Mechanisms of mitochondrial apoptosis induced by peripheral benzodiazepine receptor ligands in human colorectal cancer cells

Specific ligands of the peripheral benzodiazepine receptor (PBR) have been shown to induce apoptosis in gastrointestinal cancers. The aim of this study was to characterize the signaling pathways of PBR ligand-induced apoptosis. FGIN-1-27 but not PK 11195-induced apoptosis was associated with a decrease of mitochondrial membrane potential and an increase of mitochondrial volume in HT29 colorectal cancer cells. However, PK 11195-elicited apoptosis was associated with a downregulation of Bcl-2, translocation of Bax to the mitochondria including subsequent oligomerization, and activation of caspase-9, indicating the involvement of mitochondria in PK 11195-induced apoptosis. Moreover, PK 11195-induced apoptosis was associated with the generation of reactive oxygen species. This study demonstrates a novel mechanism of PK 11195-induced mitochondrial apoptosis without alteration of the mitochondrial membrane potential. The characterization of signaling pathways associated with PBR ligand-induced apoptosis will build the base for a future use of these ligands in anti-neoplastic therapeutic approaches.     

Effect of PK11195, a peripheral benzodiazepine receptor agonist,on insulinoma cell death and insulin secretion

Functional role of peripheral benzodiazepine receptor on mitochondrial membrane in apoptosis and insulin secretion from insulinoma cells was studied. A prototypic peripheral benzodiazepine receptor agonist PK11195 induced insulinoma cell apoptosis, while a central benzodiazepine receptor agonist did not. Death of insulinoma cells by PK11195 was inhibited by cyclosporin A,{ a blocker of mitochondrial permeability transition pore}. Caspase inhibitors further inhibited MIN6N8 cell death. PK11195 induced dissipation of mitochondrial potential and cytochrome c translocation to cytoplasm. PK11195 induced an increase in cytoplasmic [Ca^{2 +}], which was reversed by cyclosporin A. Rhod-2 staining showed decreased mitochondrial [Ca^{2 +}] after PK11195 treatment. PK11195 potentiated glucose-induced insulin secretion probably due to the increased cytoplasmic [Ca^{2 +}]. Calpain was activated following Ca^{2 +} release, and calpain inhibitors attenuated death of insulinoma cells by PK11195. These results suggest that PK11195 induces mitochondrial potential loss, cytochrome c translocation, increased insulin secretion in conjunction with an increase in cytoplasmic [Ca^{2 +}] and calpain activation, which collectively leads to apoptosis of insulinoma cells.     

Activation of Xenopus eggs by the kinase inhibitor 6-DMAP suggests a differential regulation of cyclin B and p39(mos) proteolysis

The molecular mode of action of arsenic, a therapeutic agent employed in the treatment of acute promyelocytic leukemia, has been elusive. Here we provide evidence that arsenic compounds may act on mitochondria to induce apoptosis. Arsenite induces apoptosis accompanied by a loss of the mitochondrial transmembrane potential (Delta Psim). Inhibition of caspases prevents the arsenite-induced nuclear DNA loss, but has no effect on the Delta Psim dissipation and cytolysis induced by arsenite. In contrast, Bcl-2 expression induced by gene transfer prevents all hallmarks of arsenite-induced cell death, including the Delta Psim collapse. PK11195, a ligand of the mitochondrial benzodiazepine receptor, neutralizes this Bcl-2 effect. Mitochondria are required in a cell-free system to mediate arsenite-induced nuclear apoptosis. Arsenite causes the release of an apoptosis-inducing factor (AIF) from the mitochondrial intermembrane space. This effect is prevented by the permeability transition (PT) pore inhibitor cyclosporin A, as well as by Bcl-2, which is known to function as an endogenous PT pore antagonist. Arsenite also opens the purified, reconstituted PT pore in vitro in a cyclosporin A- and Bcl-2-inhibitible fashion. Altogether these data suggest that arsenite can induce apoptosis via a direct effect on the mitochondrial PT pore.     

The peripheral-type benzodiazepine receptor. Localization to the mitochondrial outer membrane

We have investigated the subcellular localization of the peripheral-type benzodiazepine receptor in rat adrenal gland using the high affinity ligand 3H-labeled 1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3-isoquinoline carboxamide ([3H]PK11195). The autoradiographic pattern of [3H]PK11195 binding sites in tissue sections of adrenal gland is similar to the histochemical distribution of the mitochondrial marker enzymes, cytochrome oxidase and monoamine oxidase, which are present in high concentrations only in the cortex. Subcellular fractionation studies of homogenates of adrenal gland indicate that the recovery and enrichment of [3H]PK11195 binding sites in the nuclear, mitochondrial, microsomal, and soluble fractions correlate closely with cytochrome oxidase activity, but not with markers for the nuclei, lysosomes, peroxysomes, endoplasmic reticulum, plasma membrane, or cytoplasm, indicating an association of the peripheral-type benzodiazepine receptor with the mitochondrial compartment. Titration of isolated mitochondria with digitonin results in the simultaneous release of the peripheral-type benzodiazepine receptor and of monoamine oxidase, but not cytochrome oxidase, indicating association of the peripheral-type benzodiazepine receptor with the mitochondrial outer membrane. Scatchard analysis and drug displacement studies of the binding of [3H] PK11195 to intact mitochondria and to the outer membrane-enriched digitonin extract further confirm the localization of the peripheral-type benzodiazepine receptor to the mitochondrial outer membrane.     

Bcl-2 inhibits apoptosis induced by mitochondrial uncoupling but does not prevent mitochondrial transmembrane depolarization

Bcl-2 overexpression protects cells from apoptosis induced by many cytotoxic agents. In this study, we investigated the effects of uncoupling mitochondrial electron transport in both HL60 wild-type and Bcl-2-overexpressing cells using the protonophore carbonyl cyanide m-chlorophenylhydrazone. We found that uncoupling mitochondrial electron transport induced apoptosis in wild-type, but not in Bcl-2-overexpressing cells. To investigate the mechanism of action of Bcl-2-mediated inhibition of cyanide m-chlorophenylhydrazone-induced apoptosis, we measured the mitochondrial transmembrane potential (DeltaPsi(m)) after uncoupling mitochondrial electron transport and found that both HL-60 wild-type and Bcl-2-overexpressing cells similarly depolarize following cyanide m-chlorophenylhydrazone exposure. Western blot analysis demonstrated that Bcl-2 overexpression did not completely block cytochrome c release from mitochondria after uncoupling mitochondrial electron transport. Since Bcl-2 may act as an antioxidant, we studied the effect of altering the cellular redox state prior to uncoupling mitochondrial electron transport in Bcl-2-overexpressing cells. Depletion of mitochondrial (but not cytosolic) glutathione induced apoptosis in Bcl-2-overexpressing cells and negated the protective effect of Bcl-2. Furthermore, following glutathione depletion, Bcl-2-overexpressing cells were sensitized to undergo cyanide m-chlorophenylhydrazone-induced apoptosis. These data suggest that the action of Bcl-2 is dependent, in part, on the cellular and mitochondrial redox state.     

PK11195 inhibits mitophagy targeting the F1Fo-ATPsynthase in Bcl-2 knock-down cells

The pharmacological agent 1-(2-Chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195) is the prototypical ligand of the 18-kDa Translocator Protein (TSPO) but at μM concentrations deactivates the oncoprotein Bcl-2 increasing the efficiency of chemotherapeutic agents and promoting the Ca2+-dependent macro-autophagy (or autophagy). In this paper, we report that PK11195, in HeLa cells, modifies the mitochondria-targeted type of autophagy--hereafter referred to as mitophagy--and the associated resizing of the mitochondrial network but does so exclusively in absence of the oncoprotein Bcl-2 (Bcl-2 Kd cells). This is consequence of a "side" targeting of the mitochondrial F1Fo-ATPsynthase enzyme, since identical outcome is mimicked by the antibiotic Oligomycin, of which PK11195 matches the effect on: i) mitochondrial membrane potential (ΔΨm), ii) ATP homeostasis and iii) Reactive Oxygen Species (ROS) generation. Taken together, these data highlight a novel TSPO-independent biological effect for PK11195 and provide evidences for a hitherto uncovered Bcl-2-dependent role of the F1Fo-ATPsynthase in mitochondrial quality control.     

The bovine peripheral-type benzodiazepine receptor: a receptor with low affinity for benzodiazepines.

The density of bovine peripheral-type benzodiazepine receptors (PBR) in four tissues was highest in adrenal cortex. The adrenal cortex PBR cofractionated with a mitochondrial membrane marker enzyme and could be solubilized with intact ligand binding properties using digitonin. The membrane bound and soluble mitochondrial receptors were pharmacologically characterized and showed the rank order of potency to inhibit [3H]PK 11195 binding was PK 11195 greater than protoporphyrin IX greater than benzodiazepines (clonazepam, diazepam, or Ro5-4864). [3H]PK 11195 binding to bovine adrenal mitochondria was unaffected by diethylpyrocarbonate, a histidine residue modifying reagent that decreased binding to rat liver mitochondria by 70%. [3H]PK 14105 photolabeled the bovine PBR and the Mr was estimated under nondenaturing (200 kDa) and denaturing (17 kDa) conditions. These results demonstrate the bovine peripheral-type benzodiazepine receptor is pharmacologically and biochemically distinct from the rat receptor, but the receptor component photolabeled by an isoquinoline ligand has a similar molecular weight.     

The mitochondrial benzodiazepine receptor: Evidence for association with the voltage-dependent anion channel (VDAC)

Specific, high-affinity receptors for numerous drugs have recently been localized to mitochondrial membrane proteins. This review discusses the association of the mitochondrial receptor for benzodiazepines (mBzR) with the voltage-dependent anion channel (VDAC), indicating a possible auxiliary role for VDAC as a putative drug binding protein. The proposed subunit composition of the purified mBzR complex isolated from rat kidney mitochondria includes VDAC, which functions as a recognition site for benzodiazepines (e.g., flunitrazepam), the adenine nucleotide carrier (ADC), and an 18 kDa outer membrane protein identified by covalent labelling with the mBzR antagonists isoquinoline carboxamides (e.g., PK 14105).Abbreviations and chemical names: Ro5-4864: 7-chloro-1,3-dihydro-1-methyl-5-(p-chlorophenyl)-2H-1,4-benzodiazepin-2-one; Ro15-1788: ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-]-[1,4]benzodiazepine-3-carboxylate; AHN-086: (1-(2-isothiocyanatoethyl-7-chloro-1,3-dihydro-5-(4-chlorophenyl)-2H-1,4-benzodiazepin-2-one hydrochloride;) PK11195: 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-isoquinoline-3-carboxamide; PK14105: 1-(2-fluoro-5-nitrophenyl)-3-isoquinoline-carboxylic acid.     

Functional characterization and expression of PBR in rat gastric mucosa: stimulation of chloride secretion by PBR ligands

Previous studies have demonstrated that gastric mucosa contained high levels of the polypeptide diazepam binding inhibitor, the endogenous ligand of the peripheral-type benzodiazepine receptor (PBR). However, the expression and function of this receptor protein in these tissues have not been investigated. Immunohistochemistry identified an intense PBR immunoreactivity in the mucous and parietal cells of rat gastric fundus and in the mucous cells of antrum. Immunoelectron microscopy revealed the mitochondrial localization of PBR in these cells. Binding of isoquinoline PK 11195 and benzodiazepine Ro5-4864 to gastric membranes showed that fundus had more PBR-binding sites than antrum, displaying higher affinity for PK 11195 than Ro5-4864. In a Ussing chamber, PK 11195 and Ro5-4864 increased short-circuit current (I(sc)) in fundic and antral mucosa in a concentration-dependent manner in the presence of GABA(A) and central benzodiazepine receptor (CBR) blockers. This increase in I(sc) was abolished after external Cl(-) substitution and was sensitive to chloride channels or transporter inhibitors. PK 11195-induced chloride secretion was also 1) sensitive to verapamil and extracellular calcium depletion, 2) blocked by thapsigargin and intracellular calcium depletion, and 3) abolished by the mitochondrial pore transition complex inhibitor cyclosporine A. PK 11195 had no direct effect on H(+) secretion, indicating that it stimulates a component of Cl(-) secretion independent of acid secretion in fundic mucosa. These data demonstrate that mucous and parietal cells of the gastric mucosa express mitochondrial PBR functionally coupled to Ca(2+)-dependent Cl(-) secretion, possibly involved in the gastric mucosa protection.     

Proapoptotic Bid binds to monolysocardiolipin,a new molecular connection between mitochondrial membranes and cell death

Recent evidence indicates that the mitochondrial lipid cardiolipin may be instrumental in the proapoptotic action of Bcl-2 family proteins on mitochondrial membranes, leading to the release of apoptogenic factors. However, contrasting evidence indicates that progressive loss of cardiolipin occurs during apoptosis. Here we show that Bid, a crucial proapoptotic protein that integrates the action of other Bcl-2 family members, exhibits discrete specificity for metabolites of cardiolipin, especially monolysocardiolipin (MCL). MCL, normally present in the remodelling of mitochondrial lipids, progressively increases in mitochondria during Fas-mediated apoptosis as a by-product of cardiolipin degradation, and also enhances Bid binding to membranes. MCL may thus play a crucial role in connecting lipid metabolism, relocation of Bid to mitochondria and integrated action of Bcl-2 proteins on mitochondrial membranes. We propose that Bid interaction with MCL 'primes' the mitochondrial outer membrane via segregation of lipid domains, facilitating membrane discontinuity and leakage of apoptogenic factors.     

Binding and Release of Cytochrome c in Brain Mitochondria Is Influenced by Membrane Potential and Hydrophobic Interactions with Cardiolipin

Factors influencing the release and anchorage of cytochrome c to the inner membrane of brain mitochondria have been investigated. Metabolic activity of mitochondria caused a decrease in the membrane potential Δψ_m, accompanied by detachment of the protein from the inner membrane. In a model system of cytochrome c reconstituted in cardiolipin (CL) liposomes, phosphate was used to breach the hydrophilic lipid-protein interactions. About 44% cytochrome c was removable when heart CL (80% 18:2n-6) was employed, whereas the remaining protein accounted for the tightly bound conformation characterized by hydrophobic lipid-protein interactions. Cytochrome c release from brain CL liposomes was higher compared to heart CL, consistent with lower polyunsaturated fatty acid content. The release was even higher with CL extracted from metabolically stressed mitochondria, exhibiting more saturated fatty acid profile compared to control (30% vs.17%). Therefore, weakening of the hydrophobic interactions due to saturation of CL may account for the observed cytochrome c release from mitochondria following metabolic stress. Moreover, mitochondria enriched with polyunsaturated CL exhibited higher Δψ_m, compared to less unsaturated species, suggesting that CL fatty acid composition influences Δψ_m. Mitochondria incorporated exogenous cytochrome c without protease-sensitive factors or Δψ_m. The internalized protein anchored to the inner membrane without producing swelling, as monitored by forward and side light scattering, but produced Δψ_m consumption, suggesting recovery of respiratory activity. The Δψ_m decrease is ascribed to a selected mitochondrial population containing the incorporated cytochrome c.This revised version was published online in August 2005 with a corrected cover date.     

Genetic analysis of dTSPO,an outer mitochondrial membrane protein,reveals its functions in apoptosis,longevity, and Aβ42‐induced neurodegeneration

The outer mitochondrial membrane (OMM) protein, the translocator protein 18 kDa (TSPO), formerly named the peripheral benzodiazepine receptor (PBR), has been proposed to participate in the pathogenesis of neurodegenerative diseases. To clarify the TSPO function, we identified the Drosophila homolog, CG2789/dTSPO, and studied the effects of its inactivation by P‐element insertion, RNAi knockdown, and inhibition by ligands (PK11195, Ro5‐4864). Inhibition of dTSPO inhibited wing disk apoptosis in response to γ‐irradiation or H₂O₂ exposure, as well as extended male fly lifespan and inhibited Aβ42‐induced neurodegeneration in association with decreased caspase activation. Therefore, dTSPO is an essential mediator of apoptosis in Drosophila and plays a central role in controlling longevity and neurodegenerative disease, making it a promising drug target.     

High-affinity peripheral benzodiazepine receptor ligand, PK11195, regulates protein phosphorylation in rat brain mitochondria under control of Ca(2+)

The effects of PK11195, a high-affinity peripheral benzodiazepine receptor (PBR) ligand, on protein phosphorylation in isolated purified rat brain mitochondria were investigated. The isoquinoline carboxamide ligand of PBR, PK11195, but not the benzodiazepine ligand Ro5-4864, in the nanomolar concentration range strongly increased the phosphorylation of 3.5 and 17 kDa polypeptides. The effect of PK11195 was seen in the presence of elevated Ca(2+) levels (3 x 10(-7) to 10(-6) m), but not at very low Ca(2+) levels (10(-8) to 3 x 10(-8) m). This indicates that PBR involves Ca(2+) as a second messenger in the regulation of protein phosphorylation. Staurosporine, an inhibitor of protein kinase activity was able to suppress the PK11195-promoted protein phosphorylation. When the permeability transition pore (PTP) was opened by threshold Ca(2+) load, phosphorylation of the 3.5-kDa polypeptide was diminished, but strong phosphorylation of the 43-kDa protein was revealed. The 43-kDa protein appears to be a PTP-specific phosphoprotein. If PTP was opened, PK11195 did not increase the phosphorylation of the 3.5 and 17-kDa proteins but suppressed the phosphorylation of the PTP-specific 43-kDa phosphoprotein. The ability of PK11195 to increase the protein phosphorylation, which was lost under Ca(2+)-induced PTP opening, was restored again in the presence of calmidazolium, an antagonist of calmodulin and inhibitor of protein phosphatase PP2B. These results show a tight interaction of PBR with the PTP complex in rat brain mitochondria. In conclusion, a novel function of PBR in brain mitochondria has been revealed, and the PBR-mediated protein phosphorylation has to be considered an important element of the PBR-associated signal transducing cascades in mitochondria and cells.     

Ornithine decarboxylase prevents methotrexate-induced apoptosis by reducing intracellular reactive oxygen species production

Methotrexate (MTX), a folate antagonist, was developed for the treatment of malignancies, and is currently used in rheumatoid arthritis (RA) and other chronic inflammatory disorders. It has been proven in short-term and long-term prospective studies that low doses of MTX (0.75 mg/Kg/week) are effective in controlling the inflammatory manifestations of RA. Low-concentrations of MTX achieve apoptosis and clonal deletion of activated peripheral T cells. One of the mechanisms of the anti-inflammatory and immunosuppressive effects may be the production of reactive oxygen species (ROS). However, the drug resistance of MTX in malignancies remains poorly understood. Ornithine decarboxylase (ODC) plays an important role in diverse biological functions, including cell development, differentiation, transformation, growth and apoptosis. In our previous studies, ODC overexpression was shown to prevent TNFα-induced apoptosis via reducing ROS. Here, we also investigated one mechanism of MTX-induced apoptosis and of drug resistance as to the anti-apoptotic effects of ODC during MTX treatment. We found MTX could induce caspase-dependent apoptosis and promote ROS generation together with disrupting the mitochondrial membrane potential (ΔΨ_m) of HL-60 and Jurkat T cells. Putrescine and ROS scavengers could reduce MTX-induced apoptosis, which leads to the loss of ΔΨ_m, through reducing intracellular ROS. Overexpression of ODC in parental cells had the same effects as putrescine and the ROS scavengers. Moreover, ODC overexpression prevented the decline of Bcl-2 that maintains ΔΨ_m, the cytochrome c release and activations of caspase 9 and 3 following MTX treatment. The results demonstrate that MTX-induced apoptosis is ROS-dependent and occurs along a mitochondria-mediated pathway. Overexpressed ODC cells are resistant to MTX-induced apoptosis by reducing intracellular ROS production.     

Lysosomal release of Cathepsin D precedes relocation of Cytochrome C and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress

Apoptosis was induced in human foreskin fibroblasts by the redox-cycling quinone naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Most of the cells displayed ultrastructure typical of apoptosis after 8 h of exposure to naphthazarin. Apoptosis was inhibited in fibroblasts pretreated with the cathepsin D inhibitor pepstatin A. Immunofluorescence analysis of the intracellular distribution of cathepsin D revealed a distinct granular pattern in control cells, whereas cells treated with naphthazarin for 30 min exhibited more diffuse staining that corresponded to release of the enzyme from lysosomes to the cytosol. After 2 h, release of cytochrome c from mitochondria to the cytosol was indicated by immunofluorescence. The membrane-potential–sensitive probe JC-1 and flow cytometry did not detect a permanent decrease in mitochondrial transmembrane potential (ΔΨ_m) until after 5 h of naphthazarin treatment. Our findings show that, during naphthazarin-induced apoptosis, lysosomal destabilization (measured as release of cathepsin D) precedes release of cytochrome c, loss of ΔΨ_m, and morphologic alterations. Moreover, apoptosis could be inhibited by pretreatment with pepstatin A.     

Inhibition of the mitochondrial F1F0-ATPase by ligands of the peripheral benzodiazepine receptor

Although PK11195 binds to the peripheral benzodiazepine receptor with nanomolar affinity, significant data exist which suggest that it has another cellular target distinct from the PBR. Here we demonstrate that PK11195 inhibits F(1)F(0)-ATPase activity in an OSCP-dependent manner, similar to the pro-apoptotic benzodiazepine Bz-423. Importantly, our data indicate that cellular responses observed with micromolar concentrations of PK11195, which are commonly attributed to modulation of the PBR, are likely a direct result of mitochondrial F(1)F(0)-ATPase inhibition.     

Differentiation between two ligands for peripheral benzodiazepine binding sites, [3H]RO5-4864 and [3H]PK 11195, by thermodynamic studies

The [3H]PK 11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinolinecarboxamide, binding sites in rat cardiac membranes are saturable, with high affinity, specific GABA-independent and correspond to the peripheral type of benzodiazepine. The order of potency of displacing agents was: PK 11195 greater than RO5-4864 greater than dipyridamole greater than diazepam greater than clonazepam. The Bmax obtained with [3H]PK 11195 was equivalent of the Bmax obtained with [3H]RO5-4864 in the same experimental conditions. However thermodynamic analysis indicates that the [3H]PK 11195 binding was entropy driven whereas the [3H]RO5-4864 binding was enthalpy driven. Consequently PK 11195 might be an antagonist of these binding sites and RO5-4864 an agonist or a partial agonist. The simultaneous use of both drugs might help to elucidate the physiological relevance of peripheral benzodiazepine binding sites.     

Solubilization and reassembly of the mitochondrial benzodiazepine receptor

We have solubilized and reassembled the peripheral-type benzodiazepine receptor, a component of the mitochondrial outer membrane, from rat adrenal gland mitochondria. The ligand binding site of this receptor undergoes denaturation during solubilization in digitonin, Triton X-100, or 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate at detergent concentrations above 0.1%, which is evident from the loss of high-affinity binding of [3H]PK11195, a ligand selective for the mitochondrial benzodiazepine receptor. The conformation of the binding site for PK11195 can be stabilized during solubilization in sodium cholate by relatively low concentrations of supplementary soybean lipid. Drug displacement studies demonstrate that the pharmacological properties of the receptor are preserved under these conditions. Electron micrographs of the solubilized preparation show a heterogeneous population of many small particles (less than 100 A) and some larger membranous aggregates (up to 500 A). Sucrose gradient centrifugation indicates that these lipoprotein complexes are of high buoyant density. They can be incorporated in liposomes via cholate dialysis in the presence of additional supplementary lipid. The behavior of the mitochondrial benzodiazepine receptor during solubilization and reassembly suggests that it is an integral protein of the outer membrane.     

Mitochondria: A novel target for the chemoprevention of cancer

The mitochondria have emerged as a novel target for anticancer chemotherapy. This tenet is based on the observations that several conventional and experimental chemotherapeutic agents promote the permeabilization of mitochondrial membranes in cancerous cells to initiate the release of apoptogenic mitochondrial proteins. This ability to engage mitochondrial-mediated apoptosis directly using chemotherapy may be responsible for overcoming aberrant apoptosis regulatory mechanisms commonly encountered in cancerous cells. Interestingly, several putative cancer chemopreventive agents also possess the ability to trigger apoptosis in transformed, premalignant, or malignant cells in vitro via mitochondrial membrane permeabilization. This process may occur through the regulation of Bcl-2 family members, or by the induction of the mitochondrial permeability transition. Thus, by exploiting endogenous mitochondrial-mediated apoptosis-inducing mechanisms, certain chemopreventive agents may be able to block the progression of premalignant cells to malignant cells or the dissemination of malignant cells to distant organ sites as means of modulating carcinogenesis in vivo. This review will examine cancer chemoprevention with respect to apoptosis, carcinogenesis, and the proapoptotic activity of various chemopreventive agents observed in vitro. In doing so, I will construct a paradigm supporting the notion that the mitochondria are a novel target for the chemoprevention of cancer.