*2.3.1 Therapeutic opportunities based on Bcl-2 family proteins modulation*

In view of the critical role of Bcl-2 proteins in regulation of mitochondrial pathway of apoptosis, targeting various members of this family have been considered amongst the most promising therapeutic strategies in cancer, a well-known dysfunctional apoptosis disorder [117]. Numerous attempts have been carried out to target the modifications in Bim expression and therefore regulate tumor cell response to apoptosis. Histone deacetylase inhibitors have been shown not only cause to up regulation of Bim in transformed cells, but also they are able to reverse silencing of Bim in cancer cells and consequently restored their sensitivity to various anticancer-agents reported in leukemia and Burkitt's lymphoma cells [118]. The proteasome inhibitors are also recognized to promote accumulation of Bim and enhance the lethality of cancer cells [119, 120]. Another approach is through diminishing its degradation by blocking its phosphorylation. Ras/Raf/MEK/ERK pathway have a key role in regulating the expression and function of Bim through its phosphorylation and triggering its proteasomal degradation. MEK1/2 Inhibitors has been applied to disrupt this process leading to accumulation of Bim and consequently apoptosis. MEK1/2 Inhibitors are also able to modify the interaction between BIM and other Bcl-2 family members contributing to cell death [118, 121, 122].

Furthermore, structure-based drug design can be applied to discover anticancer agents which are able to effectively activate a pro-apoptotic Bcl-2 protein through changing its conformation promoting cell death. Bax as a unique entry point for intrinsic apoptotic signaling is another major pro-apoptotic member of the Bcl-2 family proteins which has been greatly getting attention to be targeted in order to control apoptosis. Recent studies have revealed that direct binding and activation of Bax can be a promising approach for cancer treatment. Discovery of small-molecule functioning as a Bax activators may result in selective induction of tumor cell apoptosis and overcome chemoresistance which has been proved through invitro and invivo studies [117, 123]. Besides, some studies targeting a regulatory site in Ser184 of Bax protein have determined that its agonists SMBA1–SMBA3 can effectively bind to and trigger its oligomerization through the suppression of its phosphorylation that eventually lead to cyrochrome c release and induction of apoptosis in mouse lung cancer xenografts [124]. Similar results were also reported with other Bax agonists as promising Bax direct activators in breast cancer, glioblastoma and acute myeloid leukemia cells. These drug candidates demonstrated noteworthy in vivo efficiency inhibiting xenograft tumor growth though induction of apoptotic cell death [125–127].

The next emerging strategy in cancer drug discovery was the BH3 mimetics which are able to antagonize the function of Bcl-2 and selectively kill cancer cells. In this approach, BH3 mimetics are antagonists of the anti-apoptotic Bcl-2 proteins. These small molecules acting as the competitive inhibitors induce apoptosis though binding to their hydrophobic cleft and therefore affect the interactions between

anti- and pro-apoptotic proteins [128]. Various BH3 mimetics with different level of specificity and efficiency have been reported. For instance, TW-37 derived from phenolic aldehyde gossypol has been showing high affinity to bind MCL-1, Bcl-2 and Bcl-xL anti-apoptotic proteins and induce apoptosis in B-cell lymphomas and pancreatic cell lines along with decreasing tumor size in xenograft models [129–131]. As ABT-737 mimicked the BH3 domain of Bad protein, it was able to bind selectively to Bcl-2, Bcl-xL and Bcl-W. It also demonstrated poor affinity to other member of ani-apoptic proteins including MCL-1 and BFL-1. ABT-737 has shown efficacy in the killing of several cancer cell lines including leukemia, lymphoma, multiple myeloma, glioma and small cell lung cancer cell lines as well as primary samples. Also, these two inhibitors of Bcl-2 families are currently in clinical trials [132–134].

Another approach to antagonize the function of Bcl-2 anti-apoptotic proteins is focusing on the protein interaction among members of Bcl-2 family through their essential death domain. In this regard, peptide-based inhibitors have been significant achievements in targeting and regulating intracellular protein–protein interaction. Stapled peptides are synthetic, bioactive α-helical peptides locked into their bioactive structure that have brought new hope to target drug discovery [135, 136]. For instance, stabilized alpha-helix of Bcl-2 domains, SAHBs, is the peptide having the ability to penetrate leukaemia cells and trigger induction of apoptosis through its binding to the Bcl-xL which its function has been further confirmed though invivo mouse xenograft models of leukaemia [137]. Another research study has also revealed that exclusive MCL-1 stapled peptide inhibitor (MCL-1 SAHBD) can effectively resensitize cancer cells to caspase-mediated apoptosis through directly targeting of MCL-1 and suppress its inhibitory interaction with Bak protein [138].

### *2.3.2 Therapeutic opportunities based on caspase modulation*

Given the vital role of caspases in the regulation of apoptosis, it is not surprising that numerous therapeutic opportunities targeting caspase activity demonstrate great promise for the cancer treatment. Different strategies have been investigated to upregulate caspase-8 expression to restore its function in tumors. As hypermethylation of its promotor has been recognized as the main mechanism of silencing, one approach for its reactivation is using demethylation agents. Azacytidine, decitabine and nucleoside analogs promoting the demethylation of caspase-8 promotor have been successfully applied in neuroblastoma, medulloblastoma, breast cancer and lung carcinoma [139, 140]. Another interesting strategy is designing the small molecules that selectively and directly target and trigger caspase-8 activation. These small molecules has been reported to potentiate TRAIL-induced cell death [141]. Proteosomal inhibitors such as bortezomib has been also reported to increase total cellular caspase-8 levels apparently by blocking its degradation [111, 142]. Some studies have also reported that the use of interferons can elevate the caspase-8 expression through modification at transcriptional level. This strategy targeting interferon-sensitive response elements within the caspase-8 promoter leading to sensitize cancer cell to apoptotic cell death in cancer chemotherapy or irradiation therapy [139, 143, 144].

Besides, developing molecules that are able to directly activates caspase 3 have been of research interests as well. For this purpose, particular sequence of inactive procaspase-3 consisting of the triplet of aspartic acid residues has been targeted. In vitro studies have exhibited that PETCM, gambonic acid and its derivatives have the potential to effectively activate caspase 3 leading to apoptotic cell death in cancer cell lines [145–147]. Furthermore, procaspase-activating compound1 (PAC-1) has been shown to induce anticancer activity through promoting the procaspase-3 activation. PAC-1 exerted its effect by chelation of inhibitory labile zinc ions and currently is in phase I clinical trial for cancer treatment [148].

#### *The Role of Apoptosis as a Double-Edge Sword in Cancer DOI: http://dx.doi.org/10.5772/intechopen.97844*

In order to sensitize tumor cells to apoptotic stimuli, caspase −9 can be also regarded as a potential target in cancer therapy. There are a wide range of molecules such as protein kinase, microRNAs and heat shock protein that have been identified to modulate caspase-9 expression and hence have been getting interest as candidates for new drug development though regulating intrinsic apoptosis in cancer cells [149, 150]. Targeting caspase-9 have been also initiated in clinical trials (phase I) against several cancer including Chronic Myeloid Leukemia, non-Hodgkin's lymphoma, Acute Lymphoblastic Leukemia, [151, 152].

In addition, several attempts have also been conducted on cancer gene therapy focusing on apoptotic caspases. Gene transfer technologies may restore caspase gene expression resulting in selectively induction of apoptosis in various tumor types [153–155]. In this regard, caspase-9 and caspase-3 has been suggested for being used in gene therapy strategies. A main benefit of involving these caspases is that they start apoptosis at the downstream of the mitochondrial outer membrane potential and they will not be affected with the enhanced expression of anti-apoptotic of Bcl-2 proteins. The researchers conducted on inducible version of these caspases have shown encouraging results related to remarkable reduction in size of lung and gastric tumors, respectively [156–158].

Other than directly targeting of caspases, another area of research has focused on discovery of anticancer agents that trigger the caspases activity indirectly. In this approach, certain members of the inhibitors of apoptosis proteins (IAP) are targeted. IAPs are functioning as the endogenous caspase inhibitors and prevent apoptosis event by binding and inhibiting caspases through the degradation of active caspases or keeping them away from their substrate. In this regard, numerous researches have investigated various IAP inhibiting agents, accomplishing a breakthrough in cancer treatment [159, 160]. Some of these agents are acting as the IAP antagonist and exert their effect via suppression of their activity, while others are analogs of the endogenous IAP inhibitor Smac. Several Smac mimetics such as LCL161 and birinapant IAP inhibitors have currently being tested in phaseI/II in clinical trials, with promising outcomes [161–164]. Besides, IAP inhibitors have been reported to exert the synergistic effect in combination chemotherapy and sensitize the cancer cells to radiotherapy which is of particular interest in malignant gliomas [165–167].

## **3. Conclusion**

It is well established that the apoptosis dysfunction promotes the malignant transformation and renders the cancer cell resistant to treatment. Targeting apoptotic pathways in tumor cells has been a main clinical interest as the evasion of apoptosis is a hallmark of all cancers regardless of their causes or types. There are numerous defects found in apoptotic mechanism contributing to inhibition of cancer cell death. As demonstrated in this chapter, impaired activation of caspases and disturbance in the balance between anti-apoptotic and pro-apoptotic members of Bcl-2 family proteins are remarkably involved in tumorgenesis. The enhanced knowledge about their critical roles in apoptosis and cell fate in recent years has eventually made them promising therapeutic targets. This also has facilitated the generation of more specific anticancer agents and led to shifting in anticancer therapy form typical cytotoxic approaches to the designing and development of apoptosis-inducing drugs that particularly target the cancer cells. An exciting development in successful eradication of cancer cells involves structure-based drug design of small molecules such as BH3 mimetics, specifically targeting Bcl-2 proteins, that is currently being tested in clinical trials with promising effects of

selective induction of tumor cell apoptosis and overcoming chemoresistance as well. These inhibitor molecules are in continuous development and a great deal of effort is required to discover the most efficient ones having more specificity for individual Bcl-2 proteins and offer maximal clinical efficacy. Besides, new therapeutic applications targeting apoptotic caspases including gene therapy approaches and small molecules suppressing inhibitors of caspases are beginning to show some promise through selectively and directly targeting of individual caspases and eventually triggering their activity. Caspase-targeted approaches, epigenetic modulators and their combinations with established therapies may have the potential to overcome the limitation of previous strategies through exerting synergistic pro-apoptotic activity and may enhance the effectiveness of conventional cancer therapy, worthy of further investigation in preclinical advanced models and clinical trial. Apoptosis-targeted therapies are now remarkably advancing and remain a promising approaches in future clinical practice of oncology.
