c release and various downstream events,
such as caspase activation and DNA fragmentation,
induced by DNA damage (Lassus et al., 2002; Robert-
son et al., 2002). Caspase-2 can act indirectly on the
mitochondria, for example, by cleaving the proapoptotic
protein Bid, followed by its translocation to the
mitochondria to induce cytochrome c release (Guo
et al., 2002). Additionally, caspase-2 can directly
permeabilize the outer mitochondria membrane and
stimulate the release of cytochrome c and Smac/
DIABLO, possibly as a result of direct interaction of
processed caspase-2 with putative proteins and/or
phospholipids located in the outer mitochondrial
membrane, or at contact sites between the outer and
inner membranes (Robertson et al., 2004). Permeabili-
zation of the outer mitochondria membrane requires
processing of the caspase-2 zymogen but not the
associated proteolytic activity and occurs independently
Oncogene
of several Bcl-2 family proteins, including Bax, Bak and
Bcl-2 (Robertson et al., 2004). Further, caspase-2 was
also shown to have the surprising ability to disrupt the
association between cytochrome c and anionic phos-
pholipids, notably cardiolipin, thereby making addi-
tional cytochrome c available for release into the cytosol
(Enoksson et al., 2004). Upon DNA damage, caspase-2
has recently been reported to become activated in the so-
called PIDDosome, a complex of the p53-inducible,
death domain-containing protein PIDD, caspase-2 and
the adaptor protein RAIDD (Tinel and Tschopp, 2004)
pointing to the existence of a nucleo-mitochondrial
apoptotic pathway.
Additionally, histone H1.2 has been reported to play
an important role in transmitting apoptotic signals from
the nucleus to the mitochondria following DNA double-
strand breaks (Konishi et al., 2003). Nuclear histone
H1.2 is released into the cytoplasm through a p53-
dependent mechanism after DNA double-strand breaks
and induced cytochrome c release from isolated
mitochondria in a Bak-dependent manner (Konishi
et al., 2003). Reducing H1.2 expression enhanced
cellular resistance to apoptosis induced by X-ray
irradiation or etoposide (Konishi et al., 2003).
Moreover, the orphan nuclear receptor Nur77 (also
known as TR3) has recently been coupled with the Bcl-2
apoptotic machinery at the mitochondria (Lin et al.,
2004). Nur77 binding to the Bcl-2 N-terminal loop
region, located between its BH4 and BH3 domains,
induces a Bcl-2 conformational change that exposes its
BH3 domain, resulting in conversion of Bcl-2 from a
protector to a killer (Lin et al., 2004). Interestingly,
elevated levels of an Nur77-family member have been
associated with favorable responses to chemotherapeu-
tic agents in patients (Shipp et al., 2002).
Furthermore, upon cellular stress including che-
motherapeutic drugs, specic proapoptotic Bcl-2 family
members are activated, derepressed or induced, and
thereby act as sensors. The activity of BH3-only proteins
is kept in check by several mechanisms, keeping these
proteins away from the multidomain Bcl-2 counterparts
under normal circumstances, yet allowing their rapid
activation under stress conditions (Bouillet and Strasser,
2002). As mentioned above, Bcl-2 family protein such as
Bid, Puma or Noxa are under the transcriptional control
of the tumor suppressor p53 and hence, upregulated in
response to DNA-damaging agents (Oda et al., 2000; Yu
et al., 2001; Sax et al., 2002). The BH3-only protein Bim,
which is associated with the cytoskeleton by binding to
microtubules, is set free and activates the intrinsic
pathway following treatment with taxol, which acts on
microtubule assembly (Sunters et al., 2003). Recently,
paclitaxel has been demonstrated to induce Bim
accumulation and Bim-dependent apoptosis in epithelial
tumors in vitro and also in vivo (Tan et al., 2005). Active
BH3-only proteins bind and counteract antiapoptotic
and in some cases activate multidomain proapoptotic
Bcl-2 family members, leading to the loss of mitochon-
drial membrane permeability (Bouillet and Strasser,
2002). How Bcl-2 proteins induce disturbance of the
mitochondrial outer membrane is still a matter of debate

and may involve the pore-forming and self-oligomeriz-
ing capacity of some Bcl-2 family proteins, modulation
of the mitochondrial permeability transition pore by
Bcl-2 family proteins and/or lipid changes and lipid
protein interactions within the mitochondrial mem-
branes. Moreover, chemotherapeutic agents such as
paclitaxel cause hyperphosphorylation and inactivation
of Bcl-2, and, simultaneously, favor opening of the
permeability transition (PT) pore (Ruvolo et al., 2001).
Chemotherapeutic drugs may also induce or facilitate
permeabilization of the outer mitochondrial membrane
through changes in cellular redox potentials owing to an
enhanced generation of reactive oxygen species (or a
decrease in their detoxication), depletion of reduced
glutathione or depletion of NADPH, as the mitochon-
drial megachannel possesses several redox-sensitive sites
(Debatin et al., 2002). In addition, changes in energy
metabolism, for example depletion in ADP and ATP,
might facilitate permeability transition pore complex
(PTPC) opening, since ADP and ATP are the physiologic
ligands of the adenine nucleotide translocator, which
function as endogenous inhibitors of the PTPC (Costan-
tini et al., 2000). Also, uncoupling or inhibition of the
respiratory chain or matrix alkalinization may favor
mitochondrial membrane permeabilization. In addition,
lipid messengers such as ceramide, which is generated in
cells exposed to several apoptosis-inducing stimuli,
including cytotoxic drugs, can contribute to mitochon-
drial membrane permeabilization (Susin et al., 1997). At
high concentrations. some chemotherapeutic drugs, for
example, etoposide or paclitaxel, can also induce mito-
chondrial outer membrane permeabilization in isolated
mitochondria (Robertson et al., 2000; Kidd et al., 2002).
In addition, an increasing number of experimental
anticancer drugs, including arsenite, lonidamide, the
synthetic retinoid CD437 or the natural product
betulinic acid, have been shown to act directly on
mitochondria (Debatin et al., 2002). For example,
betulinic acid has been reported to trigger apoptosis
by directly inducing loss of mitochondrial membrane
potential in isolated mitochondria in a way that is not
affected by the caspase inhibitor Z-VAD-fmk and yet is
inhibited by BA, an inhibitor of the PTPC, or by Bcl-2
and Bcl-XL (Fulda et al., 1998b).
Strategies targeting the intrinsic pathway
Bcl-2 family proteins. Since antiapoptotic Bcl-2 pro-
teins, which potently block the intrinsic apoptosis
pathway, are found at elevated levels in human cancers
of both hematological and nonhematological origin
(Cotter, 2004), they represent promising targets for
therapeutic interventions. Consequently, several strate-
gies have been developed to target Bcl-2 proteins, for
example, antisense techniques, BH3-domain peptides or
synthetic small molecule drugs interfering with Bcl-2-
like protein function. To downregulate Bcl-2 expression,
Bcl-2 antisense oligonucleotidex (genasense) were devel-
oped by Genta Incorporated (Berkeley Heights, NJ,
USA). Genasense is a synthetic, 18-base, single-stranded
phosphorothioate oligonucleotide that selectively tar-
Apoptosis pathways in anticancer chemotherapy
S Fulda and K-M Debatin
4807
gets the rst six codons (i.e., 18 bases) of the mRNA
open reading frame encoding the Bcl-2 protein (Cotter,
2004). Treatment with genasense markedly enhanced the
antitumor activity of many chemotherapeutic drugs, for
example, taxanes, anthracyclines, alkylators or plati-
num-containing agents (Cotter, 2004). In a preclinical
model of melanoma, pretreatment with genasense
increased the chemosensitivity of human melanoma
(Jansen et al., 1998). Also in a clinical trial, genasense
was reported to act as chemosensitizer for dacarbazine
in patients with malignant melanoma (Jansen et al.,
2000). To optimize antisense-based therapy, bispecic
antisense oligonucleotides directed against a sequence,
which is highly homologous in Bcl-2 and Bcl-xL, but
missing in Bcl-xS mRNA, were subsequently designed
(Zangemeister-Wittke et al., 2000). Simultaneous down-
regulation of both Bcl-2 and Bcl-xL induced apoptosis
and enhanced chemosensitivity in various cancer cells
(Gautschi et al., 2001; Tortora et al., 2003; Milella et al.,
2004; Yamanaka et al., 2005).
Furthermore, BH3-domain peptides or synthetic small
molecule inhibitors were developed to target anti-
apoptotic Bcl-2-like proteins. The BH3 domain com-
prises a nine-amino-acid amphipathic a-helix that binds
to a hydrophobic pocket of Bcl-2-like proteins (Cory and
Adams, 2002). Similarly, BH3-domain peptides aim at
disrupting this complex, thereby sensitizing cancer cells
to apoptosi (Letai et al., 2002). Additionally, substitution
of the Bid BH3 domain with non-natural amino acids on
the surface opposite to the interacting region by
hydrocarbon stapling resulted in stabilized BH3 peptides
termed SAHBs (stabilized a-helix of Bcl-2 domains),
with improved pharmacological properties (Walensky
et al., 2004). These stabilized BH3 peptides triggered
apoptosis in a variety of leukemic cell lines and also
inhibited the growth of leukemia xenografts in mice
without adverse side effects (Walensky et al., 2004).
Also, several small molecule compounds interfering
with Bcl-2/Bcl-xL function have been identied. Screen-
ing of a chemical library for compounds able to bind to
the BH3 pocket of Bcl-2 proteins resulted in the
identication of HA14-1, a compound that competes
with Bak for the binding to Bcl-2 (Wang et al., 2000). By
screening a library of 16 320 preselected compounds for
the ability to displace a uorescent Bak BH3 peptide
from Bcl-xL in a uorescent polarization assay, Degterev
et al. (2003) identied two classes of agents called BH3
inhibitors (BH3Is), which also disrupt the Bcl-xL
complex with Bax and Bad in intact cells.
Using nuclear magnetic resonance (NMR)-based
screening, parallel synthesis and structure-based design,
a small-molecule inhibitor of the antiapoptotic proteins
Bcl-2, Bcl-X(L) and Bcl-w, ABT-737, was recently
discovered. ABT-737 was effective as a single agent
against certain lymphomas and solid tumors and
displayed synergistic cytotoxicity with chemotherapeu-
tics and radiation (Oltersdorf et al., 2005).
Smac/DIABLO agonists. For the design of potentially
therapeutic small molecules to target XIAP, the binding
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 Apoptosis pathways in anticancer chemotherapy
S Fulda and K-M Debatin
4808
groove of the BIR3 domain of XIAP, to which Smac/
DIABLO binds to after its release from mitochondria,
has attracted most attention. Structural analysis has
provided a clear rationale for the synthesis of small
compounds that can mimic the caspase-9 displacing
activity of Smac/DIABLO from XIAP BIR3 (Chai
et al., 2000; Wu et al., 2000). To enhance intracellular
delivery, Smac peptides were linked to a carrier, for
example, the protein transduction motif of the HIV Tat
protein (Fulda et al., 2002), the Drosophila antenna-
paedia penetratin sequence (Arnt et al., 2002) or a
polyarginine stretch (Yang et al., 2003b). A heptapep-
tide representing the N-terminus of mature Smac/
DIABLO, which is essential for binding to XIAP, was
reported to promote caspase activation and to sensitize
various tumor cell lines and also primary patients
derived tumor cells for apoptosis induced by death-
receptor ligation or cytotoxic drugs (Fulda et al., 2002).
Importantly, Smac peptides even enhanced the anti-
tumor activity of TRAIL in vivo in an intracranial
malignant glioma xenograft model (Fulda et al., 2002b).
Likewise, an 8-mer peptide (AVPIAQKS) fused to the
protein transduction domain of Drosophila antennapae-
dia penetratin was able to enter breast cancer cells, bind
XIAP and cIAP1, and potentiate caspase activity
induced by a number of anticancer drugs, including
paclitaxel, etoposide, 7-ethyl-10-hydroxycamptothecin
(SN-38) and doxorubicin (Arnt et al., 2002). Moreover,
on the basis of the three-dimensional structure of Smac/
DIABLO in complex with XIAP BIR3, Smac peptido-
mimetics were designed, which cooperated with TRAIL,
TNFa, cisplatin or etoposide to trigger apoptosis in
tumor cells (Li et al., 2004; Sun et al., 2004a, b, 2005).
Subsequently, non-peptidic small molecule antago-
nists of XIAP, which were derived by screening of a
phage library or polyphenylurea library, were developed
for targeting IAPs (Schimmer et al., 2004; Wang et al.,
2004). In addition, the natural product embelin from the
Japanese Ardisia herb was recently discovered as a cell-
permeable, non-peptidic, small-molecular weight inhi-
bitor of XIAP through structure-based computational
screening of a traditional herbal medicine three-dimen-
sional structure database (Nikolovska-Coleska et al.,
2004). Embelin was shown to effectively overcome the
protective effect of XIAP in prostate cancer cells with
high endogenous levels of XIAP or in Jurkat cells
transfected with XIAP through binding to the XIAP
BIR3 domain (Nikolovska-Coleska et al., 2004). Thus,
Smac agonists or low molecular weight XIAP antago-
nists may be promising candidates for cancer therapy by
potentiating the efcacy of cytotoxic therapies selec-
tively in cancer cells.
References
Adrain C, Slee EA, Harte MT, Martin SJ. (1999). J Biol Chem
274: 2085520860.
Arnoult D, Parone P, Martinou J-C, Antonsson B, Estaquier
J, Ameisen JC. (2002). J Cell Biol 159: 923929.
Arnt CR, Chiorean MV, Heldebrant MP, Gores GJ,
Kaufmann SH. (2002). J Biol Chem 277: 4423644243.
Oncogene
Conclusions
Continued efforts over many years to understand the
mechanisms controlling tumor cell proliferation and cell
death have delineated key molecular pathways involved
in cancer formation, progression and treatment resis-
tance. One of the key discoveries in cancer research has
been the recognition that anticancer chemotherapy kills
cancer cells by activating the intrinsic and/or extrinsic
apoptosis pathway. Although a considerable amount of
data support a role of the CD95 system in anticancer
drug-induced apoptosis, at least under certain circum-
stances, most cytotoxic drugs are considered to primar-
ily initiate cell death by triggering a cytochrome c/Apaf-
1/caspase-9-dependent pathway linked to mitochondria.
Collectively, these data point to a key role of the
mitochondrial pathway in chemotherapy-induced apop-
tosis, whereas the CD95 system may amplify killing by
cytotoxic drugs under certain conditions. Importantly,
this amplication of the chemoresponse may have
important clinical implications, since it may critically
affect the time required for execution of the death
program. Thus, defects in the intrinsic or extrinsic
pathway may promote tumor progression and therapy
resistance. Accordingly, targeting defective apoptosis
programs may restore sensitivity in resistant forms of
cancer. However, apoptosis does not represent the sole
killing mechanism by which cancers are eradicated, and
other modes of cell death, for example, necrosis,
autophagy, mitotic catastrophe or some forms of cell
death that cannot be easily classied at present, have
also to be considered. Further insight into the complex
signaling network activated in response to anticancer
therapy using cancer cell lines, primary tumor cells and
animal models are necessary to see to what extent the
current knowledge can be exploited for the design of
apoptosis-based cancer therapies. Also, further studies
on the role of apoptosis signaling molecules in clinical
samples using DNA or proteomic arrays are warranted
to assess the impact of these molecular parameters
on clinical outcome. Hopefully, these studies may
eventually allow the identication of novel therapeutic
targets thereby providing the basis for tailored, indivi-
dual tumor therapy.
Acknowledgements
Work in the authors laboratory was supported by grants from
the Deutsche Forschungsgemeinschaft, the Deutsche Kreb-
shilfe, Wilhelm-Sander-Stiftung, Kind-Phillipp-Stiftung and
Else-Kroner-Stiftung.
Ashkenazi A. (2002). Nat Rev Cancer 2: 420430.
Ashkenazi A, Pai RC, Fong S, Leung S, Lawrence
DA, Marsters SA et al. 1999)). J Clin Invest 104:
155162.
Bartling B, Lewensohn R, Zhivotovsky B. (2004). Exp Cell Res
298: 8395.

Belka C, Schmid B, Marini P, Durand E, Rudner J, Faltin H
et al. (2001). Oncogene 20: 21902196.
Bouillet P, Strasser A. (2002). J Cell Sci 115: 15671574.
Bratton SB, Walker G, Srinivasula SM, Sun XM, Butterworth
M, Alnemri ES et al. (2001). EMBO J 20: 9981009.
Brown JM, Attardi LD. (2005). Nat Rev Cancer 5: 231237.
Brown JM, Wilson G. (2003). Cancer Biol Ther 2: 477490.
Cain K, Bratton SB, Langlais C, Walker G, Brown DG, Sun
XM. (2000). J Biol Chem 275: 60676070.
Cande C, Cecconi F, Dessen P, Kroemer G. (2002). J Cell Sci
15: 47274734.
Cande C, Vahsen N, Kouranti I, Schmitt E, Daugas E, Spahr
C et al. (2004). Oncogene 23: 15141521.
Chai J, Du C, Wu JW, Kyin S, Wang X, Shi Y. (2000). Nature
406: 855862.
Chinnaiyan AM, Prasad U, Shankar S, Hamstra DA,
Shanaiah M, Chenevert TL et al. (2000). Proc Natl Acad
Sci USA 97: 17541759.
Chipuk JE, Kuwana T, Bouchier-Hayes L, Droin NM,
Newmeyer DD, Schuler M et al. (2004). Science 303:
10101014.
Chuntharapai A, Dodge K, Grimmer K, Schroeder K,
Marsters SA, Koeppen H et al. (2001). J Immunol 166:
48914898.
Cory S, Adams JM. (2002). Nat Rev Cancer 2: 647656.
Costantini P, Jacotot E, Decaudin D, Kroemer G. (2000).
J Natl Cancer Inst 92: 10421053.
Cotter FE. (2004). Semin Oncol 31: 1821.
Cowling V, Downward J. (2002). Cell Death Differ 9:
10461056.
Davis RJ. (2000). Cell 103: 239252.
Debatin KM, Poncet D, Kroemer G. (2002). Oncogene 21:
87868803.
Debatin KM, Stahnke K, Fulda S. (2003). Semin Cancer Biol
13: 149158.
Decaudin D, Marzo I, Brenner C, Kroemer G. (1998). Int J
Oncol 12: 141152.
Dechant MJ, Fellenberg J, Scheuerpug CG, Ewerbeck V,
Debatin KM. (2004). Int J Cancer 109: 661667.
Degterev A, Boyce M, Yuan J. (2003). Oncogene 22:
85438567.
Du C, Fang M, Li Y, Li L, Wang X. (2000). Cell 102: 3342.
Egle A, Harris AW, Bouillet P, Cory S. (2004). Proc Natl Acad
Sci USA 101: 61646169.
Eischen CM, Kottke TJ, Martins LM, Basi GS, Tung JS,
Earnshaw WC et al. (1997). Blood 90: 935943.
El-Deiry WS. (2001). Cell Death Differ 8: 10661075.
Enoksson M, Robertson JD, Gogvadze V, Bu P, Kropotov
A, Zhivotovsky B et al. (2004). J Biol Chem 279:
4957549578.
Friesen C, Fulda S, Debatin KM. (1997). Leukemia 11:
18331841.
Friesen C, Herr I, Krammer PH, Debatin KM. (1996). Nat
Med 2: 574577.
Fulda S, Debatin KM. (2004). Curr Cancer Drug Targets 4:
569576.
Fulda S, Los M, Friesen C, Debatin KM. (1998a). Int J Cancer
76: 105114.
Fulda S, Meyer E, Debatin KM. (2000). Cancer Res 60:
39473956.
Fulda S, Meyer E, Debatin KM. (2002a). Oncogene 21:
22832294.
Fulda S, Meyer E, Friesen C, Susin SA, Kroemer G, Debatin
KM. (2001). Oncogene 20: 10631075.
Fulda S, Scafdi C, Susin SA, Krammer PH, Kroemer G,
Peter ME et al. (1998b). J Biol Chem 273: 3394233948.
Apoptosis pathways in anticancer chemotherapy
S Fulda and K-M Debatin
4809
Fulda S, Sieverts H, Friesen C, Herr I, Debatin KM. (1997).
Cancer Res 57: 38233829.
Fulda S, Wick W, Weller M, Debatin KM. (2002b). Nat Med
8: 808815.
Gautschi O, Tschopp S, Olie RA, Leech SH, Simoes-Wust A
P, Ziegler A et al. (2001). J Natl Cancer Inst 93: 463471.
Gliniak B, Le T. (1999). Cancer Res 59: 61536158.
Green DR, Kroemer G. (2004). Science 305: 626629.
Guo Y, Srinivasula SM, Druilhe A, Fernandes-Alnemri T,
Alnemri ES. (2002). J Biol Chem 277: 1343013437.
Hakem R, Hakem A, Duncan GS, Henderson JT, Woo M,
Soengas MS et al. (1998). Cell 94: 339352.
Hao C, Beguinot F, Condorelli G, Trencia A, Van Meir EG,
Yong V et al. (2001). Cancer Res 61: 11621170.
Hao Z, Duncan GS, Chang CC, Elia A, Fang M, Wakeham A
et al. (2005). Cell 121: 579591.
Hemann M, Zilfou JT, Zhao Z, Burgess DJ, Hannon GJ,
Lowe SW. (2004). Proc Natl Acad Sci USA 101: 93339338.
Hengartner MO. (2000). Nature 407: 770776.
Houghton JA, Harwood FG, Tillman DM. (1997). Proc Natl
Acad Sci USA 94: 81448149.
Huang Y, Rich RL, Myszka DG, Wu H. (2003). J Biol Chem
278: 4951749522.
Hyer ML, Voelkel-Johnson C, Rubinchik S, Dong J, Norris
JS. (2000). Mol Ther 2: 348358.
Ichikawa K, Liu W, Zhao L, Wang Z, Liu D, Ohtsuka T et al.
(2001). Nat Med 7: 954960.
Igney FH, Krammer PH. (2002). J Leuk Biol 71: 907920.
Jansen B, Schlagbauer-Wadl H, Brown BD, Bryan RN, van
Elsas A, Muller M et al. (1998). Nat Med 4: 232234.
Jansen B, Wacheck V, Heere-Ress E, Schlagbauer-Wadl H,
Hoeller C, Lucas T et al. (2000). The Lancet 356: 17281733.
Jin Z, McDonald ER, Dicker DT, El-Deiry WS. (2004). J Biol
Chem 279: 3582935839.
Jo M, Kim TH, Seol DW, Esplen JE, Dorko K, Billiar TR
et al. (2000). Nat Med 6: 564567.
Karin M, Cao Y, Greten FR, Li ZW. (2002). Nat Rev Cancer
2: 301310.
Kasibhatla S, Brunner T, Genestier L, Echeverri F, Mahboubi
A, Green DR. (1998). Mol Cell 1: 543551.
Kataoka T, Schroter M, Hahne M, Schneider P, Irmler M,
Thome M et al. (1998). J Immunol 161: 39363942.
Keane MM, Rubinstein Y, Cuello M, Ettenberg SA, Banerjee
P, Nau MM et al. (2000). Breast Cancer Res Treat 64:
211219.
Kidd JF, Pilkington MF, Schell MJ, Fogarty KE, Skepper JN,
Taylor CW et al. (2002). J Biol Chem 2: 65046510.
Kinoshita H, Yoshikawa H, Shiiki K, Hamada Y, Nakajima
Y, Tasaka K. (2000). Int J Cancer 88: 986991.
Kischkel FC, Hellbardt S, Behrmann I, Germer M, Pawlita M,
Krammer PH et al. (1995). EMBO J 14: 55795588.
Kitada S, Pedersen IM, Schimmer AD, Reed JC. (2002).
Oncogene 21: 34593474.
Knudson C, Johnson GM, Lin Y, Korsmeyer SJ. (2001).
Cancer Res 61: 659665.
Konishi A, Shimizu S, Hirota J, Takao T, Fan Y, Matsuoka Y
et al. (2003). Cell 114: 673688.
Krammer PH. (2000). Nature 407: 789795.
Krueger A, Baumann S, Krammer PH, Kirchhoff S. (2001).
Mol Cell Biol 21: 82478254.
Lacour S, Hammann A, Grazide S, Lagadic-Gossmann D,
Athias A, Sergent O et al. (2004). Cancer Res 64: 35933598.
Lassus P, Opitz-Araya X, Lazebnik Y. (2002). Science 297:
13521354.
Lawrence D, Shahrokh Z, Marsters S, Achilles K, Shih D,
Mounho B et al. (2001). Nat Med 7: 383385.
Oncogene
 Apoptosis pathways in anticancer chemotherapy
S Fulda and K-M Debatin
4810
LeBlanc HN, Ashkenazi A. (2003). Cell Death Differ 10:
6675.
Letai A, Bassik MC, Walensky LD, Sorcinelli MD, Weiler S,
Korsmeyer SJ. (2002). Cancer Cell 2: 183192.
Li L, Thomas RM, Suzuki H, De Brabander JK, Wang X,
Harran PG. (2004). Science 305: 14711474.
Li W, Srinivasula SM, Chai J, Li P, Wu JW, Zhang Z et al.
(2002). Nature 9: 436441.
Lin B, Kolluri SK, Lin F, Liu W, Han YH, Cao X et al.
(2004). Cell 116: 527540.
Longley DB, Wilson TR, McEwan M, Allen WL, McDermott
U, Galligan L et al. (2006). Oncogene 25: 838848.
Maecker HL, Yun Z, Maecker HT, Giaccia AJ. (2002). Cancer
Cell 2: 139148.
Makin G, Dive C. (2001). Trends Cell Biol 11: S22S26.
Marks P, Rifkind RA, Richon VM, Breslow R, Miller T, Kelly
WK. (2001). Nat Rev Cancer 1: 194202.
Martins LM, Iaccarino I, Tenev T, Gschmeissner S, Totty NF,
Lemoine NR et al. (2002). J Biol Chem 277: 439444.
McDonnell TJ, Korsmeyer SJ. (1991). Nature 349: 254256.
McNeish IA, Bell S, McKay T, Tenev T, Marani M, Lemoine
NR. (2003). Exp Cell Res 286: 186198.
Meng RD, El-Deiry WS. (2001). Exp Cell Res 262: 154169.
Meng RD, McDonald ER, Sheikh MS, Fornace AJ, El-Deiry
WS. (2000). Mol Ther 1: 130144.
Micheau O, Hammann A, Solary E, Dimanche-boitrel MT.
(1999). Biophys Res Commun 256: 603611.
Mihara M, Erster S, Zaika A, Petrenko O, Chittenden T,
Pancoska P et al. (2003). Mol Cell 11: 577590.
Milella M, Trisciuoglio D, Bruno T, Ciuffreda L, Mottolese
M, Cianciulli A et al. (2004). Clin Cancer Res 10: 77477756.
Moll UM, Wolff S, Speidel D, Deppert W. (2005). Curr Opin
Cell Biol 17: 631636.
Muller M, Strand S, Hug H, Heinemann EM, Walczak H,
Hofmann WJ et al. (1997). J Clin Invest 99: 403413.
Muller M, Wilder S, Bannasch D, Israeli D, Lehlbach K,
Li-Weber M et al. (1998). J Exp Med 188: 20332045.
Nagane M, Pan G, Weddle JJ, Dixit VM, Cavenee WK,
Huang HJ. (2000). Cancer Res 60: 847853.
Nagata S. (2000). Exp Cell Res 256: 1218.
Ng CP, Bonavida B. (2002). Mol Cancer Ther 1: 10511058.
Nikolovska-Coleska Z, Xu L, Hu Z, Tomita Y, Li P, Roller
PP et al. (2004). J Med Chem 47: 24302440.
Nitsch R, Bechmann I, Deisz RA, Haas D, Lehmann TN,
Wendling U et al. (2000). Lancet 356: 827828.
Oda E, Ohki R, Murasawa H, Nemoto J, Shibue T, Yamashita
T et al. (2000). Science 288: 10531058.
Okada H, Mak TW. (2004). Nat Rev Cancer 4: 592603.
Okada H, Suh WK, Jin J, Woo M, Du C, Elia A et al. (2002).
Mol Cell Biol 22: 35093517.
Okano H, Shiraki K, Inoue H, Kawakita T, Saitou Y,
Enokimura N et al. (2003). Int J Mol Med 12: 2528.
Olsson A, Diaz T, Aguilar-Santelises M. (2001). Leukemia 15:
18681877.
Oltersdorf T, Elmore SW, Shoemaker AR, Armstrong RC,
Augeri DJ, Belli BA et al. (2005). Nature 435: 677681.
Pai SI, Wu GS, Ozoren N, Wu L, Jen J, Sidransky D et al.
(1998). Cancer Res 58: 35133518.
Petak I, Danam RP, Tillman DM, Vernes R, Howell SR,
Berczi L et al. (2003). Cell Death Differ 10: 211217.
Pitti RM, Marsters SA, Lawrence DA, Roy M, Kischkel FC,
Dowd P et al. (1998). Nature 396: 699703.
Pollack IF, Erff M, Ashkenazi A. (2001). Clin Cancer Res 7:
13621369.
Rampino N, Yamamoto H, Ionov Y, Li Y, Sawai H, Reed JC
et al. (1997). Science 275: 967969.
Oncogene
Ricci JE, Munoz-Pinedo C, Fitzgerald P, Bailly-Maitre B,
Perkins GA, Yadava N et al. (2004). Cell 117: 773786.
Rich T, Allen RL, Wyllie AH. (2000). Nature 407: 777783.
Robertson JD, Enoksson M, Suomela M, Zhivotovsky B,
Orrenius S. (2002). J Biol Chem 277: 2980329809.
Robertson JD, Gogvadze V, Kropotov A, Vakifahmetoglu H,
Zhivotovsky B, Orrenius S. (2004). EMBO Rep 5: 643648.
Robertson JD, Gogvadze V, Zhivotovsky B, Orrenius S.
(2000). J Biol Chem 275: 3243832443.
Rohn TA, Wagenknecht B, Roth W, Naumann U, Gulbins E,
Krammer PH et al. (2001). Oncogene 20: 41284137.
Roth W, Isenmann S, Nakamura M, Platten M, Wick W,
Kleihues P et al. (2001). Cancer Res 61: 27592765.
Ruvolo PP, Deng X, May WS. (2001). Leukemia 15: 515522.
Saelens X, Festjens N, Walle LV, van Gurp M, van Loo G,
Vandenabeele P. (2004). Oncogene 23: 28612874.
Sax JK, Fei P, Murphy ME, Bernhard E, Korsmeyer SJ,
El-Deiry WS. (2002). Nat Cell Biol 4: 842849.
Scafdi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli
KJ et al. (1998). EMBO J 17: 16751687.
Schimmer AD, Welsh K, Pinilla C, Wang Z, Krajewska M,
Bonneau MJ et al. (2004). Cancer Cell 5: 2535.
Sheikh MS, Huang Y, Fernandez-Salas EA, El-Deiry
WS, Friess H, Amundson S et al. (1999). Oncogene 18:
41534159.
Shipp MA, Ross KN, Tamayo P, Weng AP, Kutok JL, Aguiar
RCT et al. (2002). Nat Med 8: 6874.
Srinivasula SM, Hegde R, Saleh A, Datta P, Shiozaki E, Chai
J et al. (2001). Nature 410): 112116.
Stassi G, Garofalo M, Zerilli M, Ricci-Vitiani L, Zanca C,
Todaro M et al. (2005). Cancer Res 65: 66686675.
Strasser A. (2005). Nat Rev Immunol 5: 189200.
Sun H, Nikolovska-Coleska Z, Chen J, Yang CY, Tomita Y,
Pan H et al. (2005). Bioorg Med Chem Lett 15: 793797.
Sun H, Nikolovska-Coleska Z, Yang CY, Xu L, Liu M,
Tomita Y et al. (2004a). J Am Chem Soc 126: 1668616687.
Sun H, Nikolovska-Coleska Z, Yang CY, Xu L, Tomita Y,
Krajewski K et al. (2004b). J Med Chem 47: 41474150.
Sunters A, Fernandez de Mattos S, Stahl M, Brosens JJ,
Zoumpoulidou G, Saunders CA et al. (2003). J Biol Chem
278: 4979549805.
Susin SA, Zamzami N, Castedo M, Daugas E, Wang HG,
Geley S et al. (1997). J Exp Med 186: 2537.
Suzuki Y, Imai Y, Nakayama H, Takahashi K, Takio K,
Takahashi R. (2001). Mol Cell 8: 613621.
Suzuki Y, Takahashi-Niki K, Akagi T, Hashikawa T,
Takahashi R. (2004). Cell Death Differ 11: 208216.
Tagawa H, Karnan S, Suzuki R, Matsuo K, Zhang X, Ota A
et al. (2005). Oncogene 24: 13481358.
Takimoto R, El-Deiry WS. (2000). Oncogene 19: 17351743.
Talos F, Petrenko O, Mena P, Moll UM. (2005). Cancer Res
65: 99719981.
Tan TT, Degenhardt K, Nelson DA, Beaudoin B, Nieves-
Neira W, Bouillet P et al. (2005). Cancer Cell 7: 227238.
Tinel A, Tschopp J. (2004). Science 304: 843846.
Tortora G, Caputo R, Damiano V, Caputo R, Troiani T,
Veneziani BM et al. (2003). Clin Cancer Res 9: 866871.
Traver D, Akashi K, Weissman IL, Lagasse E. (1998).
Immunity 9: 4757.
Trencia A, Fiory F, Maitan MA, Vito P, Barbagallo APM,
Perfetti A et al. (2004). J Biol Chem 279: 4656646572.
Tsujimoto Y, Finger LR, Yunis J, Nowell PC, Croce CM.
(1984). Science 226: 10971099.
van Loo G, van Gurp M, Depuydt B, Srinivasula SM,
Rodriguez I, Alnemri ES et al. (2002). Cell Death Differ 9:
2026.

van Noesel MM, van Bezouw S, Salomons GS, Voute PA,
Pieters R, Baylin SB et al. (2002). Cancer Res 62: 21572161.
Varfolomeev EE, Schuchmann M, Luria V, Chiannilkulchai
N, Beckmann JS, Mett IL et al. (1998). Immunity 9:
267276.
Vaux DL, Silke J. (2003). Biochem Biophys Res Commun 304:
499504.
Verhagen AM, Ekert PG, Pakusch M, Silke J, Connolly LM,
Reid GE et al. (2000). Cell 102: 4353.
Villunger A, Egle A, Kos M, Hartmann BL, Geley S, Koer R
et al. (1997). Cancer Res 57: 33313334.
Walczak H, Bouchon A, Stahl H, Krammer PH. (2000).
Cancer Res 60: 30513057.
Walczak H, Krammer PH. (2000). Exp Cell Res 256: 5866.
Walensky LD, Kung AL, Escher I, Malia TJ, Barbuto S,
Wright RD et al. (2004). Science 305: 14661470.
Wang JL, Liu D, Zhang ZJ, Shan S, Han X, Srinivasula SM
et al. (2000). Proc Natl Acad Sci USA 97: 71247129.
Wang S, El-Deiry WS. (2003). Proc Natl Acad Sci USA 100:
1509515100.
Wang Z, Cuddy M, Samuel T, Welsh K, Schimmer A, Hanaii
F et al. (2004). J Biol Chem 279: 4816848176.
Waterhouse NJ, Goldstein JC, von Ahsen O, Schuler M,
Newmeyer DD, Green DR. (2001). J Cell Biol 153: 319328.
Apoptosis pathways in anticancer chemotherapy
S Fulda and K-M Debatin
4811
Wu G, Chai J, Suber TL, Wu JW, Du C, Wang X et al. (2000).
Nature 408: 10081012.
Yamanaka K, Rocchi P, Miyake H, Fazli L, Vessella B,
Zangemeister-Wittke U et al. (2005). Mol Cancer Ther 4:
16891698.
Yang L, Cao Z, Yan H, Wood WC. (2003a). Cancer Res 63:
68156824.
Yang L, Mashima T, Sato S, Mochizuki M, Sakamoto H,
Yamori T et al. (2003b). Cancer Res 63: 831837.
Yeh WC, Pompa JL, McCurrach ME, Shu HB, Elia AJ,
Shahinian A et al. (1998). Science 279: 19541958.
Yoshida H, Kong YY, Yoshida R, Elia AJ, Hakem A, Hakem
R et al. (1998). Cell 94: 739750.
Yu J, Zhang L, Hwang PM, Kinzler KW, Vogelstein B. (2001).
Mol Cell 7: 673682.
Zangemeister-Wittke U, Leech SH, Olie RA, Simoes-Wust
AP, Gautschi O, Luedke GH et al. (2000). Clin Cancer Res
6: 25472555.
Zhao J, Jin J, Zhang X, Shi M, Dai J, Wu MY et al. (2006).
Cancer Gene Ther 13: 420427.
Zinkel SS, Ong CC, Ferguson DO, Iwasaki H, Akashi K,
Bronson RT et al. (2003). Genes Dev 17: 229239.
Zou H, Henzel WJ, Liu X, Lutschg A, Wang X. (1997). Cell
90: 405413.
Oncogene