The Role of the BCL2 Family in Targeted Anticancer Response as Apoptotic Mediators

2.1. Location of the BCL-2 Family Gene within the Genome

The BCL-2 family of proteins is central to the regulation of apoptosis. Since the discovery of BCL-2 in the 1980s in association with B-cell lymphoma [16], a broad array of related proteins has been identified. The intrinsic apoptotic pathway is controlled by approximately 20 members of the BCL-2 gene family, which collectively maintain the critical balance between cell survival and programmed cell death [28]. In response to apoptotic stimuli, several BCL-2 family members translocate from the cytoplasm to the mitochondria. Moreover, the BCL-2 gene itself is expressed across various intracellular membranes, including the mitochondria and endoplasmic reticulum, underscoring its fundamental role in cellular homeostasis.

The BCL-2 gene (B-cell lymphoma 2) was originally identified at the t(14;18) chromosomal translocation breakpoint in B-cell follicular lymphomas, where the immunoglobulin heavy chain gene enhancer and promoter on chromosome 14 drive aberrant transcriptional activation of BCL-2 (Fig. 1) [29-31]. This strong association between BCL-2 and the follicular lymphoma translocation provided early evidence that BCL-2 functions as an oncogene [32]. The translocation results in the fusion of the BCL-2 gene on chromosome 18 with regulatory elements of the immunoglobulin heavy chain gene on chromosome 14, leading to overexpression of the BCL-2 protein [21]. Approximately 90% of follicular lymphomas and 30% of diffuse large B-cell lymphomas harbor this characteristic translocation [33]. Members of the BCL-2 family are defined by the presence of conserved BCL-2 homology (BH) domains (see Table 1) [29]. Importantly, the oncogenic role of the BCL-2 translocation is not due to enhanced proliferation but rather the inhibition of apoptosis, thus contributing to tumorigenesis by impairing programmed cell death [30].

2.2. Structures of BCL-2 Family Members

The three-dimensional structures of BCL-2 family proteins are characterized by six to seven amphipathic α-helices of varying lengths, along with two core predominantly hydrophobic α-helices. These are separated by a long, unstructured loop between the first two helices. Notably, the overall structural fold of BCL-2 proteins closely resembles that of the pore-forming domains found in certain bacterial toxins [28, 44]. BCL-2 family members contain conserved BCL-2 homology (BH) domains, regions of sequence similarity that define their classification [45]. The multi-domain BCL-2 proteins, which possess multiple BH motifs, include both antiapoptotic (e.g., BCL-2, BCL-XL, Bcl-w, Mcl-1, A1, and Bcl-B) and proapoptotic (e.g., BAX and BAK) members. In contrast, the proapoptotic BH3-only proteins, such as Bim, Bad, tBid, Bmf, Bik, Noxa, Puma, and Hrk, contain only the BH3 domain. This BH3 motif, essential for proapoptotic function, is present in nearly all proapoptotic BCL-2 proteins but may be absent in antiapoptotic counterparts (Fig. 2) [1].

The BCL-2 family proteins share four conserved regions known as BCL-2 homology (BH) domains, BH1, BH2, BH3, and BH4, which represent key sites of structural homology among family members and are critical for their function. These domains are essential for mediating protein-protein interactions, including heterodimerization between pro- and anti-apoptotic members, as demonstrated by studies involving domain-specific mutations and deletions [36]. Approximately 44% of the amino acid sequence of BCL-XL (also known as BCL-2L1, BCL-2-like 1) is homologous to BCL-2, and it exists in multiple isoforms beyond the well-characterized BCL-XL and BCL-XS variants [16]. The BH domains in BCL-XL are composed of eight α-helices (α1-α8), which are essential for its tertiary structure. A hydrophobic pocket formed by the BH1–BH3 regions serves as the binding site for the BH3 domain of pro-apoptotic proteins, triggering oligomerization [46]. Although the precise structure of BCL-XS remains unresolved, the loss of both BH1 and BH2 domains would likely result in significant disruption of the hydrophobic binding cleft [16]. Structural studies of BAK and BAX monomers reveal a globular conformation with a hydrophobic core surrounded by eight α-helices [21]. In BAK, the carboxyl-terminal transmembrane helix (helix 9) is constitutively inserted into the mitochondrial outer membrane (MOM). In contrast, BAX is primarily cytoplasmic due to helix 9 being sequestered within the hydrophobic groove formed by the BH3 domain-binding interface [17]. Bid, another pro-apoptotic family member, contains six amphipathic helices surrounding two central, largely hydrophobic α-helices, with the most notable conformational changes occurring in the N-terminal region of the protein [44].

2.3. Gene Expression of BCL-2 Family Protein

A variety of cytotoxic stimuli can harm cells, but BCL-2 expression can stop this damage. Hodgkin's lymphoma (NHL), myeloma, lung, prostate, and breast cancer, melanoma, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and non-lymphoma all have BCL-2 overexpression [29, 47]. MicroRNAs (miRNAs) control the downregulation/deletion of genes that cause BCL-2, chromosome translocation, and gene amplification. BCL-2 levels may rise due to a variety of processes, including RNA degradation (See Table 2) [29].

In CLL, the most prevalent leukemia in humans, BCL-2 overexpression was caused by the absence of regulation of miRNA 15/16 [48]. Differentiating cells have exceptional amounts of BCL-XL expression, which persists throughout neural ontogeny at greater levels. Ablation of the BCL-XL gene, BCL-2l1, causes significant apoptosis in the developing neurological and hematological systems as well as embryonic death by E13.5 [13]. Tumor development is significantly influenced by BCLXL. Previous studies have demonstrated that the BCL-2 L1 gene, which codes for BCLXL, is amplified in many solid tumors [21].

A poor prognosis for patients with human cervical neoplasms and ovarian cancer has been linked to MCL-1 expression. Through a PI3K/Akt-dependent mechanism, IL-6 inhibits cellular apoptosis and encourages the oncogenesis of cervical cancer [33]. Antiapoptotic protein overexpression can result in treatment resistance in addition to lowering normal cell death. Overexpression of BCL-XL and other anti-apoptotic proteins has been associated with therapeutic resistance, as it interferes with the apoptotic pathways targeted by many anticancer agents [49]. In various malignancies, including hepatocellular carcinoma, multiple myeloma, chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), elevated expression of anti-apoptotic proteins, particularly BCL-XL and Mcl-1, has been correlated with treatment resistance and poor clinical outcomes [33, 47].

By increasing the transcriptional factor specificity protein 1's expression, BCLW stimulates tumor invasion and the subsequent generation of matrix metalloproteinase 2 [21]. BCL-2L10 (also called BCL-B or DIVA) is the most important maternally inherited mRNA, according to an analysis of early mRNA expression in early embryos. It is highest in mature eggs but decreases during division [13].

As a result of diverse regulatory mechanisms, BH3-only proteins tend to be underproduced or functionally impaired in both tumor cells and actively proliferating normal cells. While some BH3-only proteins are constitutively expressed, others are transcriptionally induced in response to cellular stressors, such as DNA damage or hypoxia [60]. These observations support the rheostat model, which posits that cell fate is determined by the relative levels of proapoptotic and antiapoptotic BCL-2 family proteins. The discovery of BAK (BCL-2 antagonist/killer), a proapoptotic protein structurally more similar to BCL-2 than to BAX, yet functionally analogous to BAX, further demonstrated that multiple proteins beyond BCL-2 can exert antiapoptotic control [50].

The BAX promoter is broadly active in both normal and malignant tissues and is a direct transcriptional target of the tumor suppressor TP53 [21]. Additionally, BCL-2A1 (also known as A1) was found to be overexpressed in gastric cancer relative to normal tissue, suggesting its potential role in the development of solid tumors [37]. Although PUMA expression is typically low under basal conditions, it can be strongly induced by transcription factors, such as p53, p73, E2F1, and FOXO3a, which may initiate an apoptotic response [61].

3.1. The Role of the BCL-2 Family in Apoptosis

Apoptosis is a strictly controlled type of cell death with unique genetic and biochemical processes that are necessary for healthy tissue development and homeostasis. It helps metazoan organisms maintain the proper balance between cell death and survival by assisting in the removal of unwanted and superfluous cells [58, 74]. Both internal cell damage and signals from the cell surface can cause apoptosis [67]. It has been determined that the BCL-2 family is essential for either triggering or inhibiting the apoptotic process [75].

Transcriptional and/or posttranscriptional activation of BH3-only proteins triggers the BCL-2-regulated apoptotic pathway in response to various upstream signalling events [76]. BH3-only proteins activate BAK and BAX. By competing with pro-survival family members, it can either directly or indirectly activate BAX and BAK [77]. Homodimers and heterodimers produced by activated BAX and BAK interact to create pore-like structures in the outer mitochondrial membrane. This process, called mitochondrial outer membrane permeabilization (MOMP), enables the release of cytochrome c and other apoptogenic substances into the cytosol, which triggers caspase activation and apoptosis [78].

MOMP (mitochondrial outer membrane permeabilization) not only causes mitochondrial malfunction, leading to depletion of energy reserves, but also results in cell death even when caspases are inactive; thus, recovery from MOMP is possible only under exceptional circumstances, making it a critical event preceding cell death [79]. The induction of apoptosis by BH3-only proteins varies, with some, such as BIM, PUMA, and BID, being more potent inducers than others, such as BAD, NOXA, and BMF [77]. The interactions between proapoptotic BH3-only proteins and prosurvival proteins differ: BIM and PUMA are promiscuous, binding with high affinity to all prosurvival BCL-2 family members, whereas BAD selectively binds to BCL-2, BCL-XL, and BCL-W but not MCL-1 or BFL-1, and NOXA primarily interacts with MCL-1 [80]. This selectivity is also reflected in the design of drugs aimed at promoting apoptosis, which often mimic these specific interactions [76, 80].

3.2. BAX and BAK Activation Models

In healthy cells, BAK is localized to the mitochondria and contains a transmembrane domain (α9) that spans the outer mitochondrial membrane [45]. Conversely, BAX is primarily found in the cytosol, with its transmembrane domain concealed within a hydrophobic groove in its inactive form. Upon encountering cytotoxic signals, BAX translocates to the mitochondria, where it contributes to apoptosis [45]. The mechanisms underlying the activation of BAX and BAK have long been considered a central challenge, often described as the “holy grail” in apoptosis research. During apoptosis, both proteins undergo conformational changes that transform them from harmless precursors into potent effectors capable of permeabilizing the mitochondrial outer membrane [10].

Three hypotheses have been proposed to explain the activation of BAX and BAK: direct activation, indirect activation, and membrane-mediated permissive mechanisms [21, 26]. Some BH3-only proteins are capable of directly activating BAX and BAK; however, while these proteins readily interact with prosurvival BCL-2 family members, detecting early interactions with BAX and BAK has been challenging [81]. In the direct activation model, BH3-only proteins are categorized as either sensitizers or activators. Sensitizer BH3-only proteins bind to and inactivate antiapoptotic BCL-2 proteins, rather than directly activating BAX and BAK [82]. Conversely, activator BH3-only proteins bind directly to BAX and BAK, inducing conformational changes that promote oligomerization and activation of these effectors [21].

According to the indirect activation model, BH3-only proteins neutralize anti-apoptotic proteins, thereby enabling BAK and BAX activation. This process allows apoptosis to be mediated indirectly via BAK and BAX. Under this mechanism, antiapoptotic BCL-2 family members bind to BAK and BAX to inhibit mitochondrial outer membrane permeabilization (MOMP). Instead of directly interacting with BAK and BAX, the primary function of BH3-only proteins is to bind to and displace BAK and BAX from the anti-apoptotic proteins. This mechanism is also referred to as the displacement model [83].

Another hypothesis for BAX and BAK activation is the membrane-mediated permissive paradigm, which involves the mitochondrial outer membrane (MOM). In healthy cells, BAX and BAK typically exist as inactive monomers or dimers. During apoptosis, antiapoptotic BCL-2 family proteins bind to and are neutralized by activated BH3-only proteins on the mitochondrial outer membrane. This neutralization increases the likelihood of spontaneous activation of BAX and BAK, leading to homo-oligomerization and the formation of cytochrome c-releasing pores [84]. Both BAX and BAK are anchored to the membrane via helix 9, which facilitates their spontaneous activation within the lipid bilayer of the MOM. In this context, the primary role of BH3-only proteins is to inactivate anti-apoptotic BCL-2 proteins, aligning with the indirect activation paradigm. However, the membrane-mediated permissive model posits that BH3-only proteins can directly activate BAX and BAK within the membrane [10, 29]. Although BH3-only proteins are considered the main activators of BAX and BAK, other proteins, such as p53, Casp8p41, and retinoblastoma protein, have also been shown to directly activate these effectors. Recent evidence further suggests that active BAX and BAK can, in turn, act as activators of each other [21].

3.3. Formation of Cellular Membrane Pores by BAX and BAK

Cellular membranes serve as essential platforms for storing and transmitting biochemical information required for cell signaling and function. To maintain this functionality, membranes must support controlled exchange between the intracellular and extracellular environment. This is achieved through the presence of specific proteins, such as pore-forming proteins, ion pumps, and cell adhesion molecules, all of which interact with the dynamic properties of the lipid bilayer. Pore-forming proteins (PFPs) are found in all life forms and modify membrane permeability, allowing the passage of ions, small molecules, and occasionally larger biomolecules. In mammals, the BCL-2 family plays a regulatory role in this process during apoptosis and bacterial defense mechanisms [85]. Specifically, the proteins BAX and BAK contribute to mitochondrial outer membrane (MOM) permeabilization during apoptosis, resulting in the release of various mitochondrial proteins into the cytoplasm [86].

In normal physiological conditions, BAX and BAK exist as monomeric proteins with a compact globular structure composed of α-helices. The hydrophobic α5 helix is surrounded by amphipathic helices, while the α9 helix at the C-terminal acts as a transmembrane segment. This α9 domain secures BAK within the MOM under resting conditions. In contrast, BAX remains primarily in the cytosol and only integrates into the MOM after receiving apoptotic stimuli, inserting its α9 region into the membrane. Conformational changes during activation expose the BH3 domain (α2), which later re-engages with activated BAX or BAK molecules. This leads to the formation of symmetric dimers via α6 helix interaction, which then assemble into larger oligomeric complexes [87, 88].

BAX and BAK disrupt the lipid bilayer by forming toroidal pores, where both proteins and lipids contribute to the pore wall. The physical properties of the membrane and the conformational states of BAX and BAK influence the size and stability of these pores [89]. In artificial lipid systems, the pores expand rapidly upon formation and then stabilize at diameters ranging from 3 to 8 nanometers, depending on the concentration and configuration of the proteins. Two categories of BAX-induced pores have been identified: one is voltage-sensitive, small in diameter, selectively permeable to cations, and incapable of protein translocation; the other is voltage-independent and sufficiently large to permit proteins, such as cytochrome c to pass through the membrane [90].

Various structural models explain how BAX and BAK insert into mitochondrial membranes to create toroidal pores [91]. The umbrella model suggests that helices α5 and α6 insert into the lipid bilayer as a hairpin structure that spans the membrane [89, 92]. Another explanation, known as the in-plane model, proposes that α4 and α5 helices lie flat on the membrane surface, causing local membrane curvature and tension, which facilitates pore formation. In this configuration, α9 is fully inserted into the membrane, whereas α5 and α6 are only partially embedded [91].

The clamp model describes BAX oligomers forming dimers in which helices α2 through α5 form a dimerization domain that lines the pore edge, in association with lipid head groups. Since the pore-forming process does not involve full transmembrane insertion of the entire protein, this mechanism may require translocation of BAX monomers to the opposite side of the membrane. In this model, the critical conformational shift involves partial opening of the hairpin formed by the α5 and α6 helices, thereby creating a clamp-like configuration. Helix α5 is directly exposed to the membrane interior, while helix α6 remains at the lipid interface [93]. These structures are essential for mitochondrial outer membrane permeabilization, a key step in apoptotic signaling.

3.4. Apoptotic Pathway Stimulation by BH3-only Proteins

Members of the BCL-2 protein family are defined by the presence of conserved BCL-2 homology (BH) domains, including BH1, BH2, BH3, and BH4. BH3-only proteins, as the name implies, express only the BH3 domain and function either as activators or sensitizers. In contrast, anti-apoptotic and effector proteins contain multiple BH domains [94]. BH3-only proteins function as molecular sensors that detect a variety of stress signals, such as developmental cues or deprivation of growth factors. These stimuli can initiate apoptosis, a tightly regulated process that eliminates damaged or unneeded cells [95].

Eight BH3-only proteins have been extensively investigated in mammalian systems. These include BIM, BAD, BMF, BID, NOXA, BIK, PUMA, and HRK [96]. Their individual functions and overlapping roles have been clarified through gene disruption studies [45]. These proteins either directly activate proapoptotic proteins like BAX or indirectly promote apoptosis by inhibiting anti-apoptotic members of the BCL-2 family. Anti-apoptotic proteins and their viral homologs contain hydrophobic binding grooves formed by their BH domains. BH3-only proteins interact with these sites, and their ability to bind a range of targets is regulated by transcriptional and post-translational mechanisms [97].

BH3-only activators, such as BID, PUMA, and BIM, are capable of directly binding to and activating BAX and BAK. Their activation leads to oligomerization and formation of macropores in the mitochondrial outer membrane. In contrast, BH3-only sensitizers, which include BAD, BIK, NOXA, HRK, and BMF, do not directly activate BAX or BAK. Instead, they bind to antiapoptotic proteins, displacing BH3-only activators or previously activated forms of BAX and BAK, thereby indirectly promoting cell death [98]. The pro-survival activity of BAX and BAK is controlled by six known mammalian anti-apoptotic proteins: BCL-2, BCL-XL, BCL-W, MCL-1, A1, and BCL-B. These proteins are targets of BH3-only inhibition [99].

The primary step in the apoptotic pathway is the oligomerization of BAX and BAK at the mitochondrial outer membrane, leading to membrane permeabilization (MOMP). This allows the release of proapoptotic factors, including cytochrome c, from the mitochondrial intermembrane space, thereby initiating a caspase-dependent proteolytic cascade [99, 65]. The upregulation of BH3-only protein expression frequently triggers MOMP and marks the onset of apoptosis [100].

Among BH3-only proteins, PUMA and NOXA are direct transcriptional targets of p53 and play pivotal roles in apoptosis induced by genotoxic stress, including gamma irradiation and chemotherapeutic drugs [65]. BID must be unfolded and cleaved by caspases to become active. The resulting tBID form acts as a membrane-targeting ligand for BAK and facilitates cytochrome c release. BID, BIM, and PUMA are all capable of directly activating BAX and BAK, while the remaining BH3-only proteins function primarily as derepressors by blocking antiapoptotic interactions [95].

BIM and PUMA have high binding affinities for all members of the anti-apoptotic BCL-2 family, making them effective across a broad range of targets. Other BH3-only proteins exhibit selective binding preferences, sometimes differing by as much as 1000-fold [97]. BH3 mimetics are synthetic molecules developed to mimic BH3-only proteins. These compounds bind with high affinity and specificity to the hydrophobic grooves of anti-apoptotic proteins, blocking their function [94, 98]. ABT-737 was the first BH3 mimetic developed. It binds strongly to BCL-2, BCL-XL, and BCL-W, but shows lower affinity for MCL-1 and A1/BFL1 [101]. ABT-263 (navitoclax) is another potent inhibitor that has shown therapeutic potential in treating chronic lymphocytic leukemia (CLL) [98]. Both ABT-737 and ABT-263 have demonstrated significant proapoptotic effects in experimental models using primary tumor cells [94].