Anti-Cancer Agents 2012

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: [email protected]

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

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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.


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].


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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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


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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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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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


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

Anti-Cancer Agents 2012

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