How (or why) is cyt c released from mitochondria during Apoptosis?

Answer 1

In certain apoptotic settings, cytochrome c (cyt c) redistributes from inside the mitochandria to the cytosol leading to the activation of a downstream capsase cascade. These capsases are involved in activation of the death sequence in apoptosis.

Answer 2

(1) Programmed cell death (apoptosis) is used by multicellular organisms during development and to maintain homeostasis within mature tissues. One of the first genes shown to regulate apoptosis was bcl-2. Subsequently, a number of Bcl-2-related proteins have been identified. Despite overwhelming evidence that Bcl-2 proteins are evolutionarily conserved regulators of apoptosis, their precise biochemical function remains controversial. Three biochemical properties of Bcl-2 proteins
have been identified: their ability to localize constitutively and/or inducibly to the outer mitochondrial, outer nuclear and endoplasmic reticular membranes, their ability to form heterodimers with proteins bearing an amphipathic helical BH3 domain, and their ability to form ion-conducting channels in synthetic membranes. The discovery that mitochondria can play a key part in the induction of apoptosis has focused attention on the role that Bcl-2 proteins may have in regulating either mitochondrial physiology or mitochondria-dependent caspase activation. Here we attempt to synthesize our current understanding of the part played by mitochondria in apoptosis with a consideration of how Bcl-2 proteins might control cell death through an ability to regulate mitochondrial physiology.

(2) A crucial amplificatory event in several apoptotic cascades is the nearly complete release of cytochrome c from mitochondria. Proteins of the BCL-2 family which include both anti- and proapoptotic members control this step. Here, we review the proposed mechanisms by which proapoptotic BCL-2 family members induce cytochrome c release. Data support a model in which the apoptotic pathway bifurcates following activation of a "BH3 only" family member. BH3 only molecules induce the activation of the multidomain proapoptotics BAX and BAK, resulting in the permeabilization of the outer mitochondrial membrane and the efflux of cytochrome c. This is coordinated with the activation of a distinct pathway characterized by profound changes of the inner mitochondrial membrane morphology and organization. This mitochondrial remodelling insures complete release of cytochrome c and the onset of mitochondrial dysfunction that is a typical feature of many apoptotic deaths.

(3) Cytochrome c release is a central step in the initiation of caspase-dependent apoptosis (10-12). It has been demonstrated that overexpression of either mitochondria- or endoplasmic reticulum (ER)1-specific Bcl-2 prevents the induction of apoptosis by inhibiting the mitochondrial release of cytochrome c (13, 14). Although the molecular mechanism governing the cytochrome c release regulated by Bcl-2 has yet to be fully characterized, what does appear to be important is the heterodimerization of Bcl-2 with proapoptotic proteins. Such heterodimerization directly or indirectly inhibits the apoptotic function, especially the homo-oligomerization of multidomain proapoptotic Bax or Bak that corresponds with the release of cytochrome c (15-18). Heterodimerization of Bcl-2 with proapoptotic proteins or BH3 domain-derived peptides from proapoptotic members such as Bax, Bak, or Bad has been demonstrated by NMR, cell-free protein-protein binding, and yeast two-hybrid studies (19-21). The anti-cytochrome c releasing effect of Bcl-2 can be suppressed by the overexpression of proapoptotic proteins such as Bax (22). Conversely the cytochrome c releasing activity of proapoptotic proteins can be limited by the overexpression of Bcl-2 (16). The ratio rather than the amount of antiapoptotic versus proapoptotic proteins determines whether apoptotic signaling can proceed (15, 17, 23). Normally Bax exists as a soluble monomer in cytosol or is loosely associated with mitochondria. However, upon apoptotic stimulation, Bax translocates to mitochondria where it forms oligomers that are inserted into the outer mitochondrial membrane (24, 25). The signals that trigger the translocation of Bax to mitochondria remain largely unknown. In synthetic membranes, Bcl-2 has been shown to directly inhibit the channel or pore forming activity of Bax (16), which is consistent with the ability of Bcl-2 in preventing Bax oligomerization in outer mitochondrial membranes (22, 26).


The antiapoptotic function of Bcl-2 is thought to be primarily derived from Bcl-2 presented in the mitochondria (10, 27). However, growing evidence has indicated that Bcl-2 expressed on ER plays an important role in the inhibition of cytochrome c release and apoptosis (28). An exclusive ER-targeted form of Bcl-2, Bcl-2/cb5 has been shown to prevent apoptosis in many types of cells induced by c-Myc, tunicamycin, brefeldin A, radiation, or Bax overexpression (13, 14, 29-31). The protective effect of Bcl-2/cb5 in cells suggests the presence of cross-talk between ER and mitochondria. However, the mediators that connect ER Bcl-2 and mitochondrial cytochrome c release remain to be identified. What does appear to be important is intracellular Ca2+ signaling (32, 33). It has been shown that Bcl-2 overexpression blocks Ca2+ entry into or release from ER in response to many apoptotic stimuli. For example, overexpression of Bcl-2 decreases the ER Ca2+ load (34, 35) and prevents or delays the depletion of Ca2+ from ER after cells have received a treatment with apoptosis inducers such as thapsigargin, 2,5-di-(tert-butyl)-1,4-benzohydroquinone, cyclopiazonic acid, or ceramide (36-38).

The involvement of Ca2+ signaling in apoptosis has been suggested by a number of recent studies (39, 40). With transient increases in cytosolic Ca2+, many intracellular enzymes including phospholipases, proteases, and endonucleases can be activated, while prolonged or unregulated cytosolic Ca2+ elevation can further lead to apoptosis or cell death (41, 42). Although the molecular mechanism underlying apoptosis mediated by Ca2+ in vivo remains to be fully defined, accumulation of mitochondrial Ca2+ or activation of caspase-12 or other Ca2+-dependent intracellular enzymes may be the possible mechanisms (39, 43). ER and mitochondria are two crucial organelles in the regulation of intracellular Ca2+ homeostasis in cells. They are physiologically connected (44, 45). After certain stimulations, rapid Ca2+ accumulation in mitochondria always occurs immediately after ER Ca2+ release (46, 47). Such a rapid influx of Ca2+ into mitochondria causes mitochondrial PTP to open and subsequent mitochondrial membrane permeability, mitochondrial metabolic alteration, reactive oxygen species (ROS) generation, and cytochrome c release. ROS is another potent promoter of cytochrome c release. In addition to regulating the cytochrome c-mediated caspase-dependent apoptosis, both Ca2+ and ROS are important mediators of caspase-independent apoptosis or necrosis (39, 43).

Apoptosis, or programmed cell death, is a very important phenomenon in cytotoxicity induced by anticancer treatment. However, chemoresistance occurs when antiapoptotic protein Bcl-2 is overexpressed (48, 49). Therefore, directly targeting Bcl-2 might overcome such chemoresistance. Based on the evidence that the heterodimerization of Bcl-2 and proapoptotic proteins is mediated by the BH3 domain of the proapoptotic proteins, we recently discovered a novel organic molecule, ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (HA14-1) (C17H17BrN2O5,), that binds to the proapoptotic protein BH3 domain binding sites on the surface pocket of Bcl-2 (8). HA14-1 is a synthetic cell-permeable small molecule with a molecular weight of 409. It specifically competes with Bak BH3 domain-derived peptide in binding Bcl-2 with an IC50 of 9 µM (8). HA14-1 alone or in combination with other anticancer agents has been shown to induce apoptosis effectively in many types of cancer cells including Ara-C-resistant HL-60 cells (8, 50-52). Cytochrome c release, caspase-9/-3 activation, and DNA fragmentation are evident in HA14-1-induced apoptosis. However, the molecular mechanism upstream of cytochrome c release upon HA14-1 treatment remains unclear. In this study, we investigated and identified several upstream signals in both ER and mitochondria that are critical for cytochrome c release in response to HA14-1.