*4.2.2. Antisense oligonucleotides*

The single-stranded antisense oligodeoxynucleotides (AS ODNs) are short stretches of synthetic DNA, approximately 12–30 nucleotides long, and are complementary to a specific mRNA strand. Hybridization of the AS ODNs to the mRNA by Watson–Crick base pairing prevents the target gene from being translated into protein, thereby blocking the action of the gene, and resulting in the degradation of the target mRNA. The specificity in the AS ODN approach is based on the fact that any sequence of approximately 13 bases in RNA and 17 bases in DNA is estimated to be represented only once in the human genome. AS ODNs targeting survivin expression in human lung adenocarcinoma cell lines decreased survivin protein levels in a dose-dependent manner, induced apoptosis, stimulated higher levels of caspase-3 activation, and increased the sensitivity of cells to chemotherapeutics. XIAP AS ODNs effectively down-regulated both specific mRNA and protein levels in human non-small cell lung cancer growth both in vitro and in vivo. XIAP AS ODNs effectively induced apoptosis on their own and sensitized the tumor cells to the cytotoxic effects of several chemotherapeutics, including Taxol, etoposide and doxorubicin [26]. Furthermore, the administration of XIAP AS ODNs in a xenograft model of human nonsmall cell lung cancer results in a significant down-regulation of XIAP protein [124].

Apoptosis as a Therapeutic Target in Cancer and Cancer Stem Cells: Novel Strategies and Futures Perspectives 129

Smac interacts with IAPs via its N-terminal tetrapeptide, Ala1- Val2-Pro3-Ile4 (AVPI). Using this structural information, several groups designed small molecules mimicking AVPI to derive proteolytically stable compounds. Monovalent Smac mimetics were designed to mimic the Smac AVPI-binding motif and so target the XIAP-BIR3 domain. They exhibit high affinities, not only to XIAP-BIR3 but also to cIAP1, cIAP2 and MLIAP proteins. Bivalent Smac mimetics, containing two AVPI-binding motifs, bind to XIAP-BIR2–BIR3 with an extremely high affinity, exceeding that of Smac protein. Preclinical profiling studies have shown that Smac mimetics effectively sensitize cancer cells to other therapeutic agents, but they are also capable as single agents of inducing apoptosis in some but not all human cancer cell lines. To date, two Smac mimetics have reached phase I clinical development, and approximately ten are in an advanced preclinical development stage and are expected

Smac mimetic binds to XIAP and induces cIAP degradation. There are two cellular events that result from cIAP degradation: (1) activation of the non-canonical NF-κB pathway and subsequently increased production of autocrine TNF-α and (2) release of RIP1 from the TNF-α receptor complex, leading to caspase-8 activation, which requires TNF-α receptor to be activated in the first place. Accumulating evidence suggests that whether or not Smac mimetic induces autocrine TNF-α production is the key factor in deciding the cell's fate upon Smac mimetic treatment. Depending on their response to Smac mimetics cells can be grouped into three general classes: in class I cells, Smac mimetic induces autocrine TNF-α production and massive cell loss; in class II cells, Smac mimetic still induces autocrine TNFα production but this does not have a major effect on the cell population; and in class III cells, Smac mimetic has no effect on autocrine TNF-α production or on cell death. The ability of cells to produce TNF-α is necessary but not sufficient for Smac mimetic to induce cell

The miRNAs, is a class of endogenous, small, non-coding RNAs of 18–25 nucleotides in length, that negatively regulates gene expression by degradation of mRNA or suppression of mRNA translation. Mature miRNA products are formed in from a longer primary miRNA (pri-miRNA) transcript through sequential processing by the ribonucleases Drosha and Dicer1 [143,144]. miRNAs are known to repress thousands of target genes because only partial complementarity to the target mRNA is required. Thus, one miRNA may be simultaneously targeting a complexity of mRNAs as well as the expression of a single

The miRNAs are involved in normal processes, including cellular development, differentiation, proliferation, apoptosis, and stem cell self-renewal [28].The aberrant expression or alteration of miRNAs contributes to a range of human pathologies, including cancer. Furthermore, the deregulation of miRNA causes evasion of apoptosis which involved tumourigenesis and drug resistance [122]. During tumour initiation and progression, the functionality of aberrant miRNAs may act as oncogenes (OncomiRs) or tumour suppressors (TSmiRs), a numbers of

to enter human clinical testing soon [141].

death as a single agent [142].

**5. miRNA-based therapy**

mRNA may be regulated by many miRNAs [143].

AEG35156 is a 19-mer oligonucleotide targeting XIAP. Its sequence was designed to achieve maximal stability and potency and to minimize immunostimulation through avoiding CpG motifs. Phase I studies in patients with refractory malignancies established safety [136]. A phase I/II study of AEG35156 in combination with idarubicin and high-dose cytarabine in patients with relapsed or refractory AML demonstrated a dose-dependent knock-down of XIAP mRNA and protein and a promising response rate [137]. The molecule may rapidly enter randomized studies in AML while being also tested in lymphomas. The dose-limiting toxicity of an antisense oligonucleotide designed to inhibit surviving mRNA expression (LY2181308) was headache and the compound demonstrated some biological efficacy in decreasing survivin expression [138]. A phase II study has opened in solid tumours. The locked nucleic acid strategy was used to design other survivin-targeting antisense oligonucleotides, including SPC3042 [139] and EZN-3042 [140], which are currently being evaluated in Phase I clinical trials as single agents and in combination with cytotoxic drugs.

## *4.2.3. Smac-mimicking IAP antagonist*

Smac mimetics may be a useful therapeutic target as over-expression of Smac may potentiate apoptosis by neutralizing the caspase-inhibitory function of IAPs. Following the discovery that an IAP-binding motif consisting of four NH2-terminal amino acid residues was sufficient to bind to the BIR3 domain of XIAP, Smac-peptide mimetics were constructed which were capable of competing with caspase-9, displacing it from the BIR3 domain of XIAP. The three members of the IAP family, XIAP, cIAP1, and cIAP2, are structurally homologous (XIAP amino acid sequence identity to cIAP1 and cIAP2 of 36% and 39%, respectively, the amino acid sequence identity between cIAP1 and cIAP2 is 70%). In particular, the BIR3 IBM region is well conserved among the three IAPs. The XIAP BIR3 residues involved in van der Waals contacts (Val298, Lys299, and Trp310) and hydrogen bonds (Gly306, Leu307, and Trp323) with the inhibitory compounds are conserved. Minor exceptions are Leu292, replaced by Val in the cIAPs, Glu314 substituted by Asp in both cIAPs, and Gln319, which is Glu325 in cIAP1, and Gln311 in cIAP2. Finally, residues Thr308 and Asp309 that were found relevant for Smac-mimetics interaction with XIAP BIR3, are replaced by Arg314/Arg300 and Cys315/ Cys307 in cIAP1/cIAP2, respectively [127].

Smac interacts with IAPs via its N-terminal tetrapeptide, Ala1- Val2-Pro3-Ile4 (AVPI). Using this structural information, several groups designed small molecules mimicking AVPI to derive proteolytically stable compounds. Monovalent Smac mimetics were designed to mimic the Smac AVPI-binding motif and so target the XIAP-BIR3 domain. They exhibit high affinities, not only to XIAP-BIR3 but also to cIAP1, cIAP2 and MLIAP proteins. Bivalent Smac mimetics, containing two AVPI-binding motifs, bind to XIAP-BIR2–BIR3 with an extremely high affinity, exceeding that of Smac protein. Preclinical profiling studies have shown that Smac mimetics effectively sensitize cancer cells to other therapeutic agents, but they are also capable as single agents of inducing apoptosis in some but not all human cancer cell lines. To date, two Smac mimetics have reached phase I clinical development, and approximately ten are in an advanced preclinical development stage and are expected to enter human clinical testing soon [141].

Smac mimetic binds to XIAP and induces cIAP degradation. There are two cellular events that result from cIAP degradation: (1) activation of the non-canonical NF-κB pathway and subsequently increased production of autocrine TNF-α and (2) release of RIP1 from the TNF-α receptor complex, leading to caspase-8 activation, which requires TNF-α receptor to be activated in the first place. Accumulating evidence suggests that whether or not Smac mimetic induces autocrine TNF-α production is the key factor in deciding the cell's fate upon Smac mimetic treatment. Depending on their response to Smac mimetics cells can be grouped into three general classes: in class I cells, Smac mimetic induces autocrine TNF-α production and massive cell loss; in class II cells, Smac mimetic still induces autocrine TNFα production but this does not have a major effect on the cell population; and in class III cells, Smac mimetic has no effect on autocrine TNF-α production or on cell death. The ability of cells to produce TNF-α is necessary but not sufficient for Smac mimetic to induce cell death as a single agent [142].
