Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 150845, 23 pages http://dx.doi.org/10.1155/2014/150845

Review Article and Molecular Targeting Therapy in

Mohamed Hassan,1,2 Hidemichi Watari,2 Ali AbuAlmaaty,1 Yusuke Ohba,3 and Noriaki Sakuragi2

1 Biotechnology Program, Department of Zoology, Port Said University, Faculty of Science, Port Said 42521, Egypt 2 Department of Obstetrics and Gynecology, Graduate School of Medicine, Hokkaido University, Sapporo 060-8638, Japan 3 Department of Cell Physiology, Graduate School of Medicine, Hokkaido University, Sapporo 060-8638, Japan

Correspondence should be addressed to Mohamed Hassan; [email protected]

Received 4 February 2014; Accepted 11 May 2014; Published 12 June 2014

Academic Editor: Elena Orlova

Copyright © 2014 Mohamed Hassan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Apoptosis is the programmed cell death which maintains the healthy survival/death balance in metazoan cells. Defect in apoptosis can cause cancer or autoimmunity, while enhanced apoptosis may cause degenerative diseases. The apoptotic signals contribute into safeguarding the genomic integrity while defective apoptosis may promote carcinogenesis. The apoptotic signals are complicated and they are regulated at several levels. The signals of carcinogenesis modulate the central control points of the apoptotic pathways, including inhibitor of apoptosis (IAP) proteins and FLICE-inhibitory protein (c-FLIP). The tumor cells may use some of several molecular mechanisms to suppress apoptosis and acquire resistance to apoptotic agents, for example, by the expression of antiapoptotic proteins such as Bcl-2 or by the downregulation or of proapoptotic proteins such as BAX. In this review, we provide the main regulatory molecules that govern the main basic mechanisms, extrinsic and intrinsic, of apoptosis in normal cells. We discuss how carcinogenesis could be developed via defective apoptotic pathways or their convergence. We listed some molecules which could be targeted to stimulate apoptosis in different . Together, we briefly discuss the development of some promising cancer treatment strategies which target apoptotic inhibitors including Bcl-2 family proteins, IAPs, and c-FLIP for apoptosis induction.

1. Introduction the apoptotic signals help to safeguard the genomic integrity [3, 6, 7] while dysregulation of the apoptotic pathways may Apoptosis is a very tightly programmed cell death with dis- not only promote tumorigenesis but also render the cancer tinct biochemical and genetic pathways that play a critical role cell resistant to treatment. Thus, the evasion of apoptosis is in the development and homeostasis in normal tissues [1]. a prominent hallmark of cancer [8]. Cancer cells are, in fact, It contributes to elimination of unnecessary and unwanted harboring alterations that result in impaired apoptotic signal- cells to maintain the healthy balance between cell survival ing, which facilitates tumor development and metastasis [6– and cell death in metazoan [2, 3]. It is critical to animals 8]. especially long-lived mammals that must integrate multiple Here, we provide an overview of mechanisms by which physiological as well as pathological death signals. Evidence the main regulatory molecules govern apoptosis in normal indicates that insufficient apoptosis can manifest as cancer or cells and describe models of apoptotic dysregulation based autoimmunity, while accelerated cell death is evident in acute on alterations in their function that facilitate the evasion and chronic degenerative diseases, immunodeficiency, and of apoptosis in cancer cells. We will also briefly discuss the infertility. Under many stressful conditions like precancerous development of some promising cancer treatment strategies lesions, activation of the DNA damage checkpoint pathway basedontargetingtheapoptoticinhibitortostimulatethe can serve to remove potentially harmful DNA-damaged cells apoptoticsignals.Wewillshedsomelightonthisvery via apoptosis induction to block carcinogenesis [4, 5]. Thus, active field of endeavor that witnessed many breakthroughs 2 BioMed Research International over the last few years. Notably, insights from proposed 2.1. Induction of Apoptosis. Apoptosis is caused by pro- target molecules have included different portions of the death teases, known as “caspases,” which specifically target cysteine pathway depending on the type of cancer. Applications of aspartyl [17, 18]. Upon receiving specific signals instructing many of such targeted therapy in different cell types and the cells to undergo apoptosis a number of distinctive changes signals studied in them have emphasized selected control occurinthecell.Afamilyofproteinsknownascaspases points for each targeted therapy. is typically activated in the early stages of apoptosis. These proteins cleave key cellular components that are required 2. Apoptosis Defect and Cancer for normal cellular function including structural proteins in the cytoskeleton and nuclear proteins such as DNA repair Defects in programmed cell death (apoptosis) mechanisms enzymes. The caspases can also activate other degradative play important roles in tumor pathogenesis, allowing neo- enzymes such as DNases, which begin to cleave the DNA in plastic cells to survive over intended lifespans, subverting the the nucleus. need for exogenous survival factors and providing protection During the apoptotic process, apoptotic cells display from oxidative stress and hypoxia as the tumor mass expands. distinctive morphology. Typically, the cell begins to shrink That gives time for accumulation of genetic alterations that following the cleavage of lamins and actin filaments in the deregulate , interfere with differentiation, cytoskeleton. The apoptotic breakdown of chromatin in the promote angiogenesis, and increase invasiveness during nucleus often leads to nuclear condensation and/or a “horse- tumor progression [9]. Apoptosis defects are now considered shoe” like appearance. Cells continue to shrink, packaging an important complement of protooncogene activation, as themselves into a form that allows for their removal by many deregulated oncoproteins that drive also macrophages. These phagocytic cells are responsible for trigger apoptosis (e.g., Myc, E1a, and Cyclin-D1) [10]. On clearing the apoptotic cells from tissues in a clean and tidy the other hand, the noncancerous cells have a DNA repair fashion that avoids many of the problems associated with machinery. Defects in DNA repair and/or seg- necrotic cell death. In order to promote their phagocytosis regation normally trigger cell suicide as a defense mechanism by macrophages, apoptotic cells often undergo plasma mem- for eradicating genetically unstable cells and thus such suicide brane changes that trigger the macrophage response. One of mechanism’s defects permit survival of genetically unstable such changes is the translocation of phosphatidylserine from cells, providing opportunities for selection of progressively theinsideofthecelltotheoutersurface.Theendstages aggressive clones and may promote tumorigenesis [11]. of apoptosis are often characterised by the appearance of There are varieties of molecular mechanisms that tumor membrane blebs or blisters process and small vesicles called cells use to suppress apoptosis. Tumor cells can acquire apoptotic bodies. resistance to apoptosis by the expression of antiapoptotic The real workers in apoptosis mechanism are caspases. proteins such as Bcl-2 or by the downregulation or mutation Caspases family is composed of intracellular cysteine pro- of proapoptotic proteins such as BAX. Since the expression teases (𝑛=11in humans), which collaborate in proteolytic ofbothBcl-2andBAXisregulatedbythep53tumor cascades. These caspases activate themselves and each other. suppressor gene [12], some forms of human B-cell lymphoma Within these proteolytic cascades, caspases can be posi- have Bcl-2 overexpression. That example represents the first tioned as either upstream “initiators” or downstream “effec- andstrongestlinesofevidencethatfailureofcelldeath tors” of apoptosis. Several pathways for activating caspases contributes to cancer [13]. exist. Apoptosis defects may allow epithelial cells to survive First, there are thirty members of the tumor necrosis in a suspended state, without attachment to extracellular factor-(TNF-)familyreceptors;eightcontainaso-called matrix which facilitate metastasis [14]. They also promote death domain (DD) in their cytosolic tail [19]. Several resistance to the immune system, including many weapons of these DD-containing TNF-family receptors use caspase ofcytolyticTcells(CTLs)andnaturalkiller(NK)cellsused activation as a signaling mechanism, including TNFR1/ for attacking tumors that depend on integrity of the apoptosis CD120a, Fas/APO1/CD95, DR3/Apo2/Weasle, DR4/TrailR1, machinery [15]. Cancer-associated defects in apoptosis play DR5/TrailR2, and DR6. Ligation of these receptors at the a role in treatment resistance with conventional therapies cell surface results in the recruitment of several intracellu- like chemotherapy and radiotherapy, increasing the threshold lar proteins, including certain procaspases, to the cytoso- for cell death and thereby requiring higher doses for tumor lic domains of these receptors, forming a “death-inducing killing agents [16]. Thus, dysregulated or defective apoptosis signaling complex” (DISC) that triggers caspase activation, regulation is a fundamental aspect of the tumor biology. constituting the so-called “extrinsic” pathway for apoptosis Successful eradication of cancer cells by nonsurgical means is [20]. The caspase-8 and, in some cases, caspase-10 are the ultimately approached via induction of apoptosis. Therefore, specific caspases summoned to the DISC. These caspases all the cancer drug designers try either to activate the contain so-called death effector domains (DEDs) in their N- inactivated apoptotic mechanism or rectify a defective one. terminal prodomains that bind to a corresponding DED in Hence, all cytotoxic anticancer therapies currently in clinical the adaptor protein, FADD, thus linking them to the TNF- use, when they work, induce apoptosis of malignant cells. family death receptor complexes. Hence, deeper understanding of the molecular mechanisms Second is the intrinsic pathway, in which mitochondria of apoptosis and its defective status opens the gate for a new induces apoptosis by releasing cytochrome-c (cyt-c) into the class of targeted therapeutics. cytosol. The released cytochrome c assembles a multiprotein BioMed Research International 3 caspase-activating complex, referred to as the “apoptosome” and Bfl-1/A1), which display sequence homology in all BH1- [21]. The central component of the apoptosome is Apaf1, a BH4 domains, promote cell survival, whereas proapoptotic caspase-activating protein that oligomerizes upon binding proteins mediate receptor-, mitochondria-, or endoplasmic cyt-c and then binds procaspase-9 via interaction with its reticulum (ER) stress-dependent apoptosis. The latter group caspase recruitment domain (CARD). The intrinsic pathway is subdivided into multidomain or BH3-only proteins. The is activated by myriad stimuli, including growth factor former consists of Bax and Bak, which are essential for apop- + deprivation, oxidants, Ca2 overload, oncogene activation, tosis [28]. The BH3-only proteins are further divided into two DNA-damaging agents, and microtubule targeting drugs. subclasses: “activators” (e.g., Bim and tBid), which directly In addition to cyt-c, mitochondria also releases endonucle- activate Bax/Bak to induce mitochondrial outer membrane ase G, AIF (a death modulating flavor protein), and IAP permeabilization (MOMP), and “sensitizers/derepressors” antagonists SMAC (DIABLO) and OMI (HtrA2). Some of (e.g., Bad, Bik, Bmf, Hrk, Noxa, and Puma), which do not these molecules may promote caspase-independent (non- activate Bax/Bak directly but instead neutralize antiapoptotic apoptotic) cell death [22, 23]. proteins [29, 30]. The central role that Bax/Bak play in A third pathway for apoptosis induction is specific to apoptosis is supported by evidence that BH3-only proteins CTL and NK cells, which spray apoptosis-inducing protease, fail to trigger apoptosis in Bax/Bak-deficient cells [31, 32]. granzyme B (GraB), onto target cells. GraB then piggybacks Antiapoptotic proteins block death signaling by antagonizing into cells via mannose-6-phosphate receptors (IGFR2) and the actions of Bax/Bak through partially known mechanisms. enters effective cellular compartments via perforin channels As recently summarized, antiapoptotic proteins prevent [24]. GraB is a serine protease but, similar to the caspases, it Bax/Bak activation by sequestering/inhibiting “activator” cleaves substrates at Asp residues, including several caspases BH3-only proteins and/or directly inhibiting Bax/Bak activa- andsomecaspasesubstrates.Thefourthpathwayisacaspase tion [33]. “Sensitizer” BH3-only proteins displace “activator” activation pathway. This pathway is proposed to be linked to BH3-only proteins from antiapoptotic proteins, leading to endoplasmic reticulum (ER)/Golgi stress [17]; however, many Bax/Bak activation. Alternatively, BH3-only proteins may mechanistic details are lacking. Finally, a nuclear pathway for directly neutralize/inhibit antiapoptotic proteins, releasing apoptosis regulation was proposed. That pathway depends on their inhibition of Bax/Bak. discrete nuclear organelles, called Pml oncogenic domains Overexpression of antiapoptotic Bcl-2 or Bcl-xL probably (PODs) or nuclear bodies (NBs). Ablation of the pml gene occurs in more than half of all cancers [34], rendering tumor in mice results in general resistance to apoptosis through cells resistant to myriad apoptotic stimuli, including most unknown mechanisms. Several proteins that can promote cytotoxic anticancer drugs. For example, forced expression apoptosis have been localized to PODs, including Daax, Zip of Bcl-2 protein from plasmid vectors, in contrast, abrogates kinase, and Par4, and defects in assembly of these nuclear sensitivity to the apoptosis promoting effects of antiestro- structures are documented in cancers [25].HowPODsare gens in breast cancer lines, while antisense Bcl-2 prevents linked to caspase activation pathways is unknown. Several estrogen-mediated apoptosis suppression, thus establishing endogenous antagonists of the caspase-activation pathways a direct functional connection between ER and Bcl-2 and have been discovered and examples of dysregulation of their suppression of apoptosis [35]. expression or function in cancers have been obtained. These apoptosis mediators help cells to decide successful apoptosis 2.4. Carcinogenesis via Extrinsic Pathway Signaling Defect. or unsuccessful one. They sometimes act as targets for drug The extrinsic pathway is activated in vivo by TNF family discovery, with the idea that abrogating their cytoprotective functions may restore apoptosis sensitivity to tumor cells. ligands that engage DD-containing receptors, resulting in activation of DED-containing caspases. The “death ligands” 2.2. Resistance to Apoptosis in Cancer. Cancer is an exam- are expressed on CTLs, NK cells, and other types of immune- ple where the normal mechanisms of regulation relevant cells (activated monocytes/macrophages and den- are dysfunctional, with either an over-proliferation of cells dritic cells) and are used as weapons for eradication of trans- and/or decreased removal of cells [26]. In fact, suppression formed cells [19]. Mice-based studies on genetic alterations of apoptosis during carcinogenesis is thought to play a central in genes encoding death ligands or their receptors, as well as role in the development and progression of some cancers [27]. use of neutralizing antibodies and Fc-fusion proteins, have There is a variety of molecular mechanisms that tumor cells provided evidence of important roles in tumor suppression by use to suppress apoptosis. cellular immune mechanisms. Fas ligand (FasL) is important for CTL-mediated killing of some tumor targets, and TRAIL 2.3. Carcinogenesis via Intrinsic Signaling Defects. Mitochon- (Apo2 ligand) is critical for NK-mediated tumor suppression. dria-dependent apoptosis is one of the most important Some tumor cells resist the response of the death receptor pathways for apoptosis induction. That is why disturbing pathway to FasL produced by T cells to evade immune this pathway is an effective way to inhibit apoptosis. Bcl-2 destruction. Many tumor cell lines display intrinsic resistance family proteins (𝑛=24in humans) are central regulators to TRAIL even though they express the necessary cell surface of the intrinsic pathway, which either suppress or promote receptors confirming that, during evolution of tumors in vivo, changes in mitochondrial membrane permeability required selection occurs for malignant clones capable of withstanding forreleaseofcyt-candotherapoptogenicproteins[21, 24]. immune attack. This could occur in a variety of ways Antiapoptotic proteins (e.g., Bcl-2, Bcl-xL, Bcl-W, Mcl-1, including downregulation of the Fas receptor, expression of 4 BioMed Research International nonfunctioning Fas receptor, and secretion of high levels of a are required for binding and suppressing caspases-3 and -7, soluble form of the Fas receptor. That will directly sequester while the third BIR domain binds caspase-9. Thus, different the Fas ligand or expression of Fas ligand on the surface of domains in the multi-BIR containing IAPs are responsible tumor cells (reviewed, [36]). Some tumor cells are capable of a for suppression of different caspases. IAP family member Fas ligand-mediated “counter attack” that results in apoptotic Livin (ML-IAP) contains a single BIR and inhibits caspase- depletion of activated tumor infiltrating lymphocytes [37]. 9 but not caspase-3 and -7.Survivin also contains a single BIR Thus, successful biological therapy depends on restoring which associates with caspase-9. competency of the extrinsic pathway. IAPs are overexpressed in many cancers, while more Another molecule is the c-FLIP, cellular FADD-like detailed information is needed about the exact deregulation interleukin-1beta-converting enzyme inhibitory proteins of the 8 members of this gene family in specific types of long c-FLIP(L), which regulates caspase-8 activation. Despite cancer. Those IAPs elevations found in cancers are impor- theidentifieddualfunctionalityofc-FLIPLasapro-or tant for maintaining tumor cell survival and resistance to antiapoptotic factor in normal tissues, c-FLIPL has generally chemotherapy as confirmed by antisense-mediated reduc- beenshowntoactasakeynegativeregulatorofapoptosisin tions experiments. Targeting IAP-family members XIAP, human cancer cells [38, 39]. Increased expression of c-FLIP cIAP1, Survivin, or Apollon can induce apoptosis in tumor has been detected in many human malignancies, including cell lines in culture or sensitize cells to cytotoxic anticancer melanoma, hepatocellular carcinoma [40], nonsmall cell drugs. lung carcinoma [40], and endometrial [41], colon [42], Endogenous antagonists of IAPs keep these apoptosis and prostate cancer [43–45]. While its overexpression suppressors in check, promoting apoptosis. Two of these has been associated with cancer progression and/or poor naturally occurring IAP-antagonists, SMAC (Diablo) and prognosis in BL, HCC, and ovarian, endometrial, colon, HtrA2 (Omi), are sequestered inside mitochondria, becom- and prostate cancer [38, 39, 42, 46], elevated expression ing released into the cytosol during apoptosis [51]. SMAC and of c-FLIP blocks caspase-8 and renders cells resistant to HtrA2 have N-terminal leader sequences that are truncated cell receptor-mediated apoptosis [38]. Overexpression of byproteolysisuponimportintomitochondria,exposinga c-FLIPL has been reported to bind proteins involved in these novel tetrapeptide motif that binds the BIR domains of IAPs. signaling pathways, including TRAF1, TRAF2, RIP, and These IAP antagonists compete with caspases for binding to 𝜅𝛽 RAF1, and thereby promotes the activation of NF- and IAPs, thus freeing caspases from the grip of the IAPs and ERK as downstream molecules [39]. promote apoptosis. Thus, some modern synthetic peptides- In TRAIL-resistant nonsmall cell lung cancer (NSCLC) based therapeutics that mimic SMAC and HtrA2 trigger cells, c-FLIP and RIP, have been shown to be essential for apoptosis or sensitize tumor cell lines to apoptosis induced TRAIL-induced formation of the DISC in nonraft domains by cytotoxic anticancer drugs or TRAIL in vitro and even in of the plasma membrane and consequent activation of NF- some tumor xenograft models52 [ ]. 𝜅𝛽 and ERK cell survival signals. In contrast, knockdown of c-FLIP has been found to redistribute the DISC to lipid 2.6. How the Cancer Cells Overcome the Therapeutic Agents- rafts and switch DISC signaling to TRAIL-induced apoptosis Induced Apoptosis. The success of each therapeutic strategy 𝜅𝛽 [47]. NF- constitutive activation has been linked to the depends mainly on the ability of the therapeutic tool to pathogenesis of many human cancers [48], and inhibition of induce apoptosis either by targeting the overexpressed anti- 𝜅𝛽 NF- -induced transcription of c-FLIP has been shown to apoptotic proteins or by stimulating the expression of the sensitize cells to death receptor-mediated apoptosis [49, 50]. proapoptotic molecules. However, many of the therapeutic 𝜅𝛽 NF- -mediated upregulation of c-FLIP is implicated as an agents are still challenged by maneuvering(s) from the cancer important factor in the evasion of cell death by cancer cells. cells to survive treatments. Alterations in the expression levels or mutation of a chemotherapeutic drug’s target(s) may have 2.5. Carcinogenesis via Convergence Pathway Inhibition. The animpactonapoptosisbysuchdrug.Here,welistsomeof intrinsic and extrinsic pathways for caspase activation con- the mechanisms by which cancer cell can overcome three of verge on downstream effector caspases. Mechanisms for the standard therapeutic agents. suppressing apoptosis at this distal step have been revealed, andtheirrelevancetocancerisbecomingprogressively 2.6.1. 5-Fluorouracil (5-FU). The fluoropyrimidine 5-fluor- clear. In this regard, the apoptosis-inhibiting proteins (IAPs) ouracil (5-FU) is widely used in cancer treatment includ- represent a family of evolutionarily conserved apoptosis ing colorectal and breast cancer treatments [53, 54], but suppressors (𝑛=8in humans), many of which function resistance to that drug remains a major clinical obstacle; 5- as endogenous inhibitors of caspases. All members of this is part of its antitumor activity and FU targets the tumor family, by definition, contain at least one copy of a so-called suppressor and subsequently triggers the cell cycle. 5- BIR (baculovirus iap repeat) domain, a zinc binding fold, FU-induced apoptosis is p53-dependent; however, apoptosis which is important for their antiapoptotic activity, present in can also occur in p53 mutant cell lines by a mechanism 1–3 copies. Caspases-3 and -7, as well as caspase-9 (intrinsic still unknown [55–57]. 5-FU is an analogue of uracil with a pathway), are directly affected by the human IAP family fluorine atom at the C5 position of the pyrimidine ring. Inside members, XIAP, cIAP1, and cIAP2. For XIAP, the second cells, 5-FU is converted into different active metabolites, BIR domain and the linker region between BIR1 and BIR2 including fluorodeoxyuridine monophosphate (FdUMP), BioMed Research International 5 fluorodeoxyuridine triphosphate (FdUTP), and fluorouri- mechanisms of resistance to antimicrotubule agents remains dine triphosphate (FUTP). These metabolites have been to be determined. implicated in both global RNA metabolism due to the incor- poration of the ribonucleotide FUMP into RNA and DNA 2.6.3. Cisplatin. The therapeutic activity of cisplatin and car- metabolism due to thymidylate synthase (TS) inhibition or boplatin is mediated by an active species, formed by aqueous direct incorporation of FdUMP into DNA, leading to a wide hydrolysis as the drug enters the cell. This active species range of biological effects which trigger apoptotic cell death. interacts with DNA, RNA, and protein, but the cytotoxic The inhibition of TS is believed to be the primary anticancer effect seems to be primarily mediated via the formation of activity of 5-FU [58, 59]; however, 5-FU has been shown to DNA interstrand and intrastrand crosslinks. These platinum- acutely induce TS expression in both cell lines and tumors DNA adducts are recognized by a number of proteins, [60]. This induction of TS seems to be due to inhibition including those involved in nucleotide excision repair (NER), of a negative feedback mechanism in which ligand-free TS mismatch repair (MMR), and high-mobility group proteins binds to its own mRNA and inhibits its own translation (suchasHMG1andHMG2)[78]. Platinum-induced DNA [61]. When stably bound by FdUMP, TS can no longer damage is normally repaired by the NER pathway, whereas bind its own mRNA and suppress translation, resulting in the MMR pathway seems to trigger apoptosis [79, 80]. The increased protein expression. This constitutes a potentially mechanism by which damage recognition results in apoptosis important apoptosis resistance mechanism, as acute increases is unclear; however, in vitro datasupportaroleforcisplatin- inTSwouldfacilitaterecoveryofenzymeactivity.Thus,TS mediated activation of the c-ABL and JNK/SAPK pathway in expression acted as a key determinant of 5-FU sensitivity apoptotic signalling in MMR-proficient cells [81]. In addition, [62] while in vitro studies have validated the association the induction of p53, possibly by the ATM-CHK2 pathways, between TS expression and apoptosis 5-FU [63, 64] while following DNA strand breaks leads to apoptosis via the the improved response rates of 5-FU-based chemotherapy intrinsic pathway and also contributes to the cytotoxicity of were observed in patients with low tumour TS expression cisplatin [82]. [65, 66]. Recently, genotyping studies have found that patients Bcl-2 members are localized in the mitochondria and homozygous for a particular polymorphism in the TS pro- have either proapoptotic (Bax, Bak, Bid, and Bim) or anti- moter (TSER3/TSER3) that increases TS expression are less apoptotic (Bcl-2, Bcl-xL, and Bcl-W) functions [8, 83, 84]. likely to respond to 5-FU-based chemotherapy than patients These members are involved in the downstream action by who are heterozygous (TSER2/TSER3) or homozygous for cisplatin. These proteins form either homodimers (such as the alternative polymorphism (TSER2/TSER2) [67]. Col- Bcl-2/Bcl-2) or heterodimers (e.g., Bcl-2/Bax) depending lectively, many evidences indicate that high TS expression on the levels present of each component. Excess level of correlates with increased 5-FU resistance. homodimers can either inhibit (e.g., Bcl-2/Bcl-2) or induce (e.g., Bax/Bax) apoptosis. Although it is not confirmed 2.6.2. Antimicrotubule Agents (AMA). Microtubules are whether cisplatin can directly modulate levels of the anti- highly dynamic cytoskeletal fibres composed of tubulin sub- apoptotic protein or not, there is evidence that cisplatin units (most commonly 𝛼-and𝛽-tubulin) that have crucial significantly transactivates the bax gene by wild-type p53. roles in maintaining cell shape, in cell signalling, in cell divi- Thus, an increase in the Bax to Bcl-2 ratio by cisplatin- sionandmitosis,andinthetransportofvesiclesandmito- inducedp53hasbeenreportedtoactivatetheapoptotic chondria and other cellular components throughout the cell process [85]. However, extrapolating experimental results [68]. The rapid polymerization/depolymerization dynamics to the clinic results needs much caution, for instance, the of microtubules are critical for proper spindle function and demonstration that experimental overexpression of bcl-2 in accurate chromosome segregation during mitosis. tumors leads to the expected cisplatin resistance [86–88]. That was opposite to a clinical study, which reported that Taxanes, such as paclitaxel and docetaxel, and vinca cisplatin surprisingly improved survival of ovarian cancer alkaloids, such as vinblastine and vincristine, suppress micro- patients with increased bcl-2 gene expression [87]. Now, it is tubule polymerization dynamics, which results in the slowing understood that proapoptotic homodimers affect cisplatin- or blocking of mitosis at the metaphase-anaphase boundary induced apoptosis by first stimulating the mitochondria to [69]. These compounds can block or slow mitosis and sub- release cytochrome c, which in turn activates a series of sequently cells eventually die by apoptosis [70]. Microtubule proteases that includes caspase-1, -3, and -9 [84, 89–91]which polymer levels and dynamics are regulated by many factors, are the final effectors of drug mediated apoptosis. including expression of different tubulin isotypes [71]. Cells can resist paclitaxel and vinca alkaloids by changing the microtubules dynamics and levels of tubulin isotypes [72–74]. 3. Molecular Targeting Sometimes, a reduction in total intracellular tubulin levels Therapies and Apoptosis could be observed in paclitaxel-resistant cells. Therefore, the development of resistance to antimicrotubule agents Expression of inhibitors of apoptosis as well as inactivation may involve changes in the microtubule polymer mass and of apoptosis promoters is observed in human cancers [92]. expression of different tubulin isotypes. Furthermore, specific Moreover, functional defects in apoptotic signal transduction 𝛽-tubulin that alter sensitivity to paclitaxel have may translate into drug resistance of cancer [86, 93]. Over been reported in vitro [75–77]. The clinical relevance of these the past decade, significant advances have been made in 6 BioMed Research International discovery and validation of several types of novel cancer interacting with retinoid receptors and other transcription therapeutics. Such novel therapeutics tries to prime the factors. These inhibitors can modulate expression of Bcl-2 apoptotic machinery to act as promising apoptosis-inducing or Bcl-xL in some tumor lines [51]. These observations por- agents, bearing high hopes for the management of cancers tend opportunities for exploiting endogenous transcriptional resistant to conventional treatments. [94]. This therapeutics pathways for suppressing expression of antiapoptotic Bcl-2- canbeusedeitheraloneorincombinationwithsomeofthe family genes in cancer, together with the idea of employing conventional therapies. Several review articles have shed light them as chemo- or radiosensitizers, rather than relying on to the great potential of apoptosis-targeted therapies [95, 96]. their antitumor activity as single agents. The genetic char- Some of which focus on Bcl-2 family proteins [97–99], IAPs acteristics of cancer cells that dictate response or resistance [100], or c-FLIP [101] and the approaches that have been to SRTF-ligands, as well as the complex pharmacological used to modify their activity to reactivate apoptosis and thus interplay between this class of agents and conventional eradicate cancer cells. cytotoxic drugs, need to be investigated more. As below, we refer readers to these reviews for specific Because Bcl-2 antisense enhances sensitivity to cytotoxic details regarding the benefits of some novel therapeutic anticancer drugs in vitro and in xenograft models, most agents directed against some antiapoptotic targets, which clinical trials combine the antisense agent with conventional have been shown to demonstrate enhanced apoptotic killing chemotherapy. More precisely, antisense oligonucleotides and sensitize resistant cancer cells to antineoplastic agents. targeting the Bcl-2 mRNA have been introduced to Phase III Nevertheless, we show below a few of the most promising clinical trials for melanoma, myeloma, CLL, and AML and therapeutic strategies which target the induction of apoptosis. Phase II activity underway for a variety of solid tumors. [102]. Moreover, Phase II data suggest a benefit from adding Bcl-2 antisense to conventional therapy, but definitive proof awaits 3.1. Targeting Antiapoptotic Bcl-2 Family Members. The com- the Phase III results. plex interplay between proapoptotic and antiapoptotic mem- Beside the antisense oligodeoxynucleutides (e.g., G3139 bers of the Bcl-2 family plays a crucial role in cellular [103]), various drugs molecules that target Bcl-2 and Mcl- fate determination. Attempts to overcome the cytoprotective 1 have been tested for their abilities to induce apoptosis effects of Bcl-2 and Bcl-xL in cancer include three strategies: in cancer cells [104]. Understanding how cells keep these (1) shutting off gene transcription, (2) inducing mRNA antiapoptotic proteins controlled is the base on which the degradation with antisense oligonucleotides, and (3) directly therapeutic potency for targeting the Bcl-2 and Bcl-xL pro- attacking the proteins with small-molecule drugs. Herein, teins by these molecules is established. In this regard, a large we will provide some of the most promising strategies for family of endogenous antagonists of Bcl-2 and Bcl-xL has modulating the activity of apoptosis genes and their proteins been revealed (so-called “BH3-only proteins”), possessing a for cancer therapy. conserved BH3 domain that binds a hydrophobic crevice Some members of the steroid/retinoid superfamily of on the surface of Bcl-2 and Bcl-xL [100]. Synthetic BH3 ligand-activated transcription factors (SRTFs) represent peptides generated as small-molecule drugs that occupy potentially “drugable” modulators of Bcl-2 and Bcl-xL gene the BH3 binding site on Bcl-2 or Bcl-xL, abrogating their transcription although they are not components of the “core” cytoprotective functions, have been validated [105]. Many apoptosis machinery. For example, expression of Bcl-2 is research groups have presented preclinical data regarding estrogen-dependent in the mammary gland. Consequently, BH3-mimicking compounds. The optimal structure-activity antiestrogens, such as tamoxifen, inhibit endogenous Bcl-2 relation (SAR) profile of these compounds had been deter- expression in breast cancer cell lines, promoting sensitivity to mined. These molecules showed broad-spectrum activity cytotoxic anticancer drugs such as doxorubicin. In addition against the various antiapoptotic members of the Bcl-2- to antiestrogens in estrogen receptor- (ER-) positive breast family (Bcl-2, Bcl-xL, Mcl-1, Bcl-W,Bfl-1, and Bcl-B), in terms cancers, expression of Bcl-2 or Bcl-xL can be downregulated of balancing antitumor efficacy. Of these molecules, which in specific types of cancer and leukemia cells by small target Bcl-2, are the small molecule Bcl-2 inhibitors (e.g., molecule drugs that modulate the activity of retinoic acid HA14-1; [103]) and BH3 peptidomimetics [106]. The most receptors (RAR), retinoid X receptors (RXR), PPAR, vitamin prominent molecules among them are the Bcl-2 antagonists D receptors (VDR), and certain other members of the SRTF ABT-737 and ABT-263 [107, 108]. That novel Bcl-2/Bcl- superfamily. RAR and RXR ligands are already approved for xL/Bcl-W inhibitor (ABT-737) has been developed. It showed treatment of some types of leukemia and lymphoma and are enhanced activity of chemotherapeutic agents or ionizing in advanced clinical testing for solid tumors. PPAR mod- radiation in the preclinical studies. That compound displays ulators have demonstrated antitumor activity in xenograft impressive antitumor activity in human tumors including models of breast and prostate cancer [10], sometimes dis- follicular lymphoma, chronic lymphocytic leukemia, and playing synergy with retinoids, probably due, in part, to smallcelllungcancer(SCLC)in vitro and in vivo [107]and the fact that PPAR binds DNA as a heterodimer with RXR. in nonsmall lung cancer (NSCLC) [109]. Moreover, troglitazone, a potent PPAR agonist, can lower ABT specifically bind to Bcl-2, Bcl-xL, and Bcl-W flipping serum PSA in men with advanced prostate cancer; however, them from Bak and Bax. ABT-737 mimics the BH3-only its proapoptotic features are not confirmed in vivo yet. protein, Bad, by docking to the hydrophobic groove of Compounds that inhibit histone deactylases (HDACs), antiapoptotic proteins, thereby disabling their capacity to called HDAC inhibitors, act as transcriptional repressors by antagonize the actions of proapoptotic proteins. Therefore, BioMed Research International 7

ABT-737ishighlypotent(i.e.,atnanomolarconcentrations) transcription-independent role of p53 in apoptosis has been in killing tumor cells which are dependent upon Bcl-2 for attributed to its ability to block the antiapoptotic function survival [110]. Although ABT targets almost all the proapop- of Bcl-2 and Bcl-xL on the mitochondria [121, 122]. p53 totic family members of Bcl-2 except Mcl-1, it has been promotes, directly or indirectly, the apoptotic activities of found that its combination with deoxyglucose has efficiently proapoptotic members of the Bcl-2 family, Bax and Bak eliminated cancer cells in vitro and in vivo [111]. ABT-737 [121, 123]. Moreover, after targeting DNA by some damaging was modified at three sites to create improved ABT-263 for agents, p53 contribute as a transcription factor. p53 transcrip- oral bioavailability without the loss of affinity to the Bcl-2 tionally upregulates genes such as those encoding p21WAF- family of proteins [108].Asasingleagent,however,ABT- 1/CIP-1 and GADD45, which induce cell cycle arrest in 263/737 (ABT) induces apoptosis only in limited tumor types, response to DNA damage [124, 125]. However, p53 can trigger such as lymphomas and some small cell lung carcinomas elimination of the damaged cells by promoting apoptosis [107, 108, 112]. through the upregulation of proapoptotic genes such as Bax, Other strategies for countering Bcl-2 and Bcl-xL in cancer NOXA, TRAIL-R2 (DR5), and Fas (CD95/Apo-1) [12, 55, 126, include over-expression of opposing proapoptotic family 127]. However, the cell can choose between p53-dependent members such as Bax with p53 adenovirus (in Phase III trials) cell cycle arrest or apoptosis. How cells choose between or with Mda7 (IL-24) adenovirus gene therapy (completed this p53-mediated cell cycle arrest and apoptosis has three Phase I trials), as well as Bax adenovirus gene therapy for possible models. (I) One model suggests that more profound regional cancer control. Alternatively, it might be possible to DNA damage induces higher and prolonged activation of activate the Bax and Bak proteins using drugs that mimic p53, which increases the chances of apoptosis over arrest agonistic BH3 peptides [105]. [128]. (II) Another model suggests that different cell types One further strategy to counteract Bcl-2 family members might keep different p53-regulated genes in regions of active is the monoclonal antibodies which target the tetra-span chromatin, which determines the cassette of genes that membrane protein CD20 (Rituximab) but can also down- aretranscriptionallyupregulated[129]. (III) A third model regulate expression of Bcl-2 family members, Mcl-1 (intrin- proposes that the availability of transcriptional cofactors sic pathway) and XIAP (convergence pathway) in certain determines the ability of p53 to activate different subsets of leukemias, providing yet another example of unexpected genes [130, 131]. links to apoptosis pathways through receptor-mediated sig- DNA damage-inducing apoptotic factors induce the naling [113]. activation of upstream kinases such as ATM (ataxia- telangiectasia mutated), ATR (ATM and Rad-3 related), 3.2. Targeting Cell Cycle Control System for Apoptosis Induc- and DNA-PK (DNA-dependent protein kinase), which can tion. The relationship between cell cycle arrest and apoptosis directly or indirectly activate p53. Phosphorylation of p53 by is complex. Signaling pathways following apoptosis-inducing upstream kinases inhibits its negative regulation by MDM- stimuli either arrest the cell cycle following damage signals to 2, which targets p53 for ubiquitin-mediated degradation allow time for repair or switch to initiation of apoptosis after [132]. In fact, lack of functional p53 contributes to apoptosis apoptogenic agents. These agents, such as ionizing radiation resistance due to the inability to undergo p53-mediated or some chemotherapy, induce apoptosis in many cell types apoptosis sometimes due to p53 mutations. In vitro studies [114]. Apoptosis is frequently associated with proliferating have reported that loss of p53 function reduces apoptosis cells. This implies the existence of molecules in late G1 by 5-FU [57]. Other clinical studies have found that p53 and S phase, whose activities facilitate execution of the overexpression (a surrogate marker for p53 mutation) corre- apoptotic process. Once the cells are committed to cell death, lates with resistance to apoptosis by 5-FU [133, 134]. Other apoptogenic factors, including cytochrome c, are released studies have found no such correlation [135]. Such conflicting from mitochondria to initiate a caspase cascade [115]. findings may be due, at least in part, to the fact that p53 An important cell cycle regulator plays a central role in overexpression does not actually reflect p53 mutation in as the regulation of cell cycle arrest and cell death is the tumor many as 30–40% of cases [136]. A recent study has suggested suppressor, p53 [116]. It has recently been discovered that the that certain p53 mutants may increase dUTPase expression, p53 activates apoptosis through parallel pathways that may inhibiting apoptosis by 5-FU [137]. depend on transcription events or not [117]. Under many Anumberofin vitro studies have demonstrated decreased types of stress, p53 induces cell cycle arrest and apoptosis to cisplatin-induced apoptosis in p53 mutant tumor cells [138– maintain genomic integrity and prevent damaged DNA being 140].Somestudieshavereportedthatdisruptionofp53func- passed on to daughter cells. Tumor cells have inactivated p53. tion actually enhances apoptosis by cisplatin [141, 142]. This About 50% of this inactivation is achieved through mutations enhanced apoptosis was attributed to defects in p53-mediated in its sequence and the rest by disabling key components cell cycle arrest, which reduced time for DNA repair. Some that lie upstream or downstream of p53 in a common tumors with mutant p53 have a worse clinical outcome which signaling pathway [118]. In p53 wild-type tumors, p53 may means failure to induce apoptosis by therapeutic drugs [143]. be compromised, by inactivation of positive regulators of p53 In vitro data from other laboratories and others suggest that activity (such as p14ARF [119]) or over-activation of negative development of resistance to apoptosis by taxan is feasible regulators of p53 activity (such as Akt [120]). in p53 null cells [144]. The clinical relevance of p53 status Bax is one of the best known downstream p53 tran- for taxan-induced is yet to be determined. In vitro studies scriptional targets that directly affect apoptosis. A further have found that p53 status does not affect paclitaxel-induced 8 BioMed Research International apoptosis, [145–147]. On the other hand, doxorubicin was and various feedback loops exist to activate Bax and thereby more active against p53 wild-type tumor xenografts than to amplify the apoptotic cascade in hematopoietic and other p53 mutant and null cells [148]. Furthermore, apoptosis cell types. induction by doxorubicin in acute lymphoblastic leukaemia It is now clear that, through its dual role, cyclin E provides cell lines was found to be dependent on the presence of a physiological balance between cell proliferation and death wild-type p53 [149]. In the clinical setting, p53 mutations of hematopoietic cells [161]. Cyclin E has an initial limited have been correlated with lack of response to doxorubicin activation of caspase-3 perhaps through a small amount in patients with locally advanced breast cancer [150]. These of primed/activated mitochondrial Bax. Then, cyclin E is data indicate that doxorubicin is more active against p53 wild- proteolytically cleaved by caspase-3 to generate p18-cyclin E, type tumors. Fichtner et al. found no correlation between which interacts with Ku70 resulting in release of Bax from the p53statusandsensitivitytotheapoptogeniccarboplatin, Ku70-Bax complex. This contributes to a robust activation of in xenotransplanted colorectal tumors [151]. Similarly, Jacob Bax, leading to its mitochondrial targeting and amplification et al. found that p53 status did not correlate with CPT- of the intrinsic pathway of apoptosis. This is consistent with 11-induced apoptosis in a panel of colorectal cancer cell the idea that limited caspase activation is not sufficient to lines [152, 153]. Some studies have found that carboplatin release enough cytochrome c leading to significant apoptosis, induces apoptosis to a similar extent in isogenic p53 wild- at least, in hematopoietic cells. Rather, an amplification loop type and null cancer cells [118]. In general, all the above is required to trigger massive cytochrome c release from mentioned studies suggest that p53 mutant tumors may be mitochondria resulting in abundant caspase-3 activation and equally responsive to apoptosis inducers or possibly more cell death [115]. Caspase-3 and caspase-7 may participate in sensitive than p53 wild-type tumors. That led to application a feedback amplification loop to promote the mitochondrial of combinatorial drug therapy of paclitaxel and cisplatin for apoptotic pathway [162]. maximum apoptosis induction in p53 mutant ovarian tumors than p53 wild-type tumors as introduced [154]. 3.2.2. Ku70, from Cyclin E to Bax. As we previously men- tioned, the key regulatory step in the intrinsic cell death 3.2.1. Cyclines. Another important family, critically control- pathway is the activation of the proapoptotic molecule, Bax ling cell cycle, is cyclines [155, 156]. Here, we will focus on [163]. Although the mechanism of Bax activation remains an important member of this family, cyclin E, which has unclear, a recent study indicates that Bax is kept inactive direct relation with apoptosis signals. Cyclin E has been in some cells, to counteract apoptosis, by interacting with shown to have a Cdk2-independent function in regulating cytosolic Ku70 [163, 164]. Overexpression of Ku70 prevents apoptosis of hematopoietic cells [157, 158]. The larger N- Bax-mediated apoptosis, whereas targeting Ku70 causes cells terminal truncation of cyclin E that generates p18-cyclin to become more sensitive to a variety of apoptotic stim- E prevents its binding to either Cdk2 or CKIs p21Waf1 uli. Importantly, we showed that some of these apoptotic or p27Kip1. Therefore, unlike the other isoforms of cyclin stimuli which are not directly targeting DNA break like the E, p18-cyclin E cannot bind Cdk2 and therefore interferes microtubule stabilizing agents, Taxane, and hyperthermia with Cdk2-dependent (bound to cyclin E1, E2, or A) kinase stimulated caspase-dependent apoptosis after Ku70 inhibi- activity. Cleavage of cyclin E results in loss of its Cdk2 tion (unpublished data). These results demonstrated that associated kinase activity and consequently its function in Ku70 is a physiological inhibitor of Bax-induced apoptosis; the cell cycle [157]. Recently, it has been reported that p18- a finding that expands on Ku70’s previously known role cyclin E mediates p53 function-independent apoptosis by in DNA repair [163], particularly through nonhomologous regulating the relationship between Bax and Ku70 [159]. That end joining (NHEJ). Moreover, cells from Ku70 knockout study’s findings established a role for Ku70 in regulating Bax- micearehypersensitivetoagentsthatinduceapoptosisin mediated apoptosis in hematopoietic cells that generate p18- the absence of DNA damage, such as staurosporine [159]. cyclin E. By interacting with Ku70, p18-cyclin E releases Bax This suggests that Ku70 plays a role in suppressing apoptosis from Ku70, thus allowing Bax to be activated during gentoxic independently of its role in DNA repair [165]. Based on stress and thereby leading to apoptosis [159]. Therefore, these findings, nuclear Ku70 is therefore considered to be p18-cyclin E, Bax, and Ku70 emerge as key regulators of mainly responsible for repair of DNA damage, whereas the apoptotic pathway at least in hematopoietic cells. The sensitivity of these cells to genotoxic stress can be explained the cytosolic pool of Ku70 may be primarily a regulator by their ability to generate p18-cyclin E. of Bax activation [162, 164]. Therefore, Ku70 plays a dual The cleavage of cyclin E can be prevented by Bcl-2 role in NHEJ and regulation of Bax, which in turn may overexpression, indicating that Bcl-2 functions upstream of determine the balance between cell survival and programmed the mitochondrial apoptotic cascade [115, 158]. How Bax is cell death in hematopoietic cells. Ku70 may not be unique released from its complex with Ku70 is still unknown. One in assuring Bax sequestration in the cytosol. Other Bax mechanism may consist of a structural change in conforma- interactive proteins such as the peptide humanin [166], the tion of the Bax binding-domain of Ku70 upon binding of p18- apoptosis repressor with caspase recruitment domain (ARC) cyclin E at the amino terminus. Another possibility might be [167], or crystalline [168] may also function in various cellular the recruitment of histone acetyl transferases, which would systems to sequester Bax. On the other hand, after apoptotic acetylate several lysine residues at the carboxy terminus of stimulation other Bax-interacting proteins, such as p53 [169] Ku70 [160]. However, it is quite possible that other pathways and Bif- [170] and apoptosis-associated speck-like protein BioMed Research International 9

(ASC) [171], and MOAP-1 [172]promoteBaxconformational result in hypersensitivity to cisplatin, and that restoration change and mitochondrial translocation. of NER activity reduces sensitivity to more normal levels [179].NERisacomplexprocessinvolvingatleast17different 3.2.3. Nonapoptotic Function of Caspases. Although caspases proteins; however, upregulation of only a few rate limiting areclassicallyknownfortheirroleinapoptosis,their components of the NER system is necessary to increase a cell’s involvement in biological processes apart from cell death has capacity for NER [180]. One of these important rate-limiting recently received increased attention. For example, they play factors is the excision repair cross-complementing 1 protein an important role in the inflammatory signaling pathways (ERCC1), which plays a crucial role in response to apoptosis [173, 174]. The nonapoptotic functions of these proteases inducer, cisplatin. Increased expression of ERCC1 associated suggest that they may become activated independently of the with cisplatin resistance [181–183]. Clinically, high levels of apoptotic cascade, thus leading to the cleavage of specific ERCC1 are correlated with poor response to platinum-based substrates, such as kinases, cytokines, transcription factors, chemotherapy in ovarian, gastric, and nonsmall cell lung and polymerases. cancers (NSCLC) [184–186]. In addition, high mRNA expres- sion of ERCC1 and thymidin synthase (TS) has been acting Functional analysis of conditional caspase-deficient mice as predictors for poor response to combined oxaliplatin/5- or derived cells confirmed the crucial roles of caspases in cell FU treatment of advanced colorectal cancer [187]. Targeting proliferation, differentiation, and inflammation. It has been ERCC1 expression, using an antisense expression construct, reported recently that caspase-3 is a negative regulator of B- made ovarian cancer more sensitive to cisplatin than controls cell cycling. This study shows that while the apoptotic path- in cell line and xenograft models188 [ ]. Another central way is largely unaffected in caspase-3 knockout mice, splenic component of the NER machinery, B-cell proliferation is enhanced in vivo,andinresponseto group A (XPA), has been found to be overexpressed in mitogenic stimulation in vitro, hyperproliferation is seen. apoptosis resistant cancer cells under apoptosis inducer, Genetic and biochemical data demonstrate that caspase-3 cisplatin, treatment [184]. Collectively, these studies suggest is essential for the regulation of B-cell homeostasis [175]. an important role for NER in mediating resistance to DNA In contrast to caspase-3, caspase-8 is a positive regulator of damage and subsequent apoptosis by platinum. cell cycling as B-cell proliferation since caspase-8 deficient Another DNA damaging agent is 5-FU. 5-FU treatment mice fail to proliferate [176, 177]. Recently, it has been induces TS inhibition which subsequently causes nucleotide suggested that caspase-3 and caspase-7 together play a role in imbalances that severely disrupt DNA synthesis and repair development, since double knockout mice exhibit defects in [189]. TS inhibition results in the accumulation of deoxyuri- heart development, leading to death immediately after birth. dine triphosphate (dUTP), as the essential conversion process Incontrast,knockoutofeithercaspaseindividuallyonthe of deoxyuridine monophosphate (dUMP) to deoxythymi- same genetic background resulted in viable animals. dine monophosphate (dTMP) is blocked [190]. Both dUTP and another 5-FU metabolite, fluorodeoxyuridine triphos- 3.3. Targeting DNA Repair System for Apoptosis Induction. phate(FdUTP),canbemisincorporatedintoDNA.Repairof DNA repair can determine fate of a cell with or without uracil and 5-FU-containing DNA could be achieved by the treatment stress which either resists over to survive or enzyme uracil-DNA-glycosylase (UDG) respond to toxic agents. When the repair mechanisms are [191]. However, this repair mechanism is futile in the presence unsuccessful, it may cause cellular (permanent of high (F)dUTP/dTTP ratios causing false nucleotide incor- cell cycle arrest), oncogenesis, or apoptosis which plays an poration. These futile cycles of misincorporation, excision, essentialroleinsurvivaloftheorganismsbypreventingthe andrepaireventuallyleadtoDNAstrandbreaks.However, multiplication of mutated , as well as elimina- some cells show resistance to this mechanism by increased tion of indisposed cells, normal embryonic development, and dUTPase. The increased dUTPase expression has been asso- maintenance of cell homeostasis. [178]. In cancer treatments, ciated with resistance to TS inhibitors like 5-FU [192–194]. apoptosis-inducing chemotherapeutic drugs induce DNA Another mechanism is the DNA mismatch repair (MMR) damage either directly (e.g., platinum drugs) or indirectly which is an important mechanism that controls the decision (e.g., 5-FU and topoisomerase poisons). The response to from survival to apoptosis. The main function of the MMR DNA damage is either repair or cell death. Only when system is to scan newly synthesized DNA and remove single repair is incomplete, for example, when the DNA damage nucleotide mismatches that arise during replication. MMR is too extensive, cells will undergo apoptosis. Herein, we deficiency is implicated in the development of apoptosis- describe some major mechanisms through which cells can resistance against wide range of DNA-damaging agents, overcome apoptosis by damaging agents and we introduce including platinum drugs [195]. It is possible that MMR some example molecules which had successfully targeted in recognition of DNA damage may trigger an apoptotic path- sometypesofcancertostimulateorenhanceapoptosisby way or that futile cycles of DNA damage/repair mediated therapeutic agents. by the mismatch repair machinery generate lethal DNA Nucleotide excision repair (NER) is the major pathway for strand breaks [196]. MMR has a role beyond that of repair DNA repair after DNA stress. However, for example, cisplatin in response to DNA damage. Inherited defects in the DNA induces platinum-DNA adduct which could be removed MMR genes, especially hMLH1 and hMSH2, are common by NER as challenge mechanism by cancer cell to repair in certain familial forms of cancer such as hereditary non- of platinum-induced DNA damage [178]. Defects in NER polyposis colon cancer (HNPCC). Such defects were also 10 BioMed Research International observed in a variety of sporadic tumors, including colorectal, and hMSH2; thus, it plays a role in DNA repair [208]. BRCA1 breast, and ovarian cancers [197–200]. DNA MMR deficiency also plays a role in the activation of cell cycle checkpoints in gives rise to the microsatellite instability (MSI) phenotype, response to different cellular stresses including DNA damage which is detected as variations in lengths of DNA repeat and disruption of microtubule dynamics [209, 210]. Now, sequences present in the [201]. Cisplatin-induced BRCA1 has been implicated in the regulation of apoptosis apoptosis and acquisition of the drug resistance were found to [211]. It appears that, similar to p53, BRCA1 may function as be associated with hypermethylation of the hMLH1 promoter a sensor of cell stress by relaying signals to either the cell- [202]. One clinical study found that ovarian tumors that were cycle checkpoint machinery or cell death machinery. A recent initially microsatellite stable before cisplatin-based stress study by Quinn et al. found that BRCA1 acts as a differential exhibited MSI in the residual tumors after chemotherapy, and modulator of chemotherapy-induced apoptosis in breast can- that was proposed to be due to loss of hMLH1 expression cer cells [212]. They found that BRCA1 enhanced sensitivity [203]. Hence, preclinical studies strongly implicate loss of to apoptosis induced by antimicrotubule agents, such as MMR, in particular loss of hMLH1, in overcoming apoptosis paclitaxel and vinorelbine, but inhibited apoptosis induced by cisplatin. Reversal of hMLH1 promoter methylation by by DNA-damaging agents such as cisplatin and etoposide. DNA methyl transferase inhibitor, 2-deoxy-5-azacytidine Moreover, inhibitors of poly (adenosine diphosphate- (ADP-) (DAC), has been shown to resensitize cells to a range of ribose) polymerase (PARP) show the synthetically lethal apoptotic agents, including platinum drugs [202]. Thus, effect in BRCA1-defective tumors because BRCA1 encodes DAC has now entered clinical trials in combination with protein that is required for efficient homologous recombina- carboplatin in some cancers. Finally, a clinical study found tion (HR) [213]. that MMR deficiency was a predictive factor for apoptosis induction by carboplatin in advanced colorectal cancer [204]; 3.3.3. Ku Protein, from Gatekeeping to Caretaking. Double however, MMR deficiency had no effect on sensitivity to the strand break (DSB) repair includes homologous recombina- non-DNA-targeted apoptosis-inducing agents, paclitaxel and tion (HR) and nonhomologous end joining (NHEJ). DSB docetaxel. repair through HR process is an error free pathway, since it In addition to the above mentioned repair mechanisms requires an extensive region of sequence homology between and other several repair machineries, such as mutagenic the damaged and template strands, whereas NHEJ is an repair, which may become effective for the recovery of error prone alternate pathway for the repair of DSBs, which genome against constant attack of numerous genotoxins, essentially joins broken chromosomal ends independent of UV-radiation, ionizing radiation, and various chemicals are sequence homology. The NHEJ process is initiated by the responsible for most of the mutagenesis due to a process binding of specific protein to the broken ends, which may of translesion that inserts an incorrect nucleotide opposite act as end bridging factor [214]. The catalytic subunit of to the lesion and then continues elongation [205]. Targeting DNA protein kinase (DNA-PKcs) is required in mammalian any of such mechanisms could enhance apoptosis instead of NHEJ to bridge the DNA ends through their protein-protein the repair and eradicate the defectively repaired cells under interactions [215]. apoptotic inducers. Cancer susceptibility genes have been categorized into two classes, namely, caretakers and gatekeepers. Caretakers are involved in DNA repair and whose inactivation can lead 3.3.1. DNA Double Strand Break Repair and Apoptosis. to genomic instability while gatekeepers control cell death Double-strand breaks (DSBs) repair mechanism is one of and proliferation. Loss of functions in both caretaker and the widespread mechanisms which efficiently repairs double- gatekeeper genes results in an increase in cancer suscep- strand breaks and single-strand gaps in damaged DNA tibility. Ku86 and p53 are examples of such caretaker and by a series of complex biochemical reactions, as a result gatekeeper, respectfully. Their functions are closely linked of ionizing radiation, UVR, ROS, and chemotherapeutic [216]. Ku is a caretaker gene that maintains the integrity of genotoxic chemicals [205]. The lethal effects of double strand the genome by a mechanism that suppresses chromosomal breaks (DSBs) can be conquered by the existence of two rearrangements. Ku complex (a heterodimer of Ku70/Ku80 independent pathways, such as homologous recombination [≈86]) is a major end binding factor in mammalian cells, (HR) and nonhomologous end joining (NHEJ). Multiple possessing end bridging activity [217, 218]. Ku exists as a proteins are required for DSB repair by recombination, which heterodimeric DNA binding complex in eukaryotes, con- are conserved among all eukaryotes. Deficiencies in such sistingoftwoproteinsubunitsofabout70-and80-kDa, repair mechanism can develop cancer. known as Ku70 and Ku80 (in S. cerevisiae)orKu86(inhigher eukaryotes). This complex was originally identified two 3.3.2. BRCA1. BRCA1 is a that regu- decades ago as a major target of autoantibodies from Japanese lates cellular responses to DNA stress by apoptotic inducers. patients with scleroderma-polymyositis overlap syndrome Germline mutations of the BRCA1 gene account for about [219]. Functional Ku proteins are critical for the maintenance 10%ofbreastandovariancancercases,andlowerthannor- of genomic stability, DNA repair, and hence cellular and mal BRCA1 expression may be an important factor that con- organismal viability [220]. Li and colleagues reported the tributes into sporadic cancers [206, 207]. Recently, BRCA1 putative tumor suppressor function of Ku70. On the other has been found to reside in a large DNA repair complex that hand, Ku was identified as a nuclear protein involved in both includes various mismatch repair proteins including hMLH1 homologous recombination (HR) or nonhomologous end BioMed Research International 11 joining (NHEJ). Moreover, Ku proteins have been implicated resulting in the release of Bax from Ku70. Subsequently, acti- in numerous other cellular processes, including vated Bax translocates to the mitochondria. In our laboratory maintenance, antigen receptor gene arrangements (e.g., vari- we identified Ku70, particularly as a required molecule for the able (diversity) joining {V(D)J} recombination), regulation cytostatic arrest under hyperthermia treatment in nonsmall of specific gene transcription and apoptosis, regulation of lung cancer cells. In that study, small interference RNA heat shock-induced responses, and a newly identified role in against Ku70 could inhibit the cytostatic arrest and switch regulation of the G2 and M phases of the cell cycle [221]. cells to cytotoxic arrest and caspase-dependent apoptosis Moreover, an association of the DNA-PK-Ku70-Ku86v with mediated by Bax activation [232]. a number of cell cycle regulatory proteins such as p53, CDK4, and E2F-4 had been reported, suggesting a pivotal role of Ku 3.4. Targeting Death Receptors (DR). Many tumors refract to in cell cycle regulation and malignant transformation [222]. the classic apoptosis-inducing cancer therapies which pro- Recently,Ku70hasbeenshowntobindtotheproapop- voke intrinsic pathway-based apoptosis due to development totic protein BAX and inhibit BAX-mediated apoptosis in of defects in that pathway under treatment stress [233]. To vitro by preventing its relocalization to the mitochondria enhance cancer cell apoptosis and thus overcome treatment [223]. This antiapoptotic function is mediated by a domain failure, many therapeutic strategies targeting other molecules in the carboxy-terminal of Ku70 and does not require the implicated in apoptosis resistance have been developed [95, cooperative effects of Ku80. Although the exact function of 96]. Hence, alternative therapies which provoke the extrin- Ku in these processes is presently still not clear, what is sic pathway-based apoptosis were developed. For example, certain is that this protein is of fundamental importance in attempts to apply TNF as a cancer treatment were stymied cell viability especially under treatment stress [223]. Hence, by the proinflammatory effects of this cytokine, because it the idea of targeting Ku70 had emerged to enhance apoptosis induces both caspase activation pathways and NFKappaB. by different therapies. That was because tumor cells depend Fortunately, the TNF family cytokines, FasL and TRAIL, on DNA repair mechanisms to a greater extent than nor- trigger caspase activation without concomitant induction of mal cells; hence, many cancer therapies target such DNA NFKappaB giving chances for successful cancer therapy via repair machinery [224]. As DNA-PK plays a crucial role in apoptosis, where TNF failed due to toxicity. The antibodies theDSBR,thekinaseactivityoftheDNA-PKcomplexis that trigger the receptor Fas (CD95) unfortunately showed regulated through the DNA end-binding (DEB) activity of high toxicity to liver [224],whileTRAILandagonisticanti- its regulatory subunit Ku. Therefore, the regulation of Ku bodies that bind TRAIL receptors appear to be well tolerated expression and function also plays a vital role in cancer cells in vivo.Asafact,PhaseItrialinhumanswasrecentlycom- resistance to anticancer therapy. Thus, targeting Ku protein pleted using an agonistic antibody directed against TRAIL- led to changes in susceptibility to anticancer drug-induced receptor-1 (TRAIL-R1; DR4). In mouse xenograft models apoptosis [225, 226]. Most exciting are the new clinical bearing human tumor cells lines, TRAIL and its agonis- studies that target Ku as their primary strategy. Reports have tic antibodies directed against TRAIL receptors have been shown that the status of Ku or NHEJ in tumors can enhance demonstrated to possess potent antitumor activity [234], apoptosis by radiotherapy or chemotherapy, suggesting that supporting the idea of using these biological agents as a novel Ku may be a potential target to overcome resistance during approach to cancer treatment and thereby mimicking some of cancer treatment. Recently, novel therapies such as the engi- the effector mechanisms normally employed by the immune neeringofselectivesmallmolecule,DNA-PKinhibitors,that system in its defense against transformed cells and potentially enhance radiation-induced tumor control in a mouse-human bypass the defective intrinsic (mitochondrial) pathway to xenograft model of cancer have been developed and have apoptosis. shown promising results [224]. Other examples include the Synthetic triterpenoids, such as CDDO and CDDOm, use of adenovirus-mediated, heat activated antisense Ku70 sensitize solid tumor cell lines to TRAIL and induce apop- expression that radiosensitizes human tumors [227]andthe tosisassingleagentsinleukemiacells,throughacaspase- useofaKuantibodytoblockMMcelladhesionandabrogates 8-dependent mechanism that remains operative even in the protective effect of membrane Ku228 [ ]. chemorefractory cells [235–237]. Moreover, in leukemia, Further understanding of the possible link of the main the protein kinase C (PKC) modulator byrostatin induces functionsofKuindifferentcellsystemsmayalsohelpin myeloid leukemia cell lines to produce TNF, resulting answering the intriguing question of how autoantibodies in autocrine engagement of TNF-receptors and apoptosis against Ku are generated in various autoimmune diseases. induction through a mechanism that is suppressible by This provides an exciting insight into the future of anticancer TNFR-Fc fusion protein and caspase-8 dominant-negative development. It has shown that Ku70 siRNA sensitized [238]. Similarly, all-trans-retinoic acid (ATRA) induces mammalian cells to radiation and topoisomerase II inhibitor, TRAIL production and triggers autocrine induction of apop- etoposide [229]. Recently, small molecule, HDAC inhibitors, tosis in acute promyelomonocytic leukemia (APML) cells which targets Ku70 acetylation enhanced DNA damage by that harbor the RAR-PML fusion protein resulting from DNA-damaging agents in prostate cancer and they enhanced t(15; 17) chromosomal translocations [239]. radiation effect in melanoma cells230 [ , 231]. Moreover, Sub- TRAIL, a candidate for targeted therapy, which can pref- ramanian used HDAC inhibitors, which target ku70 acetyla- erentially kill cancer cells, is frequently hampered by various tion to induce apoptosis in neuroblastoma cells [163]. These mechanisms of cancer cell resistance to apoptosis [240, 241]. molecules increase the acetylation of Ku70 in the cytoplasm, As a cytokine, TRAIL induces apoptosis by binding one of 12 BioMed Research International its cognate death receptors and plays an important role in orearlyclinicaldevelopment.BivalentSMACmimetics, immune surveillance against tumors [49, 240, 241]. Neverthe- which bind to both the BIR2 and BIR3 domains within a less, in many types of cancer cells, effective antitumor activity single molecule of XIAP as well as the BIR3 domains of two of TRAIL requires the suppression of aberrantly expressed cIAP molecules, induce their dimerization, and subsequently negative regulators of apoptosis [240, 241]. In general, as trigger their proteasome mediated degradation, are more multiple mechanisms may control apoptosis, significant potent than their monovalent counterparts [257, cancer cells can employ a number of different strategies to 258]. Despite evidence indicating that SMAC mimetics show suppress a protective apoptotic response [6–8]. From other great promise for cancer therapy [257], they can induce side, survival signaling pathways are linked to the apoptotic loss of cIAP1 and cIAP2, resulting in NIK stabilization and machinery. These pathways can modulate components of the consequent NF-kB activation [48, 257]. apoptotic machinery itself or key regulatory molecules within Targeting c-FLIP appears to be a highly promising thera- thecoreapoptoticpathways[242, 243]. Notably, various peutic strategy to reduce the apoptotic threshold because its upstream signaling pathways often impinge on the same few overexpression can protect cancer cells from both TRAIL and central apoptosis control points, which involve some crucial chemotherapy-induced apoptosis, while small interfering members of the Bcl-2 family, inhibitor of apoptosis (IAP) RNA- (siRNA-) mediated downregulation of c-FLIP is able to proteins, and FLICE-inhibitory protein (c-FLIP) [6, 7, 38, 40]. sensitize cancer cells to FASL, TRAIL, and chemotherapeutic agents [39, 101]. Targeting FLIP by siRNAs in a variety of cancer cell types showed their potential as therapeutic 3.5. NF-𝜅BasaTargetforApoptosisInduction. NF-𝜅Bisa agents to induce apoptosis [101]. Nevertheless, the widespread transcription factor that was discovered in the nucleus of B use of such strategy in clinical settings depends on the cells and binds to the enhancer of the kappa light chain of safety delivery of siRNA [261]. Thus, many research efforts immunoglobulin. It is expressed widely in the cytoplasm of all have been devoted to improve RNA interference (RNAi) types of cells. NF-𝜅B is an important molecular link between therapeutics, which lead to important advances in the field chronic inflammation, cell cycle, cancer development, and [262, 263].InMay2008,thefirstPhaseIclinicaltrialfor cell death [6, 244–247]. The transactive NF-𝜅Bsuppresses thetreatmentofsolidtumorsinvolvingsystemicdelivery apoptosis by inducing the expression of some apoptosis- of siRNA via targeted nanoparticles was initiated (Clini- inhibitory genes, including IAPs, cFLIP, TNF receptor asso- calTrials.gov identifier: NCT00689065)[262]. Importantly, ciated factor 1 (TRAF1), and TRAF2 [248]. Two typical data from this trial demonstrated, for the first time, that prosurvival NF-𝜅B targets are Bcl-xL and XIAP, which can systemically delivered siRNA can reduce protein levels of a block apoptosis at multiple steps [249, 250]. Importantly, specific gene through an RNAi mechanism in humans [264]. Chuang et al. demonstrated that a wide range of cytotoxic This finding opens the gate for future application of siRNA drugs (5-FU, doxorubicin, paclitaxel, and cisplatin) activated as a gene-specific tool for cancer treatment, clinically. These NF-𝜅Binapanelofcancercelllines[251]. This suggests that applications facilitate targeting the antiapoptotic genes’ prod- NF-𝜅B activation is a general feature of cancer cell response ucts to kill cancer cell either alone or in combination with to chemotherapy. For instance, increased NF-𝜅Bactivity classic therapies. Recently, c-FLIP and other antiapoptotic in patients with oesophageal cancer has been correlated proteins are promising targets for the development of RNAi with reduced response to neoadjuvant chemotherapy and therapeutics [101]. As well as RNAi-mediated downregulation radiotherapy [252]. Now it is believed that NF-𝜅Bappears of c-FLIP, RNAi-based reduction of Bcl-2, Bcl-xL, XIAP, and to be a critical determinant of drug resistance; its activation survivin has also been shown to sensitize cancers cells to a reduces apoptosis by chemotherapy [247]. A number of in range of cancer therapies, including chemotherapeutic drugs vitro studies demonstrated that inhibition of NF-𝜅Bsensitizes and TRAIL [265]. cancer cells to chemotherapy-induced apoptosis [253–255]. Furthermore, NF-𝜅Bisbelievedtobeamajortargetfor proteasome inhibitors, as proteasome inhibition prevents 3.7. Targeting Heat Shock Chaperons degradation of I𝜅B, blocking NF-𝜅B nuclear translocation 3.7.1. HSP90. Heat shock proteins 90 (Hsp90s) are abun- [255, 256]. Clinical trials with proteasome inhibitors such as dantly and ubiquitously expressed proteins required for the bortezomib are underway to inhibit NF-𝜅B signalling and energy-driven stabilisation, conformation, and function of enhance drug-induced apoptosis in cancers. a large number of cellular proteins, named clinets [266, 267]. Several key Hsp90 proteins are involved in the pro- 3.6. Targeting Multiple Pathways Regulators. Like antiapop- cesses characteristic to the malignant phenotype, such as totic Bcl-2 proteins overexpression, IAPs overexpression invasion, angiogenesis metastasis, and treatment resistance is associated with poor prognosis and chemoresistance in [268–270]. They also contribute to leading mechanism of many cancers [257–259]. It acted as a therapeutic target mitogen-activated protein kinases (MAPK) and nuclear fac- for apoptosis-inducing strategies [260]. Antisense oligonu- tor kappa B (NF-𝜅B) induction [271–273]. Moreover, Hsp90 cleotides against XIAP and survivin have been developed stabilises Raf-1, Akt, and ErbB2 proteins [274–276], which [249]. In addition small-molecule inhibitors that bind to the are known to be associated with protection against radiation- BIR2 or BIR3 domain of XIAP, cIAP1, and cIAP2 enhance induced cell death [277–279]. Molecular studies suggested apoptosis [249]. Most of small-molecule IAP antagonists are that inhibiting HSP90 by targeted therapy could provide SMAC mimetics, several of which are in advanced preclinical a promising strategy to enhance apoptosis by conventional BioMed Research International 13 therapies [280–286]. The inhibitor of Hsp90, geldanamycin, in vivo experiments. That opened the gate for inventing the and its derivatives successfully enhance the radiosensitivity first generation ASO in which the phosphoryl oxygen of of tumor cell lines derived from different origins, including DNA was replaced with a sulphate to create a phosphothiate glioma, prostate, pancreas, and cervix [276, 280–284, 287]. backbone, ASOs, intended for cancer therapy. Recently, more However, geldanamycins have several limitations, includ- modifications were made to the ribose, the 20-position, ing poor solubility, formulation difficulties, hepatotoxicity, to improve the pharmacokinetic characteristics of second- and extensive metabolism by polymorphic enzymes, along generation phosphothioate ASOs, for example, OGX-011 with drug efflux by P-glycoprotein; therefore, efforts to [313]. design small synthetic inhibitors of Hsp90 with improved In advanced breast cancer, targeting CLU by OGX-011 bioavailability and lower toxicity are devoted [288–291]. Now, enhanced the apoptotic effect by Trastuzumab, an HER- pyrazole resorcinol compounds act as stronger inhibitors 2-targeted monoclonal antibody used in the clinical man- of Hsp90 than geldanamycin derivatives. Currently, the agement of advanced breast cancer patients. Importantly, isoxazole resorcinol NVP-AUY922 shows the highest affin- only the combination of OGX-011 and Tratuzumab leads to ity for the NH2-terminal nucleotide-binding site of Hsp90 activation of apoptosis that was not observed with either [290, 291], whereas NVP-BEP800 represents a novel fully agent alone. Phase I and phase II studies have suggested a synthetic, orally available 2-aminothieno [2,3-d] pyrimidine combined therapy using ASOs against both of CLU and Bcl-2, class Hsp90 inhibitor [292]. Both compounds have good along with Trastuzumab for advanced breast cancer patients pharmaceutical and pharmacological properties. They also [314, 315]. Targeting CLU by ASO enhanced apoptosis by exhibit strong antiproliferative activity against various tumor paclitaxel or radiation in PC-3, prostate cancer cells [314]. cell lines and primary tumors in vitro and in vivo at well- Phases I and II trials in prostate cancer have shown tolerated doses [292]. The novel inhibitors of Hsp90 can that OGX-011 significantly enhanced the apoptotic response sensitize various tumor cells to apoptosis by destabilisation andefficacyofchemotherapy,radiation,andandrogenwith- and depletion of several Hsp90 client proteins, thus causing drawal by inhibiting the expression of CLU. In other prostate the depletion of S phase and G2/M arrest by heat shock, cancer phase I trials, OGX-011 was given prior to radical increased DNA damage, and repair protraction and finally prostoctomy, while some “well tolerated” phase II trials were apoptosis [293]. established based on the biologic effectiveness of OGX-011 [315]. 3.7.2. Clusterin. Clusterin (CLU) is a cytoprotective heat In our laboratory we reported for the first time the asso- chaperoneprotein,involvedinnumerousphysiologicalpro- ciation between upregulation of the sCLU and persistence cesses important for carcinogenesis and tumor growth, against apoptosis by paclitaxel in ovarian cancer cells and including apoptotic cell death, cell cycle regulation, DNA human tumors. Importantly, when we targeted CLU either repair, cell adhesion, and theromprotection [294]. It is now by OGX-011 or RNAi we successfully enhanced caspase- accepted that the primary function of the 67–80 kDa cyto- dependent apoptosis and the cells restored their sensitivity plasmicCLU(sCLU)proteinformiscytoprotectiveasastress to paclitaxel [144]. Similarly, CLU ASO enhanced cisplatin- response gene. sCLU expression is consistently reported to induced apoptosis, human bladder cancer cells, and human beincreasedwithmanyapoptosis-inducers,chemotherapyor lung adenocarcinoma both in vitro and in vivo [304]. radiotherapy, treatment as an adaptive cell survival molecule in vivo and in vitro to block apoptosis [295]. That was Conflict of Interests alsoshowninhumantumortissuesfromprostate[296, 297], kidney [298], breast [299], ovarian [300], colon [301], The authors declare that there is no conflict of interests lung [302], pancreas [303], cervix [304], glioma [305], and regarding the publication of this paper. anaplastic large cell lymphoma [306]. It has recently been shown that sCLU knockdown in References human cancer cells, using siRNA-mediated CLU gene silenc- ing, induces significant reduction of cellular growth and [1] R. A. Lockshin and C. M. Williams, “Programmed cell death-I. higher rates of spontaneous apoptosis [294]. 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