**1. Introduction**

110 Apoptosis and Medicine

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[211] Yu L, Alva A, Su H, Dutt P, Freundt E, Welsh S, et al. (2004) Regulation of an ATG7 beclin 1 program of autophagic cell death by caspase-8. Science ;304: 1500-1502. [212] Yu L, Wan F, Dutta S, Welsh S, Liu Z, Freundt E, et al. (2006) Autophagic programmed cell death by selective catalase degradation. Proc.Natl.Acad.Sci.U.S.A ;103: 4952-4957. [213] Lum JJ, DeBerardinis RJ, Thompson CB (2005) Autophagy in metazoans: cell survival

[214] Wu YT, Tan HL, Huang Q, Kim YS, Pan N, Ong WY, et al. (2008) Autophagy plays a protective role during zVAD-induced necrotic cell death. Autophagy.4: 457-466. [215] Amaravadi RK, Thompson CB (2007) The roles of therapy-induced autophagy and

[216] Lieberthal W, Triaca V, Levine J (1996) Mechanisms of death induced by cisplatin in proximal tubular epithelial cells: apoptosis vs. necrosis. Am.J.Physiol ;270: F700-F708. [217] Healy E, Dempsey M, Lally C, Ryan MP (1998) Apoptosis and necrosis: mechanisms of cell death induced by cyclosporine A in a renal proximal tubular cell line. Kidney Int.54:

[218] Chan FK, Shisler J, Bixby JG, Felices M, Zheng L, Appel M, et al. (2003) A role for tumor necrosis factor receptor-2 and receptor-interacting protein in programmed

[219] Cho DH, Jo YK, Hwang JJ, Lee YM, Roh SA, Kim JC (2009) Caspase-mediated cleavage of ATG6/Beclin-1 links apoptosis to autophagy in HeLa cells. Cancer Lett.274: 95-100. [220] Djavaheri-Mergny M, Maiuri MC, Kroemer G (2010) Cross talk between apoptosis and autophagy by caspase-mediated cleavage of Beclin 1. Oncogene ;29: 1717-1719. [221] Hou W, Han J, Lu C, Goldstein LA, Rabinowich H (2010) Autophagic degradation of active caspase-8: a crosstalk mechanism between autophagy and apoptosis. Apoptosis is an essential part of the normal development. The homeostatic balance between cell proliferation and cell death rate is critical for maintaining normal physiological processes. Aberrant regulation of apoptotic cell death mechanisms is one of the hallmarks of cancer development and progression, and many cancer cells exhibit significant resistance to apoptosis signalling [1]. Triggering of apoptosis can be achieved via the activation of two distinct molecular pathways, the extrinsic or death receptor pathway or via the intrinsic or mitochondrial apoptotic cascades. Both pathways lead to the hierarchical activation of a family of cysteine proteases called caspases [2], that cleave a series of cellular substrates which induce changes including chromatin condensation, internucleosomal DNA fragmentation, membrane blebbing and cell shrinkage [3]. Extrinsic pathway is activated from outside the cell by proapoptotic ligands that interact with specialized cell surface death receptors, including CD95 and TNF-related apoptosis-inducing ligand (TRAIL) receptors [4]. After binding to receptors apoptosis is triggered by the intracellular formation of a death-inducing signalling complex (DISC) that consists of FAS-associated death domain (FADD) and procaspase-8 and 10 [5,6]. As a result, this protein complex activates procaspase-8 and 10 inside itself, hence triggering procaspase-3 to execute the apoptosis process [7]. The mitochondria (intrinsic) pathway is activated from inside the cell by severe cell stress, such as DNA or cytoskeletal damage, inducing mitochondrial outer membrane permeabilization and transcription or post-translational activation of BH3-only proapoptotic B-cell leukemia/lymphoma 2 (Bcl-2) family proteins [4]. This permeabilization allows the release of apoptogenic proteins, including cytochrome c and second mitochondria-derived

© 2012 Marchal et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Marchal et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

activator of caspase (Smac; also known as DIABLO), from the mitochondrial intermembrane space into the cytosol [8]. Cytochrome c assembles with apoptotic protease-activating factor-1 (Apaf-1) to activate caspase 9. This caspase, in turn, activates the effector caspases 3, 6, and 7, which carry out apoptosis [4]. Smac promotes caspase activation and apoptosis by neutralization of several IAP proteins, including XIAP, c-IAP1 and c-IAP2 [9,10]

Apoptosis as a Therapeutic Target in Cancer and Cancer Stem Cells: Novel Strategies and Futures Perspectives 113

molecule approach that is based on mimicking the IAP-binding motif of the endogenous IAP antagonist Smac [25]. Other therapies involve antisense strategies and short-interfering RNA (siRNA) molecules [26,27]. There is increasing interest in therapeutic drug

MicroRNAs (miRNAs) are small RNA gene products that regulate the activity of messenger RNAs by antisense base pairing. They are involved in stem-cell self-renewal, cellular development, differentiation, proliferation, and apoptosis [28]. In cancer, miRNAs function as regulatory molecules, acting as oncogenes or tumour suppressors [29] and they are strongly related to the apoptosis. New insights indicate that many miRNAs are antiapoptotic and mediate this effect by targeting pro-apoptotic mRNAs or positive regulators of pro-apoptotic mRNAs [30]. Conversely, many pro-apoptotic miRNAs target antiapoptotic mRNAs or their positive regulators [31]. Therefore, their inhibition leads to the induction of programmed cell death, suggesting a promising miRNA treatment for cancer. Several drugs may have the ability to modulate the expression of miRNAs by targeting signalling pathways that ultimately converge on the activation of transcription factors that

Evasion of apoptosis is one of the main mechanisms involved in tumourigenesis and drug resistance. Most cancers have a small population of tumour cells, as few as 1%, with stem cell characteristics and the capacity for self-renewal, termed cancer stem cells (CSCs). A malignant tumour can be viewed as an abnormal organ in which small populations of tumourigenic CSCs have escaped the normal limits of self-renewal, giving rise to abnormally differentiated cancer cells that contribute to tumour progression and growth [32]. These CSC express high levels of ATP-binding cassette (ABC) drug transporters, providing for a level of resistance [33]; are relatively quiescent; have higher levels of DNA repair and a lowered ability to enter apoptosis . Several cancer therapy approaches targeting ABC transporters and increasing apoptosis could be employed to selectively and more

The current review will focus on recent development of several therapeutic strategies, which interfere with apoptosis and are currently used or tested for treatment of cancer. They induce cancer cell death or enhance the responsiveness of cancer cells and CSCs to certain cytotoxic drugs. Some of them such as caspases activators, indirectly modulators of apoptosis or agents targeting apoptosis-related proteins are still in their preclinical or clinical trials. We also include future approaches directed to target apoptotic pathways with

The main goal in cancer therapy is the abrogation of tumour cell growth and proliferation, and ultimately the complete elimination of tumour cells. It is commonly accepted that tumour cells treated with anticancer agents undergo apoptosis, and that cells resistant to apoptosis often do not respond to anticancer therapy [34]. Moreover, it is widely demonstrated that some oncogenic mutations suppressing apoptosis may lead to tumour

development targeting the IAP family.

regulate miRNA encoding genes.

efficiently kill CSC.

promising application in patients with cancer.

**2. Novel apoptotic markers/targets in cancer** 

Chemotherapeutic agents act by inhibiting tumour cell proliferation and survival and most of them can kill tumour cells by activating common apoptotic pathways [11]. Therefore, apoptosis plays an important role in the treatment of cancer as it is a popular target of many treatment strategies. 5-fluorouracil (5-FU), an antimetabolite analogue of uracil employed primarily in the treatment of a variety of solid malignant tumours, leads to a wide range of biological effects which can act as triggers for apoptotic cell death [12,13]. However, resistance to the drug remains a major clinical problem. Given that many of the apoptotic regulators altered in multidrug resistant tumours have been identified, one new approach to therapy is to restore apoptotic potential through genetic or pharmacological methods [14]. Moreover, since defects in the mediators of apoptosis may account for chemo-resistance, the identification of new targets involved in drug-induced apoptosis is of main clinical interest. Recently, we have identified the ds-RNA-dependent protein kinase (PKR) as a key molecular target of 5-FU involved in apoptosis induction, in a p53 -independent manner. These results suggest the clinical importance that the PKR status could play in response to chemotherapy based on 5-FU. Moreover, the effectiveness of 5-FU cytotoxic activity induced by IFNα, especially in cancer cells expressing a mutated form or lacking p53, but with a functional PKR, might have relevant clinical application in patients [15] .

The increased knowledge of some of the molecular components of the apoptosis signalling pathways has paved the way for the generation of more specific agents that target one crucial signalling component. This has allowed a change in anticancer therapy trends, from classic cytotoxic strategies to the development of new non-harmful therapies which target the apoptosis response selectively only in tumour cells. Moreover, these strategies overcome the adverse effects associated with cytotoxic drugs and increase their anti-cancer activity. Novel antitumour drugs have been synthesised such as 5-FU *O,N*-acetals and benzo-fused seven-membered *O,N*-acetal in which the 5-FU moiety was changed for the naturallyoccurring pyrimidine base uracil, which induced cell cycle-mediated apoptosis in breast and colon cancer cells [16,17,18,19]. The mechanism of action of these drugs was mainly centred on positive apoptosis regulatory pathway genes, and the repression of genes involved in carcinogenesis, proliferation and tumour invasion. In addition, these drugs were more selective against tumour cells with lower toxic effects in non-tumour cells [20,21].

As over-expression of IAP proteins frequently occurs in various human cancers and has been linked to tumour progression, chemo-resistance and poor prognosis, it is not surprising that IAP proteins are considered to be attractive targets for improve outcomes for patients with solid tumours and hematologic malignancies [22,23]. IAPs are also attractive as therapeutic targets because their inhibition does not appear to be toxic to normal adult cells [24]. Several therapeutic strategies have been designed to target IAP, including a smallmolecule approach that is based on mimicking the IAP-binding motif of the endogenous IAP antagonist Smac [25]. Other therapies involve antisense strategies and short-interfering RNA (siRNA) molecules [26,27]. There is increasing interest in therapeutic drug development targeting the IAP family.

112 Apoptosis and Medicine

activator of caspase (Smac; also known as DIABLO), from the mitochondrial intermembrane space into the cytosol [8]. Cytochrome c assembles with apoptotic protease-activating factor-1 (Apaf-1) to activate caspase 9. This caspase, in turn, activates the effector caspases 3, 6, and 7, which carry out apoptosis [4]. Smac promotes caspase activation and apoptosis by

Chemotherapeutic agents act by inhibiting tumour cell proliferation and survival and most of them can kill tumour cells by activating common apoptotic pathways [11]. Therefore, apoptosis plays an important role in the treatment of cancer as it is a popular target of many treatment strategies. 5-fluorouracil (5-FU), an antimetabolite analogue of uracil employed primarily in the treatment of a variety of solid malignant tumours, leads to a wide range of biological effects which can act as triggers for apoptotic cell death [12,13]. However, resistance to the drug remains a major clinical problem. Given that many of the apoptotic regulators altered in multidrug resistant tumours have been identified, one new approach to therapy is to restore apoptotic potential through genetic or pharmacological methods [14]. Moreover, since defects in the mediators of apoptosis may account for chemo-resistance, the identification of new targets involved in drug-induced apoptosis is of main clinical interest. Recently, we have identified the ds-RNA-dependent protein kinase (PKR) as a key molecular target of 5-FU involved in apoptosis induction, in a p53 -independent manner. These results suggest the clinical importance that the PKR status could play in response to chemotherapy based on 5-FU. Moreover, the effectiveness of 5-FU cytotoxic activity induced by IFNα, especially in cancer cells expressing a mutated form or lacking p53, but with a

neutralization of several IAP proteins, including XIAP, c-IAP1 and c-IAP2 [9,10]

functional PKR, might have relevant clinical application in patients [15] .

selective against tumour cells with lower toxic effects in non-tumour cells [20,21].

As over-expression of IAP proteins frequently occurs in various human cancers and has been linked to tumour progression, chemo-resistance and poor prognosis, it is not surprising that IAP proteins are considered to be attractive targets for improve outcomes for patients with solid tumours and hematologic malignancies [22,23]. IAPs are also attractive as therapeutic targets because their inhibition does not appear to be toxic to normal adult cells [24]. Several therapeutic strategies have been designed to target IAP, including a small-

The increased knowledge of some of the molecular components of the apoptosis signalling pathways has paved the way for the generation of more specific agents that target one crucial signalling component. This has allowed a change in anticancer therapy trends, from classic cytotoxic strategies to the development of new non-harmful therapies which target the apoptosis response selectively only in tumour cells. Moreover, these strategies overcome the adverse effects associated with cytotoxic drugs and increase their anti-cancer activity. Novel antitumour drugs have been synthesised such as 5-FU *O,N*-acetals and benzo-fused seven-membered *O,N*-acetal in which the 5-FU moiety was changed for the naturallyoccurring pyrimidine base uracil, which induced cell cycle-mediated apoptosis in breast and colon cancer cells [16,17,18,19]. The mechanism of action of these drugs was mainly centred on positive apoptosis regulatory pathway genes, and the repression of genes involved in carcinogenesis, proliferation and tumour invasion. In addition, these drugs were more MicroRNAs (miRNAs) are small RNA gene products that regulate the activity of messenger RNAs by antisense base pairing. They are involved in stem-cell self-renewal, cellular development, differentiation, proliferation, and apoptosis [28]. In cancer, miRNAs function as regulatory molecules, acting as oncogenes or tumour suppressors [29] and they are strongly related to the apoptosis. New insights indicate that many miRNAs are antiapoptotic and mediate this effect by targeting pro-apoptotic mRNAs or positive regulators of pro-apoptotic mRNAs [30]. Conversely, many pro-apoptotic miRNAs target antiapoptotic mRNAs or their positive regulators [31]. Therefore, their inhibition leads to the induction of programmed cell death, suggesting a promising miRNA treatment for cancer. Several drugs may have the ability to modulate the expression of miRNAs by targeting signalling pathways that ultimately converge on the activation of transcription factors that regulate miRNA encoding genes.

Evasion of apoptosis is one of the main mechanisms involved in tumourigenesis and drug resistance. Most cancers have a small population of tumour cells, as few as 1%, with stem cell characteristics and the capacity for self-renewal, termed cancer stem cells (CSCs). A malignant tumour can be viewed as an abnormal organ in which small populations of tumourigenic CSCs have escaped the normal limits of self-renewal, giving rise to abnormally differentiated cancer cells that contribute to tumour progression and growth [32]. These CSC express high levels of ATP-binding cassette (ABC) drug transporters, providing for a level of resistance [33]; are relatively quiescent; have higher levels of DNA repair and a lowered ability to enter apoptosis . Several cancer therapy approaches targeting ABC transporters and increasing apoptosis could be employed to selectively and more efficiently kill CSC.

The current review will focus on recent development of several therapeutic strategies, which interfere with apoptosis and are currently used or tested for treatment of cancer. They induce cancer cell death or enhance the responsiveness of cancer cells and CSCs to certain cytotoxic drugs. Some of them such as caspases activators, indirectly modulators of apoptosis or agents targeting apoptosis-related proteins are still in their preclinical or clinical trials. We also include future approaches directed to target apoptotic pathways with promising application in patients with cancer.
