Proapoptotic proteins such as Bax, undergo translocation to the mitochondria during apoptosis, where they mediate the release of intermembrane space proteins including cytochrome c. Bax binds to the voltage-dependent anion channel (VDAC). VDAC is a ␤-barrel protein located in the outer mitochondrial membrane. In planar lipid bilayers, Bax and VDAC form a channel through which cytochrome c can pass. Hexokinase II (HXK II) also binds to VDAC. HXK II catalyzes the first step of glycolysis and is highly expressed in transformed cells, where over 70% of it is bound to the mitochondria. The present study demonstrates that HXK II interferes with the ability of Bax to bind to mitochondria and release cytochrome c. Detachment of HXK II from the mitochondria-enriched fraction isolated from HeLa cells promoted the binding of recombinant Bax-⌬19 and subsequent cytochrome c release. Similarly, the addition of recombinant HXK II to the mitochondria-enriched fraction isolated from hepatocytes, cells that do not express HXK II endogenously, prevented the ability of recombinant Bax-⌬19 to bind to the mitochondria and promote cytochrome c release. Similar results were found in intact cells, in which the detachment of mitochondrial bound HXK II or its overexpression potentiated and inhibited, respectively, Bax-induced mitochondrial dysfunction and cell death.The major emphasis of apoptosis research was initially focused on the nucleus. This is understandable, given that the nucleus exhibits some of the most striking features of apoptosis, such as chromatin condensation and oligonucleosomal fragmentation of DNA. Recently, however, the involvement of mitochondria in apoptosis has come under close scrutiny. Mitochondria are the power plants of the cell, providing the bulk of ATP production for cellular metabolism. It has been demonstrated that cytochrome c, located in the intermembrane space (IMS) 1 of the mitochondria, is released to the cytosol during apoptosis and helps trigger the activation of caspases, a family of enzymes that is integral to the breakup of apoptotic cells (1-5). Subsequently, a plethora of other proteins that are located in the mitochondrial IMS and are part of the apoptotic machinery have been discovered, including apoptosis-inducing factor, SMAC/DIABLO, caspases 9 and 8, and endonuclease G (6 -8).At present, there is considerable controversy over the mode by which IMS proteins escape from that compartment and enter the cytosol, where they become activated. Disruption of the outer mitochondrial membrane is one obvious mechanism. Due to the greater surface area of the inner mitochondrial membrane compared with the outer mitochondrial membrane, excessive swelling of the mitochondrial matrix results in rupture of the outer mitochondrial membrane and the release of intermembrane space proteins. Indeed, opening of the mitochondrial permeability transition pore with subsequent mitochondrial depolarization and outer mitochondrial membrane rupture does occur in some forms of apoptosis (9). The permeability transition ...

Transformed cells are highly glycolytic and overexpress hexokinase II (HXK II). HXK II is capable of binding to the mitochondria through an interaction with the voltagedependent anion channel (VDAC), an abundant outer mitochondrial membrane protein. The binding of HXK II to mitochondria has been shown to protect against loss of cell viability. Akt activation inhibits apoptosis partly by promoting the binding of HXK II to the mitochondria, but the mechanism through which Akt accomplishes this has not been characterized. The present report shows that Akt mediates the binding of HXK II to the mitochondria by negatively regulating the activity of glycogen synthase kinase 3B (GSK3B). On inhibition of Akt, GSK3B is activated and phosphorylates VDAC. HXK II is unable to bind VDAC phosphorylated by GSK3B and dissociates from the mitochondria. Inhibition of Akt potentiates chemotherapy-induced cytotoxicity, an effect that is dependent on GSK3B activation and its attendant ability to disrupt the binding of HXK II to the mitochondria. Moreover, agents that can force the detachment of HXK II from mitochondria in the absence of Akt inhibition or GSK3B activation promoted a synergistic increase in cell killing when used in conjunction with chemotherapeutic drugs. Such findings indicate that interference with the binding of HXK II to mitochondria may be a practicable modality by which to potentiate the efficacy of conventional chemotherapeutic agents. (Cancer Res 2005; 65(22): 10545-54)

We demonstrate that the transition from a reliance on glycolysis to oxidative phosphorylation in a transformed cell line is dependent on an increase in the levels and activity of sirtuin-3. Sirtuin-3 deacetylates cyclophilin D, diminishing its peptidyl-prolyl cis-trans isomerase activity and inducing its dissociation from the adenine nucleotide translocator. Moreover, the sirtuin-3-induced inactivation of cyclophilin D causes a detachment of hexokinase II from the mitochondria that is necessary for stimulation of oxidative phosphorylation. These results might have important implications for the role of sirtuin-3 in the metabolism of some cancer cells and their susceptibility to mitochondrial injury and cytotoxicity.

SummaryTumor necrosis factor (TNF) can induce necroptosis, wherein inhibition of caspase activity prevents apoptosis but initiates an alternative programmed necrosis. The activity of receptor-interacting serine/threonine-protein kinase 1 (RIPK-1) is required for necroptosis to proceed, with suppression of RIPK-1 expression or inhibition of RIPK-1 activity with necrostatin-1 preventing TNF-induced necroptosis. Downstream from the TNF receptor, the generation of reactive oxygen species at the mitochondria has been identified as necessary for the execution of necroptosis; with antioxidants and inhibitors of mitochondrial complex I preventing TNF-induced cytotoxicity. However, components of the signaling pathway that lie between activated RIPK-1 and the mitochondria are unknown. In the study reported here we demonstrate that during TNF-induced necroptosis, STAT3 is phosphorylated on serine 727, which is dependent on RIPK-1 expression or activity. The phosphorylation of STAT3 induces interaction with GRIM-19, a subunit of mitochondrial complex I, with a resultant translocation of STAT3 to the mitochondria, where it induces an increase in reactive oxygen species production and cell death.

Alcoholic liver disease is associated with an increase in the number of necrotic and apoptotic liver parenchymal cells. Part of this injury is mediated by TNF-alpha. Ethanol exposure sensitizes cells to the cytotoxic effects of TNF-alpha. This may be due, in part, to the increased propensity of the mitochondria in ethanol-exposed cells to induction of mitochondrial permeability transition (MPT) by various agents, including the proapoptotic protein Bax. This idea is supported by the observation that increased cell death induced by TNF-alpha in ethanol-exposed cells was dependent on development of the MPT. In the present study, we elucidate the pathways through which ethanol exposure enhances TNF-alpha induction of the MPT and the resulting cytotoxicity. Specifically, ethanol-exposed cells display caspase-8- and Bid-independent cell killing during TNF-alpha treatment. Moreover, the ethanol-enhanced pathway is dependent on p38 MAPK signaling, which brings about caspase-3 activation, mitochondrial depolarization, accumulation of cytochrome c in the cytosol, and the translocation of Bax to the mitochondria. Additionally, ethanol-exposed cells display a blunting of TNF-alpha-induced Akt activation and Bcl-2 antagonist of cell death phosphorylation that may account, in part, for the increased sensitivity of the mitochondria to Bax-mediated damage.