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226 Anti-Cancer Agents in Medicinal Chemistry, 2012, 12, 226-238
Chemotherapeutic Targeting of Cell Death Pathways
Sylvia Mansilla, Laia Llovera and José Portugal#,*
Instituto de Biología Molecular de Barcelona, CSIC, Parc Científic de Barcelona, Baldiri Reixac, 10, E-08028
Barcelona, Spain
Abstract: Cell death plays an important role in cancer growth and progression, as well as in the efficiency of chemotherapy. Although
apoptosis is commonly regarded as the principal mechanism of programmed cell death, it has been increasingly reported that several anti-
cancer agents do not only induce apoptosis but other forms of cell death such as necrosis, autophagy and mitotic catastrophe, as well as
the state of permanent loss of proliferative capacity known as senescence. A deeper understanding of what we know about chemotherapy-
induced death is rather relevant considering the emerging knowledge of non-apoptotic cell death signaling pathways, and the fact that
many tumors have the apoptosis pathway seriously compromised. In this review we examine the effects that various anti-cancer agents
have on pathways involved in the different cell death outcomes. Novel and specific anti-cancer agents directed toward members of the
cell death signaling pathways are being developed and currently being tested in clinical trials. If we precisely activate or inhibit molecules
that mediate the diversity of cell death outcomes, we might succeed in more effective and less toxic chemotherapy.
Keywords: Apoptosis, Autophagy, Chemotherapy, Mitotic catastrophe, Necrosis, Senescence. #Author’s Profile: José Portugal received his PhD in Biology from the University of Barcelona, Spain in 1983. He was a post-doc at the
University of Cambridge, UK (1985-87), and he was appointed Lecturer in Biochemistry at the University of Barcelona (1987-1992). At
present, he is Research Scientist at the Institute of Molecular Biology-CSIC in Barcelona. His research is aimed at understanding the
mechanisms used by DNA-binding drugs to inhibit transcription and how transcriptional changes commit cancer cells to dying.
INTRODUCTION
Cell death is a fundamental cellular response that has a crucial
role in shaping living organisms during development and in
regulating tissue homeostasis by eliminating unwanted cells.
Moreover, cell death regulation plays an important role in cancer
growth and progression, and defects in cell death pathways are a
hallmark of cancer [1]. In response to DNA damage, cells can be
arrested at specific cell cycle checkpoints to allow for DNA repair
or, if the damage cannot be repaired, activation of programmed cell
death can occur [2-6]. DNA-damaging agents have been in use for
cancer therapy for decades. Indeed, the nitrogen mustards, the first
agents to be employed clinically in the treatment of cancer, are DNA
cross-linking drugs. The form of cell death induced by a particular
anti-cancer agent seems to depend on the cell type and its genotype as
well as the kind of DNA damage to which cells are exposed [3, 4, 7].
A key regulator of the response to DNA damage is the tumor
suppressor p53, which is activated and stabilized [2, 8]. Activated
p53 stimulates the expression of p21WAF1
, and inhibits cyclin-
dependent kinases, resulting in cell cycle arrest at both the G1 and
the G2/M phases [8]. Cells in a growing tumor have to evade
apoptosis to survive and become invasive and metastatic.
Nevertheless, there are grounds for considering that the response of
tumor cells to anti-cancer agents is not confined to apoptosis but
also includes other forms of cell death [3, 4, 7, 9-11]. Activated p53
stimulates the expression of p21WAF1
, an inhibitor of cyclin-
dependent kinases, resulting in cell cycle arrest at both the G1 and
the G2/M phases [8]. However, in about half of human cancers, the
activity of p53 is compromised [12]. In the absence of wild type
p53, the G1 checkpoint cannot be properly activated. The G2/M
checkpoint can be activated through two checkpoint kinases (Chk1
and Chk2). Chk1 is basically activated following genotoxic stress
and Chk2 is activated following double strand DNA breakage,
resulting in inactivation of cyclin-dependent kinases and cell cycle
arrest. It has been suggested that Chk1 inhibitors would abrogate
*Address correspondence to this author at the Instituto de Biología
Molecular de Barcelona, CSIC, Parc Científic de Barcelona, Baldiri Reixac, 10,
E-08028 Barcelona, Spain; Tel: +34-93- 403 4959; Fax: +34-93- 403 4979;
E-mail: jpmbmc@ibmb.csic.es
the remaining checkpoints in cancer cells lacking functional p53
and this would lead to preferential sensitization of these cancer cells
to chemotherapy over cells bearing wild-type p53 [13].
Cell death is nowadays considered to comprise a large number
of different types of cellular demise, and at least eight types of cell
death can be defined [14]. The cellular routes leading to cell death
that are generally considered in our present understanding of how
several anticancer agents exert their cytotoxic activities are
apoptosis, mitotic catastrophe and necrosis/necroptosis, while
senescence, a mode of permanent cell arrest, is usually viewed as a
fourth type of cell death in the context of chemotherapy.
Although cancer cells often have defects in a particular cell-
death pathway, they can still die because of the redundancy of cell-
death mechanisms. However, the nature of the cell-death defect
ultimately affects the clinical outcome of treatment, depending on
which mechanism is missing. In particular, lack of apoptosis in
several solid tumors in response to chemotherapy [12] does not
imply that the apoptotic response cannot be modulated to increase
sensitivity to treatments. Nevertheless, several questions remain
concerning the interactions between apoptotic and non-apoptotic
cell-death pathways, as these pathways overlap to some degree.
Anti-cancer agents designed to restore function to a key program
could restore them all (at least in theory), thus improving the efficacy
of some treatments. A promising strategy to modulate cell sensitivity
to anti-cancer agents is the combination of target-specific agents,
which might interfere with specific oncogenic processes or cellular
targets, with more conventional DNA-damaging agents [15].
APOPTOSIS
Apoptosis is a form of programmed cell death that is required
as a mechanism complementary to proliferation to ensure
homeostasis in living organisms. Apoptosis contributes to the
overall sensitivity of cells to chemotherapeutic agents as assessed
either by in vitro assays or upon some in vivo treatments [2]. Cells
undergoing apoptosis show characteristic morphological and
biochemical features, which include chromatin aggregation and
nuclear and cytoplasmic condensation.
Apoptosis in mammalian cells is mediated by a family of
cysteine proteases known as caspases [16]. To keep the apoptotic
program under control, caspases are initially expressed in cells as
1875-5992/12 $58.00+.00 © 2012 Bentham Science Publishers
Targeting Cell Death Pathways Anti-Cancer Agents in Medicinal Chemistry, 2012, Vol. 12, No. 3 227
inactive pro-caspase precursors. Inducer caspases (including
caspase-2, -8, 9 and -10) are activated by cross-cleavage after
multimerization on a scaffold protein. The effector caspases
(caspases -3, -6 and -7) are commonly activated by other proteases,
including inducer caspases. Effector caspases cleave a wide range
of cell structures and regulatory proteins leading to a set of changes
and to cell death [16]. Although caspase-2 can be considered an
initiator caspase, recent evidence suggests that caspase-2, may have
multiple roles in the DNA damage response, cell cycle regulation
and tumor suppression. One peculiar feature of caspase-2 is that
unlike the other caspases, it is found constitutively in the nucleus.
Caspase-2 is both required for p53-mediated apoptosis and down-
regulated by p53 in a p21-dependent manner [17].
Two major pathways have been described to initiate apoptosis:
the extrinsic pathway and the intrinsic (or mitochondrial) pathway
(Fig. (1)). The intrinsic apoptotic pathway is usually initiated by
stabilized p53 in response to DNA damage, and activated through
the oligomerization of the pro-apoptotic proteins Bax and Bak in
the mitochondrial membrane to activate mitochondrial outer
membrane permeabilization, thus permitting release of apoptogenic
factors such as cytochrome c (Fig. (1)). Once released, cytochrome
c binds to the apoptotic protease-activating factor 1 (Apaf-1), which
recruits pro-caspase-9, promoting its self-activation. Activated
caspase-9 cleaves the downstream effectors caspase-3 and caspase-
7, which rapidly cleave intracellular substrates. Proteins of the IAP
family can bind and inhibit the active sites of caspase-3, caspase-7,
and of caspase-9 (Fig. (1)). The anti-apoptotic Bcl-2 proteins block
oligomerization of Bax and Bak, or their associations with BH3-only
proteins, thus preventing changes in the mitochondrial membrane.
The extrinsic pathway involves activation of death receptors,
such as TNF- (tumor necrosis factor, TRAIL (TNF receptor
apoptosis-inducing ligand), DR4, and DR5 [18]. Interaction with
their respective ligands leads to a signal transduction cascade
initiated by the recruitment of some molecules and subsequent
activation of caspase-8. This caspase then catalyzes proteolytic
events that eventually result in apoptosis [12, 16]. The BH3-only
protein Bid connects the extrinsic pathway to mitochondria
(Fig. (1)). Bid is cleaved by caspase-8, resulting in a chemically
modified molecule that is then targeted to membranes where it
promotes Bax and Bak oligomerization [19].
Chemotherapy can activate a DNA damage response that
stabilizes p53 [20]. This tumor suppressor protein either arrests the
cell cycle by transcriptionally activating the cyclin-dependent
kinase inhibitor p21WAF1
, giving the cell time to repair the damage,
or else it helps to mediate apoptotic cell death. Pro-apoptotic genes
are also activated by p53, including those encoding Bax and
the BH3-only proteins [20]. Furthermore, p53 may directly alter
the mitochondrial membrane potential by binding Bcl-2 family
members and mediating Bax and Bak dimerization.
TARGETING APOPTOTIC PATHWAYS
When p53 is functional, almost any genotoxic stress caused
by DNA damage that cannot be repaired will induce apoptosis
through the p53 pathway. Therefore, almost any DNA-binding
drug, including alkylating drugs and the agents that inhibit
topoisomerases, can be classified as pro-apoptotic drugs. However,
this classical view of a direct correlation between the ability
of drugs for inducing apoptotis and the cell’s susceptibility to
chemotherapeutic agents should be considered too simplistic,
especially if we consider that mutated forms of p53 are a common
characteristic of more than 50% of human tumors [12], and that
many different routes leading to the inactivation of pro-apoptotic
signaling pathways underline tumorigenesis [1]. Needless to
say, the lack of apoptosis in many solid tumors in response
to therapy does not imply that modulation of apoptosis cannot be
used to increase the sensitivity of this tumors to chemotherapeutic
agents, because, as mentioned above, some other cell death
pathways may end in apoptosis-like death (presence of active
effector caspases and changes in mitochondrial membrane potential,
among others).
Fig. (1). Extrinsic and intrinsic apoptosis signaling pathways. The death receptor TRAIL induces cell death via the extrinsic pathway by recruiting and
activating caspases -8 and -10 to its R1 and R2 receptors. TRAIL can also activate the intrinsic pathway indirectly. The intrinsic pathway is initiated by p53
and mediated by the mitochondria. The figure shows that cytochrome c, released from the mitochondria, binds to and activates the Apaf-1, inducing the
formation of the apoptosome, and eventually mediates the activation of caspase-3 and caspase-7 effector caspases. Both apoptotic routes contain potential
targets for anti-cancer chemotherapy (see the main text and Table 1).
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228 Anti-Cancer Agents in Medicinal Chemistry, 2012, Vol. 12, No. 3 Mansilla et al.
A commonly used approach in cancer therapy has been to target
proteins involved in apoptosis, aimed at inducing cancer cell death
or to enhance the sensitivity of cancer cells to certain cytotoxic
drugs or radiation [21]. In clinical assays, there are some novel
agents (Table 1) such as bortezomib, which targets the 26S
proteasome in Ubiquitin/Proteasome system, and it is approved for
treating relapsed multiple myeloma. Moreover, some proteasome
inhibitors are under development to overcome bortezomib
resistance [22]. Imatinib, a tyrosine kinase inhibitor, does not
primarily target apoptosis but indirectly modulates it via the effect
on the Bcr-abl oncogene fusion protein that is associated with the
PI3k/AKT pathway. It is used in treating chronic myelogenous
leukemia [23] and gastrointestinal stromal tumors.
A very appealing methodology is to obtain inhibitors of the Bcl-
2 family of proteins to facilitate the regression of solid tumors
(Table 1). One of those molecules, ABT-737, does not directly
initiate the apoptotic process, but enhances the effects of death
signals, displaying synergistic cytotoxicity with some drugs and
radiation [24]. Gossypol a natural product derived from cottonseed
extracts binds to the BH3-binding regions of Bcl-2 and Mcl-1.
However, a phase II trial of this molecule in metastatic breast
cancer refractory to doxorubicin (a DNA-binding antibiotic)
and paclitaxel (which targets tubulin) produced no therapeutic
response [5]. There is also a nuclease-resistant antisense nucleotide
(oblimersen) targeting Bcl-2 (Table 1), which has shown promising
activity against melanoma [5] and chronic lymphocytic leukemia
[25]. Several reports exist about the use of oblimersen in
combination with chemotherapeutic drugs, yet not all combination
therapies produce desirable results.
Table 1. Examples of Selective Chemotherapeutic Agents Targeting Diverse Cell Death Pathways
Death Pathway Therapeutic Target(a)
Chemotherapeutic Agent(b)
Stage(c)
Oblimersen Phase II/III
Gossypol [125] Phase I Bcl-2
ABT-737 [24] Phase I
IAPs SM-164 [126] Phase I
p53/Mdm2 Nutlins [127] Pre-clinical
TRAIL (recombinant) Phase I
TNF- (recombinant) Clinical
Apomab Phase I
Death Receptors
Mapatumumab Phase II
PI3-k/AKT Imatinib [128] Clinical
Apoptosis(d)
Proteasome Bortezomib [129] Phase III
Everolimus [43] Phase III
Deferolimus [43] Phase II/III m-TOR
Rapamycin (sirolimus) [43] Clinical
Temozolomide [130] Phase II
Pro-autophagic Resveratrol [131] Phase I/II
Autophagy(d)
Anti-autophagic Hydroxychloroquine [132] Phase II
Senescence(d)
Telomerase Imetelstat Phase I/II
KSP/Eg5 Ispinesib [133] Phase II
UCN-01 [134] Phase II
XL844 [135] Phase I/II
AZD7762 [135] Phase I
CHIR-124 [135] Phase I
Chk1
PF-00477736 [135] Phase I
ZM447439 [109] Phase II
VX-680 [109] Phase II
Hesperadin [136] Phase II
MLN8054 [109] Phase I
AZD1152 [109] Phase I
Aurora kinases
PHA-739358 [137] Phase I
BI2536 [138] Phase II/III
Mitotic catastrophe(d)
PLK
ONO1910 [139] Phase II
PARP (Metabolism, ROS, Ca2+
) Photodynamic Therapy Clinical
Necrosis(d)
PARP DNA alkylating agents Clinical
(a) See main text for abbreviations.
(b) Quoted references present the chemical structure of the small molecules.
(c) Clinical trial data are summarized from: http://www.clinicaltrials.gov (last visit 16 September 2011). Other sources are indicated in the main text.
(d) The Table does not list DNA-binding drugs and spindle poisons that are in clinical use, which are discussed in the main text.
Targeting Cell Death Pathways Anti-Cancer Agents in Medicinal Chemistry, 2012, Vol. 12, No. 3 229
The specific introduction of genetic material into tumor cells
for the selective regulation of gene expression (gene therapy) and
the epigenetic restoration of lost of function of mutated proteins are
two of the most recent strategies to activate cell death pathways
with emphasis in restoring the apoptotic response. Given that
cancer cells bear widespread changes in their genomic DNA
methylation and histone modification patterns, therapies aimed to
restore normal epigenetic modification patterns through the
inhibition of epigenetic modifier enzymes are under development. Two main groups of drugs are examined to reverse epigenetic
defects in cancer. The first includes drugs that inhibit DNA
methyltransferases (DNMTs) resulting in the inhibition of DNA
methylation. This group of drugs may prove to be useful in the
treatment of cancer where hypermethylation of tumor suppressor
genes is known to lead to silencing of these genes. The other group
of drugs inhibits histone deacetylases (HDACs) resulting in the
accumulation of acetylated histones, which modify the chromatin
architecture that can mediate the anticancer effects of these drugs.
Both these drug groups have shown promising results, and some
epigenetic drugs have been approved for the treatment of subtypes
of leukemias and lymphomas [26].
Although p53 is functionally an attractive target for cancer
therapy development, there is some concern on whether this protein
can be “targeted” adequately [27], and whether it is a realistic
objective to develop tumor-specific p53 restoration therapies.
Progress in this respect has been made using adenovirus-based gene
therapy delivering a functional copy of p53 [28]. Results obtained
in clinical trials reveal that antitumor efficacy is associated with
expression of functional p53 [27].
Activation of the p53 pathway by antagonizing its negative
regulator Mdm 2 (murine double minute 2) might offer a
therapeutic strategy for the great majority of hematological
malignancies that frequently express wild-type p53 at diagnosis
[29]. Nutlins, a family of cis-imidazole analogues, have been
identified as Mdm2 inhibitors. Studies with these compounds have
strengthened the concept that selective non-genotoxic p53
activation might represent an alternative to the current cytotoxic
chemotherapy. Nutlins do not only induce apoptotic cell death
when added to primary leukemic cell cultures, but also display a
synergistic effect in combination with some chemotherapeutic drugs
commonly used for the treatment of hematological malignancies [29].
Nutlins might have therapeutic effects by two distinct mechanisms:
a direct cytotoxic effect on leukemic cells and an indirect non-cell
autonomous effect on tumor stromal and vascular cells [29]. This
later effect might be therapeutically relevant also for treatment of
some malignancies carrying p53 mutations.
Another group of molecules being developed to target death
receptors, and, therefore, basically the extrinsic apoptotic pathway,
includes several molecules in phase II and preclinical trials [18]
(Table 1), and a recombinant TNF- approved for limb perfusion
[18]. A recombinant TRAIL (TNF-related apoptosis-inducing ligand
protein), which binds to the death receptors DR4 and DR5, has also
been considered a way to selectively targeting apoptosis [5].
A small molecule mimic of Smac (a pro-apoptotic protein that
functions by relieving inhibitor-of-apoptosis protein (IAP)-
mediated suppression of caspase activity) synergizes with both
tumor necrosis factor alpha (TNF ) and TRAIL (TNF-related
apoptosis-inducing ligand) to potently induce caspase activation
and apoptosis in human cancer cells. The molecule has allowed a
temporal, unbiased evaluation of the roles that IAP proteins play
during signaling from TRAIL and TNF receptors (Fig. (1)). This
compound is a lead structure for the development of IAP
antagonists potentially useful for cancer therapy [30, 31].
AUTOPHAGY
Autophagy is a catabolic process that occurs in all eukaryotic
cells involving the degradation of their components through the
lysosomal machinery [32, 33]. It is a tightly-regulated process that
plays a normal part in cell growth, development, and homeostasis,
helping to maintain a balance between the synthesis, degradation,
and subsequent recycling of cellular products [9, 34]. Autophagy is
a major mechanism by which a starving cell reallocates nutrients
from “unnecessary” processes to more-essential processes. During
autophagy, portions of the cytoplasm are encapsulated in a double-
membrane structure referred to as autophagosome (Fig. (2)) [34,
35]. Autophagosomes then fuse with lysosomes where the contents
are delivered, resulting in their degradation (Fig. (2)). Autophagy
can promote cell adaptation and survival during stresses such as
starvation, but under some conditions cells undergo death by
excessive autophagy. A useful marker of autophagy is LC3
(autophagosome-associated protein microtubule-associated protein
1 light chain 3). LC3 exists in two forms, LC3-I and its
proteolytic derivative LC3-II, which are localized in the cytosol
(LC3-I) or in the autophagosomal membranes (LC3-II)[36].
Beclin 1, a tumor suppressor, is a core element of cellular
autophagy. At the molecular level, the signaling pathway that leads
to autophagy involves at least the activities of phosphatidylinositol
3-kinase (PI3k) and the kinase target of rapamycin (TOR). Class-III
PI3k activity is particularly important for the early stages of
autophagic vesicle formation (Fig. (2)). By contrast, TOR
negatively regulates autophagosome formation and expansion.
Consequently, inhibition of TOR by rapamycin blocks cell-cycle
progression and eventually results in autophagy [9]. Deprivation of
amino acids, nutrients or growth factors can also down-regulate
TOR signaling. Therefore, the TOR pathway coordinates signaling
pathways that are initiated by nutritional and mitogenic factors, and
also controls both protein synthesis and degradation.
Autophagy is important in the regulation of cancer development
and progression and in determining the response of tumor cells to
anticancer therapy [37]. However, the role of autophagy in these
processes is complex and may, depending on the circumstances,
have diametrically opposite consequences for the tumor. Whether
autophagy causes death in cancer cells or it protects them is a
controversial subject [35]. Some contradictory findings would
suggest that the outcome of the autophagic response can vary
depending on the type of cellular insult [38]. Because autophagy
confers stress tolerance, it limits damage and sustains viability
under adverse conditions. It has to be considered a tumor
suppression mechanism, yet it enables tumor cell survival in stress
[39]. There are also evidences that preserving cellular fitness
by autophagy may be important for tumor suppression. Hence,
there is a clear interest in establishing how the functional status of
autophagy may influence tumorigenesis and treatment response [32,
39]. Several lines of evidence have been found about a cross-talk
between autophagic and apoptotic pathways [38]. Suppression of
autophagy may contribute to the initial rapid growth of tumors,
however, in more advanced stages of cancer, autophagy may be
required to provide essential nutrients to the cells in the inner part
of solid tumors [5]. Hence, some of the recent strategies include
inducing autophagy in early-developed cancers, while inhibiting
autophagy in advanced tumors with intact autophagy response to
sensitize the cells to a variety of anti-cancer agents.
TARGETING AUTOPHAGY
Blocking autophagy in tumor cells either pharmacologically or
genetically results in increased tumor cell death. Therefore,
combining autophagy inhibitors with other cancer chemo-
therapeutics may enhance the commitment of cells to dying [40].
However, autophagy inhibitors may increase genome instability in
the surviving cancer cells and may also promote the cell non-
autonomous tumor progression, which would together accelerate
cancer relapse. Autophagy has been shown to provide a way for
breast cancer cells to avoid apoptosis and survive despite the treat-
ment with trastuzumab —a recombinant-humanized monoclonal
antibody directed to the HER-2/neu protein— [41].
230 Anti-Cancer Agents in Medicinal Chemistry, 2012, Vol. 12, No. 3 Mansilla et al.
If autophagy functions to promote survival of cancer cells by
enabling catabolism, then autophagy inhibitors may be
therapeutically useful. By carefully choosing the tumor types and
drug combinations and regimens, autophagy inhibitors could
enhance the efficacy of the existing chemotherapeutics to reduce
tumor growth. For example, chloroquine enhances the anti-cancer
effect of 5-fluorouracil in human colon cancer cells [42]. On the
other hand, promoting autophagy as a means to limit cellular
damage seems an adequate strategy for cancer prevention [33].
Alternatively, if autophagic cell death is a significant mechanism of
cancer cell elimination, then inhibition of mTOR and activation of
autophagy may be therapeutically beneficial. Accordingly, several
agents that induce autophagy through the inhibition of mTOR
are under development (Table 1) [39, 43]. Moreover, it has
been observed in an experimental model of prostate cancer that
therapeutic starvation by using 2-deoxyglucose results in autophagy
[44].
Autophagic cell death has been shown to be activated in cancer
cells in response to several chemotherapeutic agents, such as
paclitaxel and vinblastine, as well as to irradiation [9, 35].
Rapamycin (sirolimus), an m-TOR inhibitor, induces autophagy in
malignant glioma cells [45] and it presents potential clinical
benefits for patients with epithelial ovarian cancer [46], while
tamoxifen and other anti-estrogen agents induce autophagy in
MCF-7 breast cancer cells [35]. Two natural products, resveratrol
(3,5,4’-trihydroxy-trans-stilbene), present in grapes and nuts, and
soybean B-group triterpenoid saponins have been reported to
induce autophagy [47].
Inhibition of autophagy has been suggested as a therapeutic
strategy for chronic myelogeneous leukemia that is refractory to
imatinib [48]. Some clinical trials are in the way to test the efficacy
of hydroxychloroquine in combination with several anti-cancer
agents [42, 49]. In glioma, expression levels of Beclin 1 are
inversely proportional to the tumor grade and correlate with
enhanced survival of glioblastoma multiforme patients, which
indicates that inducing autophagic cell death amplifies the response
to therapy that may have prognostic importance [45]. In any case, it
is still necessary to establish the clinical utility of autophagy
inhibitors, and of autophagy induction to gain insights into the
clinical interest of targeting autophagy in patients. Because
autophagy and apoptosis share common stimuli and signaling
pathways, the final fate of cancer cells would therefore depend on
the cell response [32, 38, 45].
SENESCENCE
Cell senescence is broadly defined as a physiological program
of terminal growth arrest, which can be triggered by alterations of
telomeres or by different forms of stress. Shortening of telomeres to
a certain limit results in cell cycle arrest, sometimes referred to as
replicative senescence [50]. Under certain circumstances, tumor
cells can be readily induced to undergo senescence by genetic
manipulations or by treatment with chemotherapeutic agents [51],
which may occur in the absence of telomere shortening. Senescence
is known to contribute to the outcome of cancer therapy [52].
Although senescent cells do not proliferate, they are metabolically
active and produce secreted proteins with both tumor-suppressing
and tumor-promoting activities [51].
The induction of the lysosomal senescence-associated -
galactosidase activity (SA- -gal, a surrogate marker of senescence
[51]) by anti-cancer agents correlates partly with the functional p53
status [53, 54]. In the presence of functional p53 and p16, DNA
damage signals may result in replicative senescence. In fact, a large
number of genes involved in cell cycle regulation are also
implicated in the control of senescence, including p53, p21WAF1, Rb
and p16 [51, 55].
A pathway that comprises DNA-damage, senescence, and
faulty mitosis followed by the generation of aneuploid cells, is
referred to as neosis [56]. Neosis is defined as a parasexual somatic
Fig. (2). Induction of autophagy is activated by Beclin1 and its interacting partner class III PI3-k (phosphatidylinositol-3 -kinase). This pathway is negatively
regulated by class I PI3-k through mTOR. Induction of autophagy requires conjugation of Atg12 and Atg5, the recruitment of LC3-II and the formation of the
autophagosome. In order to accomplish degradation of the autophagosome and its contents, the autophagosome is transported to and fuses with lysosomes,
generating the autophago-lysosome. Within the autophago-lysosome, lysosomal proteases degrade the inner autophagosomal membrane and contents. Strategies for modulating autophagy are discussed in the main text.
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Targeting Cell Death Pathways Anti-Cancer Agents in Medicinal Chemistry, 2012, Vol. 12, No. 3 231
reduction division followed by the production of aneuploid cells via
nuclear budding [56, 57]. This nuclear budding can generate both
drug-sensitive and -resistant cells [57].
Senescence may have conflicting side-effects such as growth
stimulation of nonsenescent tumor cells, and de novo carcino-
genesis, which can be exacerbated, at least in part, by cyclin kinase
inhibitors [51, 58]. On the basis of these considerations, it has been
suggested that senescence-oriented therapeutics should include two
general strategies. These are the developing of agents that will
interfere with the induction of disease-promoting genes by cyclin
kinase inhibitors, and, as second strategy, obtaining molecules
aimed at inducing tumor cell senescence without up-regulating
p21WAF1
(which, unlike p16, is almost never inactivated in tumors
[51, 58]). Escape from senescence during oncogenesis has been
linked to inactivation of p53 or p16 [55]. However the requirement
for p53 in the senescence of human tumor cells might be less strict
than it appears to be in mice, while p16 may contribute to
senescence in human cells if this gene is not inactivated [58].
TARGETING SENESCENCE
Activating senescence is a frequent effect of many chemo-
therapeutic agents both in vitro and in mouse tumor xenografts [18,
51], and the propensity of tumor cells to undergo senescence
in response to damage has been demonstrated by the analysis of
the effects of chemotherapeutic drugs in different types of
human solid tumor cells [53, 59]. A wide variety of anticancer
agents might induce senescence-like morphological changes and
SA- -gal expression in tumor cells. Senescent phenotype is
observed upon treatment with several DNA-binding drugs like
doxorubicin and cisplatin. Such induction of senescence appears to
be dose-dependent, and it can be detected even at the lowest drug
concentrations that have a measurable growth-inhibitory effect.
Studies using human cancer cells have demonstrated that
senescence may be achieved by using relatively low concentrations
of antitumor drugs as compared to those required for apoptotic cell
death [11, 60, 61]. Senescence has been found to be induced by
chemotherapy in clinical samples of breast cancer [62]. However,
the ability of tumor cells to escape such low level of toxic stress and
become drug resistant must be also kept under control [57].
The anthracycline doxorubicin induces senescence in p16-
deficient HCT116 colon carcinoma cells that is associated with the
induction of several tumor suppressor genes, p21WAF1
among them.
There is, however, an undesirable effect upon drug-induced
senescence consisting of the induction of mitogenic, anti-apoptotic
and angiogenic secreted factors, which may have adverse effects.
Regulatory pathways stimulated by p21WAF1
can largely be at the
origin of the expression of these disease-associated genes in
senescent cells [58]. Further research should be directed towards a
clearer understanding of how chemotherapeutic agents induce
cellular senescence, and the identification of drug targets that could
be used in combination with other chemotherapeutic agents to
facilitate irreversible growth arrest. The ultimate goal is to reach
satisfactory levels of drug effectiveness with less toxic effects [63].
Several therapeutic strategies have been developed to exploit
the fact that a diversity of tumors display high levels of telomerase
activity [64]. The telomerase antagonist, imetelstat —a 13-mer thio-
phosphoramidate oligonucleotide complementary to the RNA
template region of human telomerase RNA— shows high resistance
to nuclease digestion in blood and tissues. It efficiently targets
glioblastoma tumor-initiating cells leading to decreased proliferation
and tumor growth. Long-term treatment with imetelstat leads
to progressive telomere shortening, and eventually cell death
[64]. Among the telomerase inhibitors under development are
also hammerhead ribozymes, which cut the RNA component of
telomerase, and some drugs that interact with DNA quadruplexes
[65].
MITOTIC CATASTROPHE
The term mitotic catastrophe is used to describe cell death
occurring during or shortly after dysregulated or failed mitosis,
which can be accompanied by morphological alterations such as
multinucleation or micronuclei [7, 66, 67]. Sometimes mitotic
catastrophe is considered as an abnormal (faulty) mitosis leading to
cell death, rather than a form of cell death [14, 68, 69]. Mitosis is a
central episode in cell proliferation that results in a doubling of the
number of cells. DNA damage activates p53 in the G1 phase of the
cell cycle, which in turns activates the expression of p21WAF1
. The
induction of p21WAF1
remains the main mechanism underlying G1
arrest after DNA damage, and it has been associated with both
transient and permanent forms of growth arrest [51]. In tumor cells
lacking active forms of p53, p21WAF1
overexpression has to follow
p53-independent routes [51]. Enhanced p21WAF1
expression leads to
cell growth arrest in G2 after DNA damage, by inhibiting the
activity of cyclin-dependent kinases [70, 71]. The inhibition or
knockout of genes in the p53 pathway, including p21WAF1
and 14-3-
3- , would facilitate entry into mitosis [70]. Therefore, mitotic
catastrophe is associated with the inability of the different cell cycle
checkpoints to arrest progression into mitosis and suppress
catastrophic events until repair has been achieved [67].
Failure to undergo complete mitosis after DNA damage can
result in polyploidy, as it is observed when tumor cells undergo
treatment with several anti-cancer agents or after radiotherapy, with
several cells bearing two nuclei or multinuclei [60, 66, 70, 72, 73].
Moderate DNA damage activates p53, and wild-type p53 appears to
promote two antiproliferative responses: apoptosis and senescence,
while it inhibits mitotic catastrophe [7]. Because tumor cells
are frequently deficient in factors controlling the cell-cycle
checkpoints, particularly functional p53, they may be predisposed
to mitotic catastrophe after chemotherapy [74]. Mitosis involves
dramatic changes in multiple cellular components, leading to
a major reorganization of the entire cell structure. During G2,
the A-type cyclins are degraded whereas the B-type cyclins are
actively synthesized. Mitotic events are initiated by activation
of the complex formed by Cdk1 (cdc2) and cyclin B (Fig. (3A)).
Chk1 and Chk2 link the monitoring of DNA integrity to the
cell-cycle molecules involved in mitosis, contributing to proper
timing of the initial steps of cell division, including mitotic spindle
formation [75]. Chk1 can phosphorylate Cdc25 and prevent it from
dephosphorylating and activating Cdk1 (Fig. (3B)). Chk1 and Chk2
phosphorylate Cdc25C and prevent it from dephosphorylating and
activating Cdk1. Chk2 is considered a negative regulator of mitotic
catastrophe [76] since it prevents premature activation of the cyclin
B-Cdk1 kinase complex [75].
The alignment of chromosomes on the metaphase spindle, as
well as the attachment of kinetochores to microtubules of the
mitotic spindle during the metaphase is monitored by the mitotic
spindle checkpoint. If anaphase is initiated before both kinetochores
of a replicated chromosome become attached to microtubules from
opposite spindle poles, daughter cells are produced that will contain
missing or extra chromosomes [77]. Activation of the anaphase-
promoting complex (APC) is induced by the cyclin B-Cdk1
complex at the beginning of mitosis [78]. Finally, the inactivation
of the cyclin B-Cdk1 complex is needed to exit from mitosis.
Furthermore, prolonged inhibition of APC, which results into
prolonged Cdk1 activation, can result in mitotic catastrophe
associated with centrosome overduplication [79]. The activity of the
polo-like kinase 1 (PLK1) –a member of the family of human polo-
like kinases- is fundamental for the precise regulation of cell
division, and the maintenance of genomic stability [80]. Although
PLK1 might promote mitotic entry, its main role is the control of
mitotic progression, first and foremost the regulation of proteins
involved in the metaphase-anaphase transition and mitotic exit. In
addition to its contribution to the spindle checkpoint, PLK1 may
232 Anti-Cancer Agents in Medicinal Chemistry, 2012, Vol. 12, No. 3 Mansilla et al.
participate in the control of mitotic exit by phosphorylating some
APC subunits.
Aurora kinases, a family of mitotic regulators, are expressed
in proliferating cells and overexpressed in some tumor cells,
thereby constituting potential anti-cancer targets [81, 82]. Aurora
kinases are regulated by phosphorylation and ubiquitin-dependent
degradation and they are required for multiple aspects of mitosis.
Aurora A localizes to centrosomes/spindle poles and is required for
spindle assembly, whereas Aurora B is required for phosphorylation
of histone H3, chromosome segregation and cytokinesis. While
Aurora A binds to centrosomes and the spindle apparatus from the
prophase until the telophase, Aurora B is present in post-mitotic
bridges during the telophase [83].
Both lethal and cytoprotective signals can be generated during
mitotic arrest [69]. Apoptosis could be initiated during mitotic
arrest by the timed degradation of an inhibitor of apoptosis that acts
upstream of caspase-9 [69]. A candidate to be degraded is Mcl-1, a
member of the Bcl-2 family [84]. Initiation of apoptosis during a
prolonged mitotic arrest is determined by Mcl-1 instability, which
is controlled by a mechanism distinct from that operating in
interphase. Stabilization of Mcl-1 would make cells resistant to
apoptosis induced by prolonged mitotic arrest. Besides, the
dynamics of mitotic catastrophe induced by DNA-damaging agents
in p53-deficient cancer cells has already been characterized [68].
Cells entering mitosis with damaged DNA have been observed to
arrest transiently at the metaphase for more than 10 h without
segregation of chromosomes, subsequently dying from metaphase
[68, 79]. In metaphase-arrested pre-catastrophic cells, the anaphase-
promoting complex appears to be inactivated, while the spindle
checkpoint is activated after DNA damage.
Although triggering apoptosis during mitotic arrest is unlikely
to be regulated by transcriptional induction, it can be affected by
the extensive shutdown of transcription during it [85]. If cells that
are halt in mitosis are not committed to dying by apoptosis or
necrosis, mitotic slippage might occur [85, 86] that can result in
micronucleation [4, 86] (Fig. (4)), which seems to depend on cyclin
B degradation. Although mitotic slippage keeps the cells away from
apoptosis, they can still die, yet preferentially via non-apoptotic
routes. The final fate of those cells seems to be determined by the
cell context [67]. Hence mitotic catastrophe in apoptotic competent
cells is usually followed by apoptotic-like cell death after mitotic
slippage, but usually there is a mixture of necrosis and apoptosis
that arises during mitosis or after multinucleation [4, 87]. When
mitotic catastrophe does not end in cell death, a population of
aneuploid cells may emerge, which can contribute to tumorigenesis
[69].
TARGETING MITOTIC CATASTROPHE
Pharmacological inhibition or genetic suppression of several
G2/M checkpoint genes can promote mitotic catastrophe [7, 67, 70,
73, 88, 89]. A plethora of DNA-damaging agents, as well as
radiation, induce mitotic catastrophe [10, 66, 72, 90]. There are data
on how some drug concentration used to treat cancer cells may
elicit a mitotic catastrophe response, while higher concentrations
would induce apoptotic cell death, especially in p53-competent
cells [11, 60, 73]. Activation of caspase-2 and caspase-3 is observed
in MDA-MB231 cells treated with the anthracycline doxorubicin,
but these protease activities are neither observed in drug-treated
MCF-7/VP cells [73] nor in Jurkat T cells treated with bis-
anthracycline WP631 [91]. Altogether, these observations imply
that differences in the contribution of caspase-dependent and
caspase-independent processes to cell death depend on both the
cytotoxic agent used and the cell type, and that activation of
caspases is not mandatory for the occurrence of cell death via
mitotic catastrophe (Fig. (4)). It seems that in apoptotic competent
cells mitotic catastrophe can be followed by apoptosis, although
apoptosis is not required for mitotic catastrophe to occur [7, 73].
In general, treatment strategies that induce DNA damage with
inhibition of its repair can induce entry into mitosis of cells bearing
damaged DNA, leading to mitotic catastrophe [92]. Intriguingly,
mitotic catastrophe induced by DNA damage appears to be
enhanced in the presence of HDAC inhibitors [93].
Drugs that target mitotic spindle assembly are commonly
used to treat a variety of human cancers [94]. Several Vinca
alkaloids (for example, vincristine and vinblastine) depolymerize
microtubules and prevent the attachment of kinetochores to spindle
Fig. (3). Control of the G2/M checkpoint. (A) Cyclin B forms complexes with Cdk1 (cdc2). Cdk1 undergoes activating (Thr 161) and inactivating (Tyr 15 and
Thr 14) phosphorylations. Dehosphorylation of Thr 14 and Tyr 15 activates the G2 to mitosis transition. The Cyclin B-Cdk1 interaction is abrogated toward
the end of mitosis by proteolysis of Cyclin B. (B) Several checkpoint proteins recognize damaged DNA, activating the Chk1 protein kinase, which
phosphorylates and inhibits Cdc25, a phosphatase required to activate Cdk1, thus preventing entry into mitosis with unrepaired DNA. Chk 1 also activates
Wee1 and Mik1, which can phosphorylate aminoacids Tyr15 and Thr 14 of Cdk1, thus keeping the kinase activity of Cdk 1 low to prevent entry into mitosis
upon DNA damage. Inhibition of Chk1 can result in unscheduled entry into mitosis and mitotic catastrophe.
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Targeting Cell Death Pathways Anti-Cancer Agents in Medicinal Chemistry, 2012, Vol. 12, No. 3 233
microtubules resulting in an inhibition of chromosome alignment
during mitosis. In contrast, taxanes and epothilones stabilize
microtubules and suppress the dynamics of the mitotic spindle
resulting also in an inhibition of chromosome alignment. Treatment
with those spindle-damaging drugs leads to the activation of the
spindle checkpoint and to an arrest during mitosis [74, 95]. Studies
on the microtubule-stabilizing agent paclitaxel (Taxol) have identified
numerous cellular and molecular effects, such as induction
of cytokines and tumor-suppressor genes, indirect cytotoxicity
due to secretion of tumor necrosis factor, large activation of
signal-transduction pathways and selective activity against cells
lacking functional p53 [10]. Paclitaxel induces mitotic arrest and
cytotoxicity at clinically relevant concentrations, as well as the
immediate activation of tyrosine kinase pathways and the activation
of gene expression at much higher concentrations. Toxicity is a
major concern when using these anti-mitotic drugs because they
also affect the division of normal cells, causing myelosuppression
[74]. Docetaxel (Taxotere) is a microtubule-stabilizing taxane,
which has higher antitumor activity than paclitaxel [96]. It has been
approved for the clinical treatment of breast and prostate cancers, in
which, following androgen depletion therapy, docetaxel has shown
to be quite effective [97].
A different approach to activate mitotic catastrophe has
centered its attention on proteins that drive the mitotic machinery,
which includes the discovery of inhibitors of the kinesin spindle
proteins (KSP) like ispinesib (Table 1), which might be used to
treat taxane-refractory tumors. Several inhibitors of KSP have
progressed into clinical trials and many others are in preclinical
development [98].
The staurosporine analogue UCN-01 abrogates DNA damage–
induced G2 arrest and selectively sensitizes p53 mutant cells to
radiation [99]. UCN-01 targets Chk1, and it appears to be a useful
therapeutic target to induce enhanced cytotoxicity in response to
DNA damage, as well as mitotic catastrophe [100]. Nevertheless,
UCN-01 also potently inhibits other kinases including Chk2 and
several cyclin-dependent kinases. Hence, the clinical effects of this
molecule cannot be considered to predict the effects that might be
seen with more specific inhibitors. A number of clinical trials with
UCN-01 in combination with a variety of DNA-damaging therapies
are on the way [99, 101]. The inhibition of Chk1 enhances the
toxicity of hydroxyurea [102], a result supporting combined
therapies using Chk1 inhibition and drugs that can induce stalled
replication forks such as gemcitabine (a nucleoside analogue), 5-
fluorouracil, and hydroxyurea. Chemotherapeutic treatments that
combine DNA damaging agents with G2 checkpoint inhibition
by UCN-01 are in clinical trials [103]. The sequential treatment
with DNA damaging agents and UCN-01 can force cells to enter a
faulty mitosis, thus augmenting cell death compared to treatments
with DNA-damaging agents alone [95, 100]. Nevertheless, although
clinical development of UCN-01 has overcome many initial
obstacles, this compound has failed to show a high level of clinical
activity when combined with chemotherapeutic agents [104].
The development of inhibitors of checkpoint kinases and
cyclin-dependent kinases is a growing field in the seeking of novel
anti-cancer agents [99, 103, 105, 106]. It seems that abrogation of
DNA damage–induced checkpoints and the induction of mitotic
catastrophe potentiate the effects of radiotherapy and chemotherapy
[95]. Several Chk1/Chk2 inhibitors are in clinical trials (Table 1).
XL-844, AZD7762 and PF-00477736 are molecules that represent
different chemical classes from both UCN-01 and each other. Every
single one is a potent inhibitor of both Chk1 and Chk2, and they
abolish DNA damage–induced cell cycle arrest. PF-00477736
Fig. (4). Schematic representation of the relationship between mitotic catastrophe and apoptosis or necrosis. Pathways leading to mitotic catastrophe and
apoptotic, or necrotic, cell death after DNA damage are represented. Several key events occurring after a faulty mitosis are presented. DNA damage, especially
in p53-deficient cells, can result in cell death occurring during mitosis, or after mitotic slippage. Molecules that facilitate entry into mitosis with damaged
DNA would facilitate mitotic catastrophe events that commit cells to dying.
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234 Anti-Cancer Agents in Medicinal Chemistry, 2012, Vol. 12, No. 3 Mansilla et al.
abrogates the arrest in S phase by gemcitabine, allowing cells to
progress through the G2 checkpoint [92], committing them to
mitotic catastrophe. The strong response seen in combination with
gemcitabine is consistent with that abrogation of DNA damage–
induced checkpoints will enhance the effects of radiotherapy and
chemotherapy. In p53-defective cells, it has been suggested that
Chk1 inhibitors can abrogate the various checkpoints and that this
would lead to preferential sensitization to chemotherapy over cells
bearing will-type p53 [13]. However, there are some reports
indicating that the status of p53 could not predict the efficacy of
Chk1 kinase inhibitors combined to DNA damaging agents [107].
Chk1 modulation in tumor samples from patients in clinical trials
is still to be fully demonstrated. Validation of target inhibition
in clinical studies involving Chk1 inhibitors presents a unique
challenge, as it requires demonstration of successful suppression of
an activated Chk1-mediated signaling event by the inhibitor [103].
While it has been shown that Chk1 inhibition potentiates the
efficacy of various DNA-damaging therapies, the context for
selective Chk2 inhibition is not well defined yet [108].
Several inhibitors of the cyclin-dependent kinases, such as
flavopiridol and roscovitine, have been characterized in the last
years in pre-clinical and clinical trials. Roscovitine shows a potent
and selective inhibition of Cyclin B, Cyclin A and Cyclin E by
competing for the ATP binding domain of the kinases, while it does
not affect cyclins D1 and D2 significantly. In general, the agents
that inhibit cyclin-dependent kinases are unselective, targeting
kinases other than those of the cell cycle, causing considerable
toxicity [105].
In view of the critical role Aurora kinases play in mitosis, and
the overexpression of both Aurora-A and Aurora-B in tumor cells
described above, they are considered outstanding targets for
anticancer therapy. Three Aurora-kinase inhibitors have recently
been described: ZM447439, hesperadin and VX-680 (Table 1).
These three chemotherapeutic agents inhibit phosphorylation of
histone H3 on serine 10, a marker of mitosis, and can inhibit cell
division. However, they do not inhibit cell-cycle progression.
Cells bearing mutant p53 might be more sensitive to Aurora
kinase inhibitors, thereby providing an advantage for healthy tissues
over tumor cells [109]. AZD1152 is a selective inhibitor of Aurora
B kinase activity. The efficacy and the toxicity of AZD1152 alone
and in combination with gemcitabine have been examined using
pancreatic tumor xenografts [110]. Inhibiting Aurora-A leads
to arrest in G2/M phase, abnormal mitotic spindle formation,
the appearance of tetraploid cells —all of them symptoms of
mitotic catastrophe—, and ultimately apoptosis [109]. VX-680 is a
powerful inhibitor of the Aurora kinases, with inhibition constant
values (Ki(app)) of 0.6, 18 and 4.6 nM for Aurora A, Aurora B and
Aurora C, respectively [109]. VX-680 shows a greater than 100-
fold in vitro selectivity for Auroras over a panel of 55 other kinases
[109], and it is in phase II clinical trials (Table 1).
Inhibition of Aurora A enhances the cytosine arabinoside-
induced mitotic catastrophe in leukemia cells [111]. Interestingly,
inhibition of Aurora A kinase by MLN8054 results in senescence
both in vitro and in vivo [112]. This example of chemotherapy-
induced senescence adds to the list of chemotherapies described
above to induce senescence.
Several PLKs (polo kinase) inhibitors are in phase I or II
clinical studies [113]. The fundamental role of PLK1 in mitosis [79,
80], as well as the current progress in generating selective
compounds is expected to provide us with a collection of novel
PLK1 inhibitors [113]. Data on the clinical evaluation of PLK1
inhibitors has been generated during the past few years. This
includes studies on BI2536, and ON01910 (Table 1). Whereas
BI2536 is an ATP-competitive inhibitor, ON01910 is not, but it
competes for the substrate binding-site of the enzyme. ON01910
produces mitotic arrest, and it also shows strong synergy with other
anti-cancer agents, often inducing complete regression of tumors
[114].
NECROSIS AND NECROPTOSIS
Necrosis describes a cellular response to severe and massive
toxic insults associated with infection, inflammation or ischemia, as
well as cellular energy depletion or nutrient starvation [4, 115]. It is
poorly defined at the molecular level and it is usually referred to as
a type of cell death that is uncontrolled and pathological [4, 18], but
there are new grounds for considering this type of cell death is
under certain cellular control [115]. Necrotic cells display increased
cytoplasmic vacuolization, organelle degeneration, and damage to
membrane lipids with cell swelling and rupture, and induction of
inflammation due to the release of cellular contents [116].
Necroptosis is used to designate one particular type of programmed
necrosis that depends on the serine/threonine kinase activity of
RIP1 (receptor-interacting protein 1) [14, 117]. Genome-wide
screens combined with multiple in silico analyses have outlined a
cellular signaling network that regulates necroptosis and the
molecular bifurcation between necroptosis and apoptosis [117,
118]. Necrosis and necroptosis represent different modes of cell
death that eventually end in similar cellular morphology that
includes rounding of the cell, cytoplasmic swelling, rupture of the
plasma membrane and spilling of the intracellular content [115,
119]. The RIP1 kinase can be activated by several stimuli including
TNF, TRAIL, or DNA damage (the latter via poly-ADP-ribose
polymerase). RIP1 can transduce signals to mitochondria and cause
mitochondrial permeability transition. Afterwards, mitochondrial
collapse would activate some proteases and phospholipases, leading
to plasma membrane destruction, a known hallmark of necrotic cell
death [120].
Necrosis in response to DNA damage requires activation of the
DNA repair protein poly(ADP-ribose) polymerase, but this
activation is not sufficient to determine the fate of cells [121].
Besides, induction of apoptosis or mitotic catastrophe can be
accompanied by necrosis [11, 60, 73]. Necrosis has been observed
as the final step in radiation-induced mitotic catastrophe [66, 90],
and after treatment with certain doses of antitumor drugs [11, 18,
73, 96], but it is not necessarily the final step of mitotic catastrophe,
particularly when caspase-3 or caspase-2 remain functional
(Fig. (4)) [67, 69].
TARGETING NECROSIS AND NECROPTOSIS
Given that the late step in cell death through mitotic catastrophe
is sometimes similar to necrosis, with absence of caspase activities
and high cell staining with propidium iodide [60, 73], the induction
of necrosis might be a key feature in the antitumor activity of a
variety of drugs [4].
Necrotic death in cancer cells has been observed after
photodynamic treatment (PDT) (Table 1). Hypericin, a natural plant
pigment, is prominent among photosensitizers. Hypericin would
exert its phototoxicity through mechanisms that implicate key
proteins and organelle membranes, leading to cell death, which
occurs by the induction of apoptosis and/or necrosis [122]. It seems
that activation of photosensitizers on lysosomes may disrupt the
lysosomal membrane and result in the release of lysosomal
proteases leading to necrosis.
The pharmacological or genetic inhibition of several key
enzymes has been shown to deeply affect the execution of
programmed necrosis. These include RIP1, cyclophilin D, and the
poly(ADP-ribose) polymerase 1 (PARP-1) [118]. Cell death in
response to DNA damage is neither impeded by Bax/Bak
deficiency nor by p53 deficiency, which suggests that there are
explanations other than apoptosis for the ability of DNA-damaging
agents to selectively kill tumor cells. A tentative explanation is the
occurrence of poly(ADP-ribose) polymerase-mediated necrosis. In
Targeting Cell Death Pathways Anti-Cancer Agents in Medicinal Chemistry, 2012, Vol. 12, No. 3 235
this context, it seems interesting to determine whether tumors
resistant to DNA alkylating agents have acquired loss of or altered
poly(ADP-ribose) polymerase activity [121].
Necroptosis participates in the pathogenesis of some diseases,
including ischemic injury and neurodegeneration, representing a
target for the avoidance of unwarranted cell death. At first glance,
this represents a completely different field in therapy. While
committing cells to dying by necrosis can be of clinical interest in
cancer, or at least a final step in other forms of cell death, as
described above, it is supposed that necroptosis is a process to be
inhibited rather than enhanced given its role in normal cell
degeneration and death. RIP1 represents the molecular target of a
class of cytoprotective agents, the necrostatins [119, 123]. In
ischemia, a condition that elevates necrotic stimuli such as reactive
oxygen species and Ca2+
overload, necrostatins have been shown to
confer in vivo neuroprotection [119].
Unlike apoptosis, necrosis elicits a pro-inflammatory response
[7]. This inflammatory response can recruit cytotoxic immune
cells to the tumor location, thereby increasing the efficacy of
chemotherapy. Unfortunately, an inflammatory response might also
damage normal tissues, or induce the production of mitogenic or
pro-survival cytokines, activate signaling pathways promoting cell
outgrowth, and even induce cell migration and associated tumor
cell metastasis [18].
CONCLUSIONS
Accumulated evidence indicates that response to chemotherapy
is not limited to apoptosis but includes other forms of cell death [3,
4, 7, 18, 32]. As a new therapeutic strategy, alternative types of cell
death might be exploited to control and eradicate cancer cells.
Autophagy, senescence, mitotic catastrophe and necrosis can be
efficiently induced by a variety of anti-cancer agents. Several
agents targeted to specific components of the cell death machinery
are now entering clinical trials. They may target more than a single
cell death pathway.
Nowadays, a common therapeutic approach is to employ
rational combinations of “target-specific” agents such as those
displayed in Table 1 and conventional DNA-damaging agents or
radiation. It has been observed that certain agents can trigger both
apoptosis and autophagy cell death routes simultaneously [5].
Nonetheless, the generalized use of such combined strategies has
been criticized because, as quoted in Ref. [124], they could be seen
as that “pharmaceutical companies developing new drugs do not
seem to fully rely in the capacity of their drugs given the now
frequent testing of the drugs in combination with standard cytotoxic
drugs”. Needless to say, this sounds a bit excessive since preclinical
evidences supports the concomitant inhibition of multiple pathways
or the activation of cell death pathways, given that single-agent
therapy may be not sufficient to control tumor growth. Targeting of
multiple pathways may be a successful strategy to deal with tumor
heterogeneity and to overcome drug resistance of tumor cells [15],
while it is likely that chemotherapy using a single agent may be not
sufficient to induce cancer cell death.
Loss of wild-type p53 activity is thought to be a major predictor
of failure to respond to radiotherapy and chemotherapy is several
human cancers [4]. Tumors with mutated p53 can be more
anaplastic, have a higher proportion of proliferating cells, be more
metastatic, and, in general, have a more aggressive phenotype than
similar tumors with wild-type p53. This can lead to a worse
prognosis for patients whose tumors have mutated p53 independent
of treatment sensitivity. Therefore, restoring wild-type p53
signaling in cancer is a therapeutic strategy currently in clinical
trials. It is noteworthy that apoptosis and senescence are regulated
largely by wild-type p53, while mitotic catastrophe is not [51, 67].
In view of the fact that apoptosis can frequently be inactivated due
to a non-functional p53, the commitment of cells to mitotic
catastrophe can be considered advantageous in cancer treatment [3].
Besides, the clinical utility of autophagy inhibitors, and of
autophagy induction, requires more data about targeting autophagy
in patients.
Furthermore, the availability of a variety of molecularly-
targeted agents that may elicit a cytostatic rather than cytotoxic
response, such as inhibitors of cyclin-dependent kinases, demands a
better understanding of delayed forms of cell death in order to
improve of the treatment with these promising new agents that
are normally associated to treatment with DNA-damaging
agents, which can more ‘directly’ activate cell death pathways.
Chemotherapeutic treatment with DNA damaging drugs together
with agents that inhibit the G2 checkpoint, such as checkpoint
kinase inhibitors, can force cells to undergo mitotic catastrophe,
thus augmenting cell death compared to treatments with DNA-
damaging agents alone [95, 100].
CONFLICT OF INTEREST
Declared none.
ACKNOWLEDGEMENTS
This work was supported by grant BFU2010-15518 from the
Spanish Ministry of Science and Innovation, and the FEDER
program of the European Community, and it was performed within
the framework of the Xarxa de Referencia en Biotecnologia of the
Generalitat de Catalunya. We apologize to authors whose work has
not been included.
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Received: September 20, 2011 Revised: November 21, 2011 Accepted: November 21, 2011
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