Table 5.

Members of the BCL-2 protein family.

BCL-2 Subfamily		Members	BH Domains	Overview
Anti-apoptotic		Bcl-2	BH1 BH2 BH3 BH4 TM	Bcl-2 is constitutively bound to mitochondrial and/or endoplasmic reticulum membranes and represents the main pro-survival member of the Bcl-2 family [41]. Bcl-2 can sequester activator and sensitizer BH3-only proteins and is also able to bind to the inositol trisphosphate receptors (InsP3R), membrane glycoprotein complexes acting as membrane calcium channels on the endoplasmic reticulum, inhibiting the initiation phase of calcium-mediated apoptosis [42]. Bcl-2 over-expression has been widely described in different types of malignancies and is related to tumor formation, progression, therapy resistance and poorer overall survival [39,43,44]. Although negative Bcl-2 expression has been proposed as a marker of good chemotherapy response in breast cancer patients [45], recent studies have shown Bcl-2 over-expression as a good prognosis factor in patients with different types of cancer including breast [46], colorectal, renal and advanced non-small cell lung cancer [47,48,49].
		B-cell lymphoma-extra large (Bcl-XL)	BH1 BH2 BH3 BH4 TM	Bcl-XL is a transmembrane protein localized in the outer mitochondrial and nucleus membranes, where it may bind to nuclear proteins and modulate transcription factors activity [36]. Bcl-XL can also sequester cytoplasmic p53, inhibiting cell death [50]. Bcl-XL, is usually over-expressed in different types of cancer and has been related to cancer cell growth, migration, invasion, maintenance of cancer stem cell phenotype, angiogenesis, enhanced aggressiveness [36] and apoptosis resistance [36,39].
		Bcl-2-like protein 2 (Bcl2l2, Bcl-W)	BH1 BH2 BH3 BH4 TM	The active Bcl-W isoform is loosely attached to mitochondria and can be neutralized by BH3 (only) proteins by enhancing the insertion of its C-terminal domain into the membrane [51]. Bcl-W over-expression has been related to different malignancies, including lymphoma, colorectal cancer and gastric cancer [52,53,54,55] and to a worse prognosis [54]. Bcl-W expression is regulated by MYC transcription factor through a specific microRNA [54] and its amount within the cell is modulated by the Akt serine-threonine kinase [56].
		Induced myeloid leukemia cell differentiation protein (Mcl-1)	BH1 BH2 BH3 BH4 TM	Mcl-1 binds to pro-apoptotic Bim [17], Bak and Bax [57] proteins to prevent apoptosis. Mcl-1-Bim complexes can be cleaved by granzyme B, allowing outer mitochondrial membrane permeabilization and apoptosis [17]. Mcl-1 is highly over-expressed in cancer cells [18] and has been related to chemotherapy-resistance [39].
		Bcl-2 related protein A1 (Bcl2A1), Bfl-1	BH1 BH2 BH3 BH4	In response to apoptotic stimuli, Bcl2A1 can translocate from the mitochondria or the cytoplasm to the nucleus [58] where its role remains unclear. Similarly to Mcl1, Bcl2A1 pro-survival ability is related to its association to pro-apoptotic BH3-only Bim, Bid and Puma proteins as well as to Bik, Hrk and Noxa [18]. Although Bcl2A1 is usually over-expressed in cancer cells [18] and contributes to the acquisition of tumor cell resistance against chemotherapy-induced apoptosis [58], the role of Bcl2A1 in both healthy and cancer cells is still under study [58]. Bcl2A1 is regulated at post-translational level by the proteasome and by transcription factors such as NFĸB [58] or retinoic acid [18].
		Bcl-B Bcl2l10	BH1 BH2 BH3 BH4 TM	Bcl-B binds to Bcl-2, bcl-XL and Bax, but not to Bak, and is able to suppress Bax-induced apoptosis in vitro [59]. Bcl-B is over-expressed in multiple-myeloma patients [60].
Pro-apoptotic	Effectors	Bcl-2-associated X protein (Bax)	BH1 BH2 BH3 TM	Along with Bak, Bax is one of the main apoptosis effectors. Bax exists as a free inactive cytosolic protein that responds to various stimuli exposing the BH3 domain to allow oligomerization [23] and then migrating and inserting into the mitochondria membrane, inducing the release of cytochrome-c [30]. Bax activity is mainly regulated by the cytosolic accumulation of the tumor suppressor protein p53 [61] as well as by other Bcl-2 family members [23].
		Bcl-2 homologous antagonist killer (Bak)	BH1 BH2 BH3 TM	Bak, is one of the main apoptosis effectors. After activation by stress signals, this integral mitochondrial membrane protein is activated by exposing the BH3 domain to allow oligomerization and outer mitochondrial membrane destabilization [23]. Bak can directly be activated by the tumor suppressor p53 by blocking the Mcl1 anti-apoptotic effect [62] and can also be regulated by other Bcl-2 family members [23].
		Bcl-2 related ovarian killer (Bok)	BH1 BH2 BH3 TM	Contrary to Bax or Bak, Bok is constitutively active and unresponsive to the inhibitory effects of Bcl-2 anti-apoptotic members [63], being able to trigger mitochondrial membrane permeabilization and apoptosis independently of Bax and Bak presence [63]. Bok activity, which is controlled by ubiquitylation and proteasomal degradation [63], is an essential mediator of p53-dependent apoptosis [64].
	Activators	BH3-interacting domain death agonist (Bid)	BH3	Bid responds to tumor suppressor p53, contributing to cell death as response to cell damage after chemotherapy [65,66]. On the other hand, Bid can also be cleaved and activated by granzyme B [17] as well as by Caspase-8 after death receptor signaling (Fas-ligation-mediated apoptosis). For these reasons, Bid has a key role as a connecting element between the intrinsic and the extrinsic apoptosis pathways [67]. After activation, Bid exposes the BH3 domain which allows its dimerization with apoptosis-effectors Bax, Bak and anti-apoptotic Bcl-2-like proteins [23], resulting in Bax and Bak activation and Bcl-2-like proteins inhibition and subsequent cell death. Once activated, Bid can also migrate from cytosol to mitochondria where it can directly promote the release of cytochrome c and other apoptogenic factors [17,68], amplifying caspase activation. Low Bid expression is related to resistance to chemotherapy [69] and TRAIL [70].
		Bcl-2-like protein 11 (Bim)	BH3 TM	Bim can appear associated to microtubules [67] or sequestered forming complexes with all pro-survival proteins [23]. These complexes can be disrupted by tumor suppressor p53 [71] as a response to cellular stress [23] and also by Granzyme B [17], allowing Bim activation and translocation to mitochondrial outer membrane to indirectly cause cell death by pro-apoptotic Bak/Bax activation [67,72,73]. Bim expression is regulated at different levels, and its abundance is controlled via the proteasome by protein kinases downstream growth factor receptor activation [67]. Bim has been reported to play a central role in regulation of tumorigenesis [74]. Indeed, Bim over-expression inhibits tumor growth and drug resistance [74], while Bim loss is associated with lymphadenopathy, autoimmunity [67] and tumor promotion [74].
		p53 upregulated modulator of apoptosis (Puma)	BH3	Similarly to Bid and Bim, Puma can directly bind and antagonize all pro-survival proteins [23,75] by directly or indirectly promoting cell death [75,76]. Puma, whose expression can be induced by nuclear p53 [50,76] after cellular stress or DNA damage [23,50,77], is able to displace cytoplasmic p53 from anti-apoptotic Bcl-xL, allowing p53 to induce cell death [50]. Puma expression can also be activated by transcription factors induced as a response to stimuli such as genotoxic stress, deregulated oncogene expression or toxins, being able to induce cell death in a p53-independent manner [75]. Puma, which is required by Bad and Noxa to induce cell death [73], can also directly activate pro-apoptotic Bax and Bak to promote mitochondrial cytochrome c release [73]. Aberrant Puma expression has been related to increased cancer risk development and therapeutic resistance [67,75].
		Bcl2 like 11, Bcl2 modifying factor (Bmf)	BH3	Similar to Bim, Bmf is bound to cytoskeletal structures [67,78]. After cellular damage or anoikis, Bmf is unleashed, being able to sequester pro-survival Bcl-2 proteins and promote cell death [78]. Bmf, which is widely expressed in lymphocytes and most hematopoietic tissues [79], is also expressed in different malignancies [80] including lung and breast [79]. Aberrant Bmf expression has been related to acquired resistance to chemotherapy [81].
	Sensitizers	Bcl-2-associated death promoter (Bad)	BH3	Bad promotes apoptosis by interacting with and inhibiting the anti-apoptotic function of Bcl-2 and Bcl-XL [82] and also, by sensitizing the cell to Bid-induced mitochondria disintegration [83]. Bad activity is determined by its hosphorylation status [82], which is modulated by protein kinases, including Akt [82] downstream growth factor receptor activation [67], allowing Bad dimerization and its sequestration away from the mitochondria [30]. As a response to death stimuli, Bad is dephosphorylated and translocated to the mitochondria to induce cell death [82]. In this regard, recent studies have shown that tumor suppressor p53 can strongly bind to dephosphorylated Bad, which determines Bad pro-apoptotic role [82]. The association between Bad and p53 also regulates Bad expression, by preventing p53 entrance into the nucleus to bind Bad promoter [82].
		Noxa (Damage in Latin)	BH3	Noxa is associated to the mitochondrial membrane [84] and can be activated, by exposing its BH3 domain and disrupting mitochondrial membrane [84] in response to cellular stress [77], such as DNA-damage [67], in a p53-dependent or -independent manner [77]. Noxa activation sensitizes the cell toward the action of other BH3-only proteins [77] and enhances the activation of Bax and/or Bak [77]. Noxa can also bind to anti-apoptotic members of Bcl-2 family [84], such as Mcl1, for proteasomal degradation [77], being also able to neutralize the pro-apoptotic effect of Bcl-XL [41].
		Harakiri (Hrk) DP5	BH3 TM	Hrk is associated to the mitochondria and is able to promote apoptosis via mitochondrial outer membrane permeabilization [85] as a response to different signals such as endoplasmic reticulum stress or cytokines [86]. Hrk is regulated by the interaction with the apoptotic inhibitors Bcl-2 and Bcl-X(L) via its BH3 domain [38,85] and its loss contributes to neoplasia and autoimmunity [85].
		Bcl-2-interacting killer (Bik)	BH3 TM	Bik localizes in the endoplasmic reticulum outer membrane [87]. As a response to stress signals and Bax activation, Bik promotes apoptosis mobilizing calcium to the mitochondria, remodeling the mitochondrial cristae [87] and provoking cytochrome c release [88]. BIK has been proposed as a new target for anti-cancer drugs that inhibit proteasomal functions as well as for the treatment of difficult cancers [87].