Bcl-2 Targeted-Therapy for the Treatment of Head ...

Recent Patents on Anti-Cancer Drug Discovery, 2011, 6, 000-000

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Bcl-2 Targeted-Therapy for the Treatment of Head and Neck Squamous Cell Carcinoma Lucas V. dos Santos1 and André L. Carvalho2;3 1

Medical Oncology Department, Barretos Cancer Hospital, Barretos -SP, 14784-400, Brazil, 2Head and Neck Department, Barretos Cancer Hospital, Barretos -SP, 14784-400, Brazil, 3Teaching and Research Institute, Barretos Cancer Hospital, Barretos -SP, 14784-400, Brazil

Received: May 12, 2010; Accepted: July 21, 2010; Revised: November 15, 2010

Abstract: Despite recent advances in treatment of head and neck cancer, survival has not been improved as expected. Bcl2 family proteins play important role in regulating apoptosis. Recently newer molecules have been developed for inhibition of Bcl-2 related proteins. We herein aim to discuss the importance of Bcl-2 family proteins to the development of head and neck cancer and how the targeted-therapy for inhibition of Bcl-2 and related proteins, based on new drugs and recent patents, could improve the efficacy of the systemic therapy.

Keywords: anticancer drugs, apoptosis, Bcl-2 family proteins, head and neck cancer, patents, targeted-therapy. INTRODUCTION Head and neck cancer is the ninth in incidence in United States, with almost 50.000 estimated new cases in 2009,[1] and more than 500.000 worldwide [2]. The most common histology is squamous cell carcinoma (SCC), which comprises more than 90% of HNSCC. The incidence of HNSCC is higher in less developed countries [2, 3]. Smoking and alcohol intake are the major risk factors, although other factors are known, including human papillomavirus (HPV) infection and occupational exposure [4, 5]. The five-year survival rate is 82% for patients with stages I and II, 53% for patients with stage III and 28% for patients with stage IV[1]. Recent advances in the management of HNSCC had increased the survival for some subsites of HNSCC in the past few decades, however this increment in survival was lower than expected [6]. Two modalities of treatment are useful for localized HNSCC: surgery and radiotherapy. Surgery and radiation therapy lead to similar survival rates for localized disease. The treatment of locally advanced disease must be multimodal: radiochemotherapy or surgery followed by radiochemotherapy [7]. Chemotherapy remains the backbone of treatment for metastatic disease [4]. Nowadays we have directed our attention to new treatment modalities: induction chemotherapy and molecular targeted therapy. Induction chemotherapy has been studied in some trials, but survival improvement with this approach has not yet been shown [8]. Cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, has *Address correspondence to this author at the Head and Neck Department, Barretos Cancer Hospital, 1331 Antenor Duarte Vilella St, Barretos -SP, 14784-400, Brazil, Teaching and Research Institute, Barretos Cancer Hospital, 1331 Antenor Duarte Vilella St, Barretos -SP, 14784-400, Brazil; Tel:+551733216600; Ext: 7009; Fax:+55173321638; E-mial: [email protected]

1574-8928/11 $100.00+.00

proven activity against metastatic or recurrent chemotherapy naïve HNSCC, but no definitive results were shown for locally advanced treatment disease [9, 10]. Bcl-2 (B-cell lymphoma/leukemia-2) family proteins play important role in regulating apoptosis [11]. Over the last few years, a new class of anticancer drugs has been developed: inhibitors of Bcl-2. The modulation of apoptosis seems to be an interesting way for enhancing the efficacy of HNSCC treatment. The aim of this article is to review the current evidence of molecular mechanisms of Bcl-2 family in development of HNSCC. We will, herein, discuss the potential effect of Bcl-2 inhibitors on the treatment of HNSCC. APOPTOTIC PATHWAY Apoptosis is a genetically programmed cell death in which the cell destroys itself via activation of endogenous proteases and endonucleases, and it is characterized by nuclear condensation, membrane blebbing, DNA fragmentation and cell shrinkage [12]. Apoptosis initiates through extrinsic or intrinsic pathways, and both converge to caspase activation. In the former, a death stimulus like tumor necrosis factor alpha (TNF-) or Fas ligand (FasL) activates a transmembrane receptor, that leads to recruitment of adaptor proteins like “Fas-associated protein with death domain” (FADD), which activates caspase-8 and thus caspase-3 and 7. This process leads to further amplification of caspase signal, which culminates to proteolysis and cell death. In the intrinsic pathway (also called mitochondrial pathway), cell stress or damage activate one or more members of BH3-only proteins family. This activation permits the efflux of mitochondrial intermembrane space proteins like cytochrome C (Cyt C). Caspase-9 is then activated, and this process implicates the activation of other caspases. Irrespective of the route of caspase activation, caspases remain the main effectors of apoptosis [13, 14]. © 2011 Bentham Science Publishers Ltd.

Santos1 and Carvalho

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The mitochondrial apoptotic pathway is controlled by the balance between pro and anti-apoptotic Bcl-2 protein family members [15, 16]. This pathway can be initiated by some factors like increasing in intracellular reactive oxygen species, DNA damage, unfolded protein response and deprivation of growth factors. These stimuli ultimately leads to increased mitochondrial outer membrane permeability, and thus permitting the release of proapoptotic proteins like cytochrome C and diablo homologues from the mitochondrial intermembrane space [17-19]. Finally, this process leads to the activation of caspase-9, which in turn, triggers the mobilization and activation of caspase-3, 6 and 7, resulting in degradation of proteins and the activation of apoptotic effectors [13, 14, 19] Fig. (1). The seminal discovery that Bcl-2 inhibits apoptosis rather than promotes cell survival contributed to the acceptance that evading apoptosis is a crucial step in tumorigenesis [20-22]. Bcl-2 family encompasses proteins with either proapoptotic or anti-apoptotic activity that share a common genetic region

called Bcl-2 homology domain (BH) [23-29]. Members with pro-survival activities such as Bcl-2 and “B cell lymphoma extra-large” (Bcl-xL) share up to four BH domains, whereas the proapoptotic proteins “Bcl-2 associated X protein” (Bax) and “Bcl-2 homologous antagonist killer” (Bak) contain only three BH domains (BH1-3). BH3-only proteins such as “BH3 interacting domain death agonist” (Bid), “Bcl-2associated death promoter” (Bad) and “Bcl-2-interacting killer” (Bik) are proapoptotic members that share only the BH3 region of homology [30-33] Fig. (2). Anti-apoptotic Proteins Bcl-2 is the anti-apoptotic protein whose functions were mostly studied. Overexpression of Bcl-2 and its relatives Bcl-xL, “Bcl-2-like protein 2” (Bcl-w), “myeloid cell leukemia 1” (Mcl-1), “Bcl-2 related protein A1” (A1) and “Bcl-2-like protein 10” (Boo) protect cell against apoptosis induced by cytotoxic stimuli [24]. These proteins share the BH1-3 domains. They also share a labile hydrophobic

Fig. (1). The interactions among Bcl-2 family proteins and the intrinsic and extrinsic apoptotic pathways. APAF-1: apoptotic proteaseactivating factor-1. Bcl-2 family proteins play important role in both apoptotic pathways, with multiple interactions and cross-talking.

Bcl-2 Targeted-therapy for HNSCC

Recent Patents on Anti-Cancer Drug Discovery, 2011, Vol. 6, No. 1

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Fig. (2). Bcl-2 family proteins and the conserved Bcl-2 homology domain. Proteins are separated according to molecular activity. TM: transmembrane domain.

groove on tertiary folding, which is essential for antiapoptotic activity and for binding of proapoptotic molecules [29, 34-37]. Multidomain Proapoptotic Bcl-2 Proteins Bax and Bak are the most important and studied members of this group, whose structures are similar to Bcl-2 [38]. These two proteins are the most important in initiating the apoptotic process in mitochondria, and both seem to have redundant function [31, 39]. BH3-only Proteins The BH3-only subset of proapoptotic proteins results in apoptosis either by inhibiting Bcl-2/Bcl-xL or activating Bax/Bak [30, 33-37, 40-42]. The BH3 domain is necessary for both activities [28]. Mutations in this domain reduce the affinity of BH3 for their targets and thus impair their proapoptotic activity [36, 37]. Bcl-2 family Proteins and Cancer Development Bcl-2 family proteins are the main regulators of the mitochondrial pathway of apoptosis [15, 16, 43-45]. Although having 3-dimensional structure similar to Bcl-2, Bax and Bak promote apoptosis rather than inhibiting it [38]. Deficiency of Bax or Bak alone causes only minor changes, but the combined loss of these two compounds not only blocks apoptosis, but also protects the cells against cytotoxic stimuli [31, 39, 46]. Proapoptotic Bcl-2 family proteins induce mitochondrial membrane permeabilization (MMP) that triggers the release of proapoptotic proteins (e.g., cytochrome c) from the intermembrane space. The MMP is a consequence of homo or hetero-oligomerization of Bax and Bak that forms pores in the mitochondrial membrane [47, 48]. Bcl-2 or other Bcl-2 family proteins with pro-survival activity protect cells against apoptosis induced by a diversity

of cytotoxic stimuli [24]. Each of them has remarkable importance depending on the nature of cell [49-60]. They block apoptosis by preventing BH3-only protein-induced oligomerization of BAX and/or BAK in mitochondrial outer membrane, by direct inhibition of Bax or Bak, or by suppressing the release of calcium by the endoplasmic reticulum [41, 61-66]. Bh3-only proteins promote apoptosis inhibiting antiapoptotic Bcl-2 family proteins or interacting with Bax and Bak [23, 62-64, 67, 68]. In the first, BH3 domain binds to the hydrophobic groove of anti-apoptotic proteins neutralizing them [34, 35, 63, 69]. In the former, BH3 domain interacts with Bax and Bak and activates them, inducing apoptosis [42, 70-72] Fig. (3). It has been proposed that BH3-only proteins act on inhibiting pro-survival Bcl-2 family proteins in a specific manner, and that these proteins do not share most of their functions [73]. For example, “Bcl-2 interacting mediator of cell death” (Bim) and “p53 upregulator of apoptosis” (Puma) have non-specific function and they can bind and inhibit all pro-survival Bcl-2 family proteins, whereas Bad or “Bcl-2 modifying factor” (Bmf) can only counteract Bcl-2 and BclxL, and Noxa (Latin from damage) essentially inhibits only Mcl-1 and A1 [63]. Some of BH3-only proteins, like Bim, tBid (the truncated and activated form of Bid) and Puma can directly induce Bax and Bak oligomerization at the same time they block pro-survival proteins [23, 31, 63]. Recent evidence suggests that both direct and indirect activation models are important for inhibition of pro-survival Bcl-2 family proteins [74]. BCL-2 FAMILY PROTEINS AND SQUAMOUS CELL CARCINOMA OF HEAD AND NECK The Bcl-2 family proteins play an important role in controlling the mitochondrial pathway. Cellular stress leads to apoptosis changing the balance between pro and antiapoptotic proteins [75]. These proteins can be modulated at



Fig. (3). The interaction among Bcl-2 family proteins and their suggested role in mitochondrial pathway. Apoptotic inducers are shown in black and apoptotic inhibitors in gray.

translational or post-translational level [12]. Reactive oxygen species (ROS) control pro and antiapoptotic proteins through phosphorilation and ubiquitination of Bcl-2 family proteins, increasing the expression of proapoptotic and decreasing the expression of antiapoptotic proteins [76]. There is an important cross-linking among proteins of many signaling pathways, like phosphatidylinositol 3-kinase (PI3K), nuclear factor kappa B (NF- B) and signal transducers and activators of transcription 3 (STAT3) pathways. All of these pathways can modulate the apoptotic pathway in HNSCC [77]. Anti-apoptotic Bcl-2 Family Proteins Smoking is more than an etiologic factor for HNSCC. It is also an adverse prognostic factor for patients with locally advanced HNSCC, and there is an association between Bcl-2 overexpression and smoking [78]. On the other hand, there is no association between Bcl-2 expression and human papillomavirus infection [79, 80]. Tumors with higher expression of anti-apoptotic Bcl-2 family proteins have lower index of apoptosis compared to those with lower Bcl-2 expression [81, 82]. Bcl-2, Bcl-xL and Mcl-1 overexpression are associated to poor differentiation and advanced stage neoplasms [81-89]. Bcl-2-associated anthanogene (Bag-1) binds to Bcl-2 protein and enhances its activity. Its overexpression, specially the nuclear pattern, is associated to high grade lesions, advanced stage, poor-response to therapy and worse prognosis [90-93]. Bcl-2 and anti-apoptotic homologue proteins decrease commitment of human SCC cells to terminal differentiation and apoptosis [94]. Also, the Bcl-2 transcript cleavage induces apoptosis and impairs cell growth [95]. Some pathways are related to Bcl-2 expression and thus control apoptosis.

Bcl-2 expression is inversely correlated to wild-type p53 expression, which enhances apoptosis modulating the expression of Bcl-2 [96, 97]. Inhibition of NF-B pathway will lead to downregulation of Bcl-2 and other pro-survival proteins leading to inhibition of cellular proliferation and inducing apoptosis in SCC cells [98]. Bcl-2 overexpression is associated with progression in experimental and observational models of carcinogenesis [99-101]. These proteins also improve resistance to DNA-damaging factors like platinum compounds and fluoropyrimidines [94, 102-107]. Paclitaxel can reverse resistance to cisplatin inducing phosphorilation of Bcl-2, and thus, facilitate apoptosis [108]. Bcl-2 is also important for angiogenesis. Bcl-2 expression in endothelial cells enhances tumor vascular density and tumor growth [109, 110]. Bcl-2 is a key component for endothelial cell cytoprotection and survival through increasing surviving expression, inhibition of p53 and downregulating the activation of p38 mitogen-activated protein kinase (MAPK) and caspase-3 [111]. Bcl-2 induces vascular endothelial growth-factor (VEGF) expression in endothelial cells through STAT3. On the other hand, VEGF induces Bcl2 and proangiogenic factors expression in HNSCC cells. Inhibition of Bcl-2 suppresses tumor growth and diminishes vascular density in HNSCC xenografted model [110]. Blockage of VEGF receptors decreases the expression of Bcl-2 in tumor-associated endothelial cells, suggesting a possible role of Bcl-2 proteins in angiogenesis [112]. Bcl-xL plays an important role in apoptosis. Suppressing Bcl-xL by silencing RNA can suppress tumor growth and induce apoptosis [113, 114]. Bcl-xL inhibits p53-induced cytochrome c release, suggesting that Bcl-xL is a key component of mitochondrial pathway of apoptosis [115]. The expression of Bcl-xL can block Fas-mediated apoptosis, and inhibition of Bcl-xL by a specific antisense oligo-


nucleotide can reverse this blockage [116]. More than that, inhibition of Bcl-xL can reverse resistance to platinum compounds and inhibit cell growth of platinum-resistant and platinum-sensitive cells [117, 118]. Combined to suppression of VEGF, telomerase reverse transcriptase (TERT) and BclxL by RNA-interference approach showed synergistic effects in reducing tumor growth and inducing apoptosis in HNSCC cells [119]. Bortezomib induces up-regulation of proapoptotic Bcl-2 family proteins and Mcl-1. Inhibition of Mcl-1 by small-interfering RNA enhances the cytotoxicity effect of bortezomib [120]. The effect of Bcl-2 expression in treated patients is inconsistent. There are conflicting results when analyzing Bcl-2 expression and prognosis of HNSCC patients. Some series with small number of cases showed no association between the overexpression of Bcl-2 and prognosis of patients with HNSCC or even a protective effect, including increased response to therapy and better survival [121-130]. However, larger studies showed that patients with Bcl-2 overexpressing HNSCC are less likely to benefit from radiation therapy, radiochemotherapy or surgery, showing lower disease control and shorter term disease-free and overall survival [78, 107, 131-137]. Also, a regulatory (938C>A) single-nucleotide polymorphism (SNP) in the inhibitory P2 BCL2 gene promoter is associated to Bcl-2 expression and worse prognosis in patients with oropharyngeal SCC [138]. On the other hand, despite of Bcl-2 being associated with poorly-differentiated and advanced stage tumors, Bcl-2 was associated to a better prognosis in large randomized trials [89, 139]. Despite these inconsistencies, there are some explanations for such disparity. Most of the studies had small number of subjects, and the analysis was retrospective, enhancing the chance of finding negative association when a positive association was expected. Methodological disparities, like tissue sampling and storage, and differences in how the expression of Bcl-2 was evaluated and quantified may also enhance the chance of finding a negative result when a positive was expected. And finally, Bcl-2 may be a more important prognostic factor depending on the primary site of tumor and the treatment used. So, analysing all of these statements, we can conclude, based on the biological rationale and the published data, that Bcl-2 expression is a negative prognostic marker, and may be used for selecting patients for more aggressive therapy in the future. Bcl-xL expression is associated to decreased response to radiation therapy and chemotherapy, worse laryngeal preservation and worse disease-free and overall survival [127, 136, 137, 140-143]. In conclusion, Bcl-2 and Bcl-xL overexpression is not only a predictive, but also a prognostic factor for HNSCC. Multidomain Proapoptotic Bcl-2 Family Proteins Bax and Bak seem to have important functions in HNSCC development. The expression of Bax and Bak is more intense in well-differentiated carcinoma than in those with poor differentiation [85, 144, 145]. A negative association was found between Bax/Bcl-2 ratio and dysplasianeoplasia progression, suggesting that loss of Bax expression and increased Bcl-2 expression are early steps in tumor


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progression [146]. More than that, low Bax expression is associated to advanced stage neoplasms, poor response to therapy and poor prognosis [136, 144, 147, 148]. Spontaneous apoptosis appears to be related to Bax and other proapoptotic proteins expression [144, 149]. Human papillomavirus proteins E6 and E7 inhibit apoptosis by downregulating Bak expression and over-expressing Bcl-2 in human laryngeal cells[150]. Bax sensitizes platinum-resistant SCC cells in vitro and in vivo to cisplatin, and increases sensitiveness to chemotherapeutic agents like cisplatin, paclitaxel and 5-fluorouracil. This suggests that Bax plays an important role in apoptosis, that low Bax expression can increase the resistance to chemotherapy and that inducing Bax expression can potentiate the chemotherapy results in patients with HNSCC [104, 151-154]. Also, chemotherapy induces both the expression and dimerization of mitochondrial Bax, suggesting that Bax is an important factor for apoptosis induced by chemotherapy [152, 155]. Bax also seems to play an important role in radiation sensitiveness [156]. Patients with Bax expression have a significantly better prognosis than those without Bax expression, especially in the absence of Bcl-2 expression [157]. BH3-only Domain BH3-only proteins interact with anti and proapoptotic Bcl-2 family proteins through BH3 domain, inhibiting the first or activating Bax and Bak, facilitating apoptosis [23, 35, 42, 63, 67]. Some of these molecules seem to play an important role in HNSCC development. Bim, for example, is downregulated in an animal model of carcinogenesis and neem (Azadirachta indica) extract upregulated Bim and prevented progression to carcinoma, suggesting that activating BH3-only proteins can be a good strategy for cancer prevention [158]. Bim expression is also a marker of effect of cytotoxic drugs and apoptosis [159, 160]. Other BH3-only proteins have important functions in controling of apoptosis. Noxa is induced by cisplatin in platinum-sensitive cells and by bortezomib in platinumresistant cells. Its induction is directly related to apoptosis [161]. Bortezomib induces apoptosis by up-regulating Bim and Bik, direct inhibitors of Bcl-2 and Bcl-Xl [120]. Puma is a BH3-only protein important to apoptosis and is also a target of p53. Puma suppresses cellular growth, induces apoptosis and diminishes resistance to cisplatin in HNSCC xenografted models. In this model, adenoviral delivery of PUMA induced apoptosis more potently than adenoviral delivery of p53 did [162]. PUMA is essential to EGFR inhibitor-induced apoptosis in HNSCC cells treated by gefitinib [163]. BCL-2 INHIBITORS FOR THE TREATMENT OF HNSCC Bcl-2 Inhibitors as Antitumor Agents Both internal and external signals can imbalance the complex equilibrium between pro and anti-apoptotic proteins. Although not totally understood, it is well recognized that Bcl-2 family proteins play an important role in many steps of HNSCC development: carcinogenesis, evasion of



apoptosis, resistance to radiation, chemotherapy and other cellular stress. Nowadays, some molecules seem to control some of those proteins, giving us the opportunity of interfering in apoptosis. Considering that HNSCC express Bcl-2/Bcl-xL and that these proteins confer resistance to radiation and chemotherapy, blocking these proteins aims to potentiate the activity of Bax and Bak, and thus, favoring apoptosis and enhancing the benefits of cancer treatment [164]. These proteins can be modulated by some strategies, including interfering with upstream pathways like NF-B, STAT3 and others [98, 165]. The aim of this article is to review strategies of inhibiting proapoptotic Bcl-2 family proteins, mainly Bcl-2 and Bcl-xL. Modulating the mitochondrial apoptotic pathway by proteins other than Bcl-2 family proteins will not be discussed. Some strategies can be used for targeting Bcl-2 family proteins. Antisense-based therapy seems to be more specific, targeting the production of pro-survival proteins. Compounds from nature can interact with Bcl-2 family proteins, but these compounds lack specificity. On the other hand, small non-peptide molecules that mimic BH3-only proteins target specific pool of pro-survival proteins, and may be good agents for further development due to inherent low antigenicity [73, 164, 166, 167] Table 1. Epigallocatechin-3 gallate (EGCG), a polyphenol derivated from green tea, increased the expression of Bax and decreased the expression of Bcl-2 and Bcl-xL, leading to activation of caspase 9 and thus, inducing apoptosis in SCC cells, suggesting that EGCG induces apoptosis through the mitochondrial pathway [168-170]. EGCG also led to inhibition of EGFR, STAT3 and extracellular regulated kinase (ERK) pathways, suggesting that EGCG inhibits growth and induces apoptosis through multiple pathways [168]. Synergistic effects in inducing apoptosis and decreasing tumor growth were observed when HNSCC cells were treated with EGCG and an EGFR inhibitor [171]. EGCG not only exhibits anti-proliferative activity but also antiangiogenic activity through inhibition of NF-B and VEGF pathways [172]. Table 1.

Gossypol is a natural product of cottonseeds and roots that binds to BH3 domain of Bcl-2 and Bcl-xL [173-176]. The gossypol enantiomer (-)-gossypol induces apoptosis in SCC cells, particularly in cells with wild-type p53. This effect seems to be due to Bcl-xL inhibition rather than Bcl-2 inhibition [177-179]. This effect was maintained in SCCNH mouse model. Those exposed to (-)-gossypol exhibited tumor growth suppression, lower mitotic rate and increased apoptosis [180]. Combined suppression of EGFR by erlotinib, STAT3 by a transcriptor factor decoy and Bcl-xL by gossypol demonstrated synergistic enhancing of cell growth inhibition and apoptosis [181]. Apogossypolone, a gossypol derivate, has similar activity to gossypol in inhibiting apoptosis, and also inhibits CD31 expression, suggesting the suppression of angiogenesis [182]. Although gossypol targets other molecules, binding to BH3 domain seems to be the main responsible to apoptosis induction [183-185]. Without other cytotoxic agents, gossypol is unable to induce tumor shrinkage in a clinical trial [186]. Antimycin, a molecule derived from Streptomyces, and its derivative methoxyantimycin bind the BH3 domain of Bcl-2 and Bcl-xL, and both have promissor activity against human cancer. Unfortunately, they have not been tested against HNSCC until now [175, 187-190]. Chelerythrine, a plant alkaloid derived from Chelidonium majus, was originaly identified as a protein kinase C (PKC) inhibitor, but it can disrupt Bcl-xL/Bax complexes, with both actions contributingto induction of apoptosis in HNSCC cells [191-193]. Chelerythrine also enhanced the sensitivity of HNSCC cells to cisplatin [194, 195]. The harmine-derivative compound 6 is a molecule with high affinity to Bcl-xL [196, 197]. By blocking Bcl-xL and inducing reactive species of oxygen (ROS) accumulation, compound 6 favors apoptosis in laryngeal cells [198]. Despite interesting activity against squamous carcinoma, it is dificult to stabilish that the anticancer activity of natural compounds are exclusively due to Bcl-2 and bcl-xL inhibition. Despite of this, the pool of available information suggests that inhibition of apoptosis is essencial for apoptosis induced by the natural compounds mentioned above.

Summary of IC50 of Bcl-2 Inhibitors for Three Antiapoptotic Bcl-2 Family Proteins. Bcl-2 Inhibitor

Origin

IC 50 (mM) Bcl-2

Bcl-xL

Mcl-1

ABT-737 (Abbott)

Designed

0.12

0.064

>20

Antimycin A

Natural

2.95

2.70

2.51

Chelerytrine

Natural

10

10

>10

EGCG

Natural

0.45

0.59

0.92

Gossypol

Natural

0.28

3.03

1.75

GX15-070 (Gemin X)†

Designed

1.11

4.69

2.90

HA14-1 (Sigma)

Designed

NR

NR

NR

TW-37 (Ascenta)

Designed

NR

NR

NR

Adapted from Zhai et al.[167]; † data for GX15, a derivative of GX15-070. NR: not reported.


ABT-737 is a potent small molecule inhibitor of Bcl-xL and Bcl-2 [199]. ABT-737 has synergistic effect with cisplatin and etoposide in activating caspase-3 and cleavage of poly(ADP-ribose) polymerase, leading to apoptosis. ABT737 up-regulates Noxa, an BH3-only protein, and inhibition of Noxa by small-interfering RNA attenuates cell death [199]. TW-37, another small-molecule Bcl-2 inhibitor, showed synergistic effects against SCC and tumor-associated endothelial cells in vivo and in vitro combined to cisplatin. It induced apoptosis and decreased angiogenesis, improving the time to tumor failure [200]. Also, other compounds as Bcl-2 inhibitors containing zinc-binding moiety based derivatives that have the enhanced and unexpected properties as inhibitors of Bcl-2 and their use in the treatment of Bcl-2 related diseases [201]. And, heterocyclic compounds that bind to Bcl proteins inhibiting the interaction of Bcl proteins with binding partners [202], have been described. A series of stapled BCL-2 family peptide helices have now been identified that target the survival protein MCL-I with high affinity and unprecedented selectivity. The MCL-I inhibitor SAHBs described target the canonical BH3 groove of MCL-I, displacing the MCL-I /BAK interaction in vitro and in situ, and sensitizing MCL-I dependent cancer cells to mitochondrial apoptosis [203]. BH3 mimetics have high affinity to pro-survival Bcl-2 family proteins and higher specificity than natural compounds. They have been synthsized in the last two decades and are promissor agents for cancer treatment [73, 204]. Computational screening strategies using the predicted structure of Bcl-2 identified some of BH3 mimetics. Cell-permeable BH3 peptides bind to Bcl-2 and Bcl-xL, inhibiting them, and inducing cytochrome c release. Bad BH3 peptides are more potent than Bax and Bak peptides [205]. Other BH3-mimetics, like obatoclax (GX15-070) and HA14-1 have been tested in many types of cancer cells and induce apoptosis by binding the BH3 domain of Bcl-2 and Bcl-xL, but none of them have demonstrated, until now, efficacy against HNSCC [206-209]. Antisense oligonucleotides (AON) against Bcl-2 and BclxL decreased both RNA and protein expression, enhanced sensitiviness to chemotherapy and increased apoptosis in SCC cell lines [210-212]. Pre-clinical studies have shown that inhibition of Bcl-xL by a specific AON can reverse the antiapoptotic effect of Bcl-xL [116]. More than that, inhibition of Bcl-xL by AON can reverse resistance to platinum compounds and inhibit cell growth of platinum-resistant and platinum-sensitive cells [117]. Most of AON, like oblimersen (G3139), were designed to target Bcl-2 and Bcl-xL [213]. These compounds have shown interesting clinical activity in phase I and II clinical trials, especially when combined to other cytotoxic agents, but there is lack of studies in HNSCC [214-220]. Anti-Mcl-1 AON decreased cell viability in vitro in combination to chemotherapy and bortezomib [120, 221]. A Bcl-2 gene inhibitory element (BIE), designated BIE1, has been described to bind a cellular factor (BBF) such as


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BBF-A. This bind can regulate the level of expression Bcl-2, thus modulate the apoptosis in a cell [222]. Moreover, the small nucleic acid molecules are useful in the treatment of cancer, in this case specific modulating Bcl2 gene expression using small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules [223, 224]. CURRENT & FUTURE DEVELOPMENTS The Bcl-2 family proteins take part in many cellular functions, but their most remarkable role is to regulate apoptosis. The balance among pro-survival and proapoptotic proteins is very important for further activation of mitochondrial events that lead to apoptosis. Understanding this complex system and how all types of Bcl-2 family proteins interact among each other were not objective of this review [13, 41, 45, 167, 225]. Herein, we discussed the importance of Bcl-2 family proteins in carcinogenesis, cancer progression, sensitivity to cytotoxic agents. Moreover, we provided the rationale for further development of inhibitors of proapoptotic Bcl-2, especially in the context of treatment of HNSCC. Some strategies for inhibiting pro-survival Bcl-2 family protein have been suggested by the medical literature, but those haven’t reached the bedside yet. All of them have specific advantages and disadvantages regarding to specificity of binding BH3, the interaction with other apoptosisregulatory proteins, bioavailability, and others. The medical knowledge about Bcl-2 inhibitors is still evolving, particularly for HNSCC. These are promissory compounds, especially for combination to other agents that induce apoptosis in cancer cells, allowing enhanced cell death. Some types of cancer exhibit an almost exclusive overexpression of one anti-apoptotic Bcl-2 protein [126, 226228]. In this setting, it seems reasonable the further development of specific Bcl-2 family proteins inhibitors instead of pan-Bcl-2 inhibitors as cited above. This could lead to individualization of cancer therapy [229]. As clinical trials have started testing pan-Bcl-2 inhibitors for solid tumors, mainly for hematological malignancies, breast and prostate cancer, testing new strategies for the treatment of HNSCC, especially in combination to platinum-based regimens, urge. Until now, the only opened clinical trial evaluates EGCG for the treatment of patients with premalignant lesions of head and neck. ACKNOWLEDGEMENTS Our sincere thanks to João Paulo da Silveira Nogueira Lima for reviewing the manuscript. CONFLICT OF INTEREST All authors declare that they do not have any potential conflict of interest. There was no funding for this work. REFERENCES [1]

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