Dual Cell Death Research Articles

Self-Illuminating Nanoagonist Simultaneously Induces Dual Cell Death Pathways via Death Receptor Clustering for Cancer Therapy.

Inducing death receptor 5 (DR5) clustering holds particular promise in tumor-specific therapeutics because it could trigger an apoptotic cascade in cancerous cells. Herein, we present a tumor microenvironment H2O2-responsive self-illuminating nanoagonist, which could induce dual tumor cell death pathways through enhancing DR5 clustering. By conjugating DR5 ligand peptides onto the surfaces of self-illuminating nanoparticles with cross-linking capacity, this strategy not only provides scaffolds for ligands to bind receptors but also cross-links them through photo-cross-linking. This strategy allows for efficient activation of DR5 downstream signaling, initiating the extrinsic apoptosis pathway and immunogenic cell death of tumor cells, and contributes to improved tumor-specific immune responses, resulting in enhanced antitumor efficacy and minimized systemic adverse effects.

ML162 derivatives incorporating a naphthoquinone unit as ferroptosis/apoptosis inducers: Design, synthesis, anti-cancer activity, and drug-resistance reversal evaluation

Activating apoptosis has long been viewed as an anti-cancer process, but recently increasing evidence has accumulated that induction of ferroptosis has emerged as a promising strategy for cancer therapeutics. Glutathione peroxidase 4 (GPX4) is one of the pivotal factors regulating ferroptosis that targeted inhibition or degradation of GPX4 could effectively trigger ferroptosis. In this study, a series of ML162-quinone conjugates were constructed by using pharmacophore hybridization and bioisosterism strategies, with the aim of obtaining more active anticancer agents via the ferroptosis and apoptosis dual cell death processes. Of these compounds, GIC-20 was identified as the most active one that exhibited promising anticancer activity both in vitro and in vivo via ferroptosis and apoptosis dual-targeting processes, without obvious toxicity compared with ML162. On one hand, GIC-20 could trigger ferroptosis in cells by inducing intracellular lipid peroxide and ROS accumulation, and destroying mitochondrial structure. In addition to GPX4 inhibition, GIC-20 can also trigger ferroptosis via proteasomal-mediated degradation of GPX4, suggesting GIC-20 may function as a molecule glue degrader. On the other hand, GIC-20 can also induce apoptosis via upregulating the level of apoptotic protein Bax and downregulating the level of anti-apoptotic protein Bcl-2 in HT1080 cells. Furthermore, GIC-20 also enhanced the sensitivity of resistant MIA-PaCa-2-AMG510R cells to AMG510, suggesting the great potential of GIC-20 in overcoming the acquired resistance of KRASG12C inhibitors. Overall, GIC-20 represents a novel dual ferroptosis/apoptosis inducer warranting further development for cancer therapeutics and overcoming drug resistance.

Selenium Donor Inhibited Hepatitis B Virus Associated Hepatotoxicity via the Apoptosis and Ferroptosis Pathways.

The serum selenium level was determined in 45 patients with HBV-positive HCC (HBV+-HCC group), 45 patients with chronic hepatitis B virus infection (CHB group), and 45 healthy cases (HC group). The sodium selenite (Na2SeO3)-treated HepG2.2.15 cells were used to observe the regulatory role of selenium on HBV replication. D-GalN/erastin-added HL7702 was used to determine the regulatory roles of Na2SeO3 on hepatotoxicity or hepatocyte ferroptosis. The wild-type (WT) C57BL/6 mice and HBx-Tg mice were received lipopolysaccharide (LPS)/D-GalN, together with or without Na2SeO3 administration for indicated period. Following euthanasia, the blood and liver tissue samples were collected, and specific markers were evaluated subsequently. The serum selenium level was downregulated in patients with HBV-positive HCC (HBV+-HCC group) (57.2 ± 22.5 μg/L vs. 91.8 ± 43.9 μg/L, P < 0.001), and its higher level could provide a better prognosis in these patients. The treatment using Na2SeO3, a selenium donor, at high concentration (5 μM), suppressed the HBV replication by about 50% in HepG2.2.15 cells (P < 0.001), through promoting apoptotic cell death and inhibiting cellular inhibitor of apoptosis proteins (cIAPs). In addition, low-dose (500 nM) Na2SeO3 could almost totally reversed the hepatotoxicity induced by hepatitis B virus X protein (HBx) (P < 0.001), which were the main causes of HCC in patients. Studies at the cellular levels showed that low-dose Na2SeO3 inhibited the HBx-related hepatotoxicity by blocking ferroptosis, and glutathione peroxidase 4 (GPX4) mediated this regulatory role. Mice model results confirmed that the treatment with Na2SeO3 could mitigated LPS/D-GalN-induced hepatic injury through ferroptosis pathways. Selenium regulated the dual cell death in different HCC stages via different signaling pathways, which could partly explain the anti-HBV and anti-HCC properties of selenium.

Medicago Sativa Defensin1 as a tumor sensitizer for improving chemotherapy: translation from anti-fungal agent to a potential anti-cancer agent.

Plant defensins including Medicago Sativa defensin 1 (MsDef1) are cysteine-rich antifungal peptides which are known for potent broad-spectrum antifungal activity against bacterial or fungal pathogens of plants. The antimicrobial activities of these cationic defensins are attributed to their capacity to bind to cell membranes to create potentially structural defects tin the cell membranes to interact with intracellular target (s) and mediates cytotoxic effects. Our earlier work identified Glucosylceramide (GlcCer) of fungus F. graminearum as a potential target for biological activity. Multi-drug resistant (MDR) cancer cells overexpress GlcCer on the surface of plasma membrane. Hence, MsDef1 may have a potential to bind to GlcCer of MDR cancer cells to induce cell death. We have characterized the three-dimensional structure of MsDef1 and the solution dynamics using of 15N-labeled MsDef1 nuclear magnetic resonance (NMR) spectroscopy which showed that GlcCer binds MsDef1 at two specific sites on the peptide molecule. The ability of MsDef1 to permeate MDR cancer cells was demonstrated by measuring the release of apoptotic ceramide in drug resistant MCF-7R cells. It was also shown that MsDef1 activated dual cell death pathways ceramide and Apoptosis Stimulating Kinase ASK1 by disintegrating GlcCer and oxidizing tumor specific biomarker thioredoxin (Trx) respectively. As a result, MsDef1 sensitizes MDR cancer cells to evoke a better response from Doxorubicin, a front-line chemotherapy for triple negative breast cancer (TNBC) treatment. The combination of MsDef1 and Doxorubicin induced 5 to10-fold greater apoptosis in vitro MDR cells MDA-MB-231R compared to either MsDef1 or Doxorubicin alone. Confocal microscopy revealed that MsDef1 facilitates a) influx of Doxorubicin in MDR cancer cells, b) preferential uptake by MDR cells but not by normal fibroblasts and breast epithelial cells (MCF-10A). These results suggest that MsDef1 targets MDR cancer cells and may find utility as a neoadjuvant chemotherapy. Hence, the extension of antifungal properties of MsDef1 to cancer my result in addressing the MDR problems in cancer.

Near-Infrared Phototheranostic Iron Pyrite Nanocrystals Simultaneously Induce Dual Cell Death Pathways via Enhanced Fenton Reactions in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is considered more aggressive with a poorer prognosis than other breast cancer subtypes. Through systemic bioinformatic analyses, we established the ferroptosis potential index (FPI) based on the expression profile of ferroptosis regulatory genes and found that TNBC has a higher FPI than non-TNBC in human BC cell lines and tumor tissues. To exploit this finding for potential patient stratification, we developed biologically amenable phototheranostic iron pyrite FeS2 nanocrystals (NCs) that efficiently harness near-infrared (NIR) light, as in photovoltaics, for multispectral optoacoustic tomography (MSOT) and photothermal ablation with a high photothermal conversion efficiency (PCE) of 63.1%. Upon NIR irradiation that thermodynamically enhances Fenton reactions, dual death pathways of apoptosis and ferroptosis are simultaneously triggered in TNBC cells, comprehensively limiting primary and metastatic TNBC by regulating p53, FoxO, and HIF-1 signaling pathways and attenuating a series of metabolic processes, including glutathione and amino acids. As a unitary phototheranostic agent with a safe toxicological profile, the nanocrystal represents an effective way to circumvent the lack of therapeutic targets and the propensity of multisite metastatic progression in TNBC in a streamlined workflow of cancer management with an integrated image-guided intervention.

Caspase-8-driven apoptotic and pyroptotic crosstalk causes cell death and IL-1β release in X-linked inhibitor of apoptosis (XIAP) deficiency.

Genetic lesions in X-linked inhibitor of apoptosis (XIAP) pre-dispose humans to cell death-associated inflammatory diseases, although the underlying mechanisms remain unclear. Here, we report that two patients with XIAP deficiency-associated inflammatory bowel disease display increased inflammatory IL-1β maturation as well as cell death-associated caspase-8 and Gasdermin D (GSDMD) processing in diseased tissue, which is reduced upon patient treatment. Loss of XIAP leads to caspase-8-driven cell death and bioactive IL-1β release that is only abrogated by combined deletion of the apoptotic and pyroptotic cell death machinery. Namely, extrinsic apoptotic caspase-8 promotes pyroptotic GSDMD processing that kills macrophages lacking both inflammasome and apoptosis signalling components (caspase-1, -3, -7, -11 and BID), while caspase-8 can still cause cell death in the absence of both GSDMD and GSDME when caspase-3 and caspase-7 are present. Neither caspase-3 and caspase-7-mediated activation of the pannexin-1 channel, or GSDMD loss, prevented NLRP3 inflammasome assembly and consequent caspase-1 and IL-1β maturation downstream of XIAP inhibition and caspase-8 activation, even though the pannexin-1 channel was required for NLRP3 triggering upon mitochondrial apoptosis. These findings uncouple the mechanisms of cell death and NLRP3 activation resulting from extrinsic and intrinsic apoptosis signalling, revealhow XIAP loss can co-opt dual cell death programs, and uncover strategies for targeting the celldeath and inflammatory pathways that result from XIAP deficiency.

Potentiating the Cytotoxic Activity of a Novel Simvastatin-Loaded Cubosome against Breast Cancer Cells: Insights on Dual Cell Death via Ferroptosis and Apoptosis.

PurposeFemale breast cancer is the most prevalent cancer worldwide. Emerging evidence shows that simvastatin (SIM) has promising anticancer activities. However, the underlying mechanisms are not fully elucidated. Increasing reports imply statins can modulate ferroptosis through disrupting reactive oxygen species (ROS) and glutathione peroxidase enzyme (GPX4) levels. However, whether ferroptosis contributes to SIM anticancer activity, especially regarding GPX4 is unclear. Moreover, poor aqueous SIM solubility hinders its delivery in adequate levels to tumor sites. Meanwhile, cubosomes are biocompatible nanocarriers that enhance lipophilic drug delivery. Therefore, in this study, we formulated a novel SIM-loaded cubosome (SIM-CB) and analyzed its cytotoxic activity on MCF-7 cancer cells in comparison with free SIM.MethodsThe present study tested the cytotoxic activity of SIM-CB on MCF-7 cells, in comparison with SIM using sulphorhodamine assay. We analyzed SIM-CB effect on apoptosis and cell cycle using flowcytometry, and investigated its effect on Bcl-2, caspase 3, ROS, reduced glutathione (GSH), lipid peroxides and GPX4 enzyme. Finally, we tested the persistence of SIM-CB apoptosis and ferroptosis activities on MCF-7 cells in presence of vitamin E, a potent antioxidant and ferroptosis inhibitor.ResultsSIM-CB was successfully formulated at the nano size. SIM-CB significantly increased simvastatin therapeutic activity, with IC50 of SIM-CB 52% lower than SIM. 95% CI [1.8, 2.7], SD = 0.34 for SIM-CB, and [4.1, 5.2], SD = 0.45 for SIM. Compared with free SIM, SIM-CB doubled total deaths and increased apoptosis (p < 0.05). Moreover, SIM-CB remarkably increased caspase-3, ROS, and lipid peroxide levels but decreased antiapoptotic Bcl-2 protein, GSH, and GPX4 compared with free SIM. Notably, SIM-CB elicited a high distinguished resistance against the inhibitory effects of vitamin E.ConclusionTo the best of our knowledge, this study is the first to present SIM-CB as a promising means to enhancing the therapeutic potential of simvastatin against breast cancer cells, through potentiating both apoptosis and ferroptosis.

Species-Specific Inhibition of Necroptosis by HCMV UL36.

Viral infection activates cellular antiviral defenses including programmed cell death (PCD). Many viruses, particularly those of the Herpesviridae family, encode cell death inhibitors that antagonize different forms of PCD. While some viral inhibitors are broadly active in cells of different species, others have species-specific functions, probably reflecting the co-evolution of the herpesviruses with their respective hosts. Human cytomegalovirus (HCMV) protein UL36 is a dual cell death pathway inhibitor. It blocks death receptor-dependent apoptosis by inhibiting caspase-8 activation, and necroptosis by binding to the mixed lineage kinase domain-like (MLKL) protein and inducing its degradation. While UL36 has been shown to inhibit apoptosis in human and murine cells, the specificity of its necroptosis-inhibiting function has not been investigated. Here we show that UL36 interacts with both human and murine MLKL, but has a higher affinity for human MLKL. When expressed by a recombinant mouse cytomegalovirus (MCMV), UL36 caused a modest reduction of murine MLKL levels but did not inhibit necroptosis in murine cells. These data suggest that UL36 inhibits necroptosis, but not apoptosis, in a species-specific manner, similar to ICP6 of herpes simplex virus type 1 and MC159 of molluscum contagiosum virus. Species-specific necroptosis inhibition might contribute to the narrow host range of these viruses.

Human cytomegalovirus protein pUL36: A dual cell death pathway inhibitor

Human cytomegalovirus (HCMV) is an important human pathogen and a paradigm of intrinsic, innate, and adaptive viral immune evasion. Here, we employed multiplexed tandem mass tag-based proteomics to characterize host proteins targeted for degradation late during HCMV infection. This approach revealed that mixed lineage kinase domain-like protein (MLKL), a key terminal mediator of cellular necroptosis, was rapidly and persistently degraded by the minimally passaged HCMV strain Merlin but not the extensively passaged strain AD169. The strain Merlin viral inhibitor of apoptosis pUL36 was necessary and sufficient both to degrade MLKL and to inhibit necroptosis. Furthermore, mutation of pUL36 Cys131 abrogated MLKL degradation and restored necroptosis. As the same residue is also required for pUL36-mediated inhibition of apoptosis by preventing proteolytic activation of procaspase-8, we define pUL36 as a multifunctional inhibitor of both apoptotic and necroptotic cell death.

Bacteroides fragilis Strain ZY-312 Defense against Cronobacter sakazakii-Induced Necrotizing Enterocolitis In Vitro and in a Neonatal Rat Model.

Cronobacter sakazakii is an important pathogen associated with the development of necrotizing enterocolitis (NEC), infant sepsis, and meningitis. Several randomized prospective clinical trials demonstrated that oral probiotics could decrease the incidence of NEC. Previously, we isolated and characterized a novel probiotic, Bacteroides fragilis strain ZY-312. However, it remains unclear how ZY-312 protects the host from the effects of C. sakazakii infection. To understand the underlying mechanisms triggering the probiotic effects, we tested the hypothesis that there was cross talk between probiotics/probiotics-modulated microbiota and the local immune system, governed by the permeability of the intestinal mucosa, using in vitro and in vivo models for the intestinal permeability. The probiotic effects of ZY-312 on intestinal epithelial cells were first examined, and the results revealed that ZY-312 inhibited C. sakazakii invasion, C. sakazakii-induced dual cell death (pyroptosis and apoptosis), and epithelial barrier dysfunction in vitro and in vivo The presence of ZY-312 also resulted in decreased expression of an inflammasome (NOD-like receptor family member pyrin domain-containing protein 3 [NLRP3]), caspase-3, and serine protease caspase-1 in a neonatal rat model. Furthermore, ZY-312 significantly modulated the compositions of the intestinal bacterial communities and decreased the relative abundances of Proteobacteria and Gammaproteobacteria but increased the relative abundances of Bacteroides and Bacillus in neonatal rats. In conclusion, our findings have shown for the first time that the probiotic B. fragilis ZY-312 suppresses C. sakazakii-induced NEC by modulating the proinflammatory response and dual cell death (apoptosis and pyroptosis).IMPORTANCE Cronobacter sakazakii is an opportunistic pathogenic bacterium that can cause necrotizing enterocolitis (NEC). However, the mechanism of pathogenicity of C. sakazakii is largely unknown. Here we have now demonstrated that apoptotic and pyroptotic stimuli are effectors of C. sakazakii-induced NEC. Previously, we isolated a novel probiotic strain candidate from fecal samples from healthy infants and characterized it as Bacteroides fragilis strain ZY-312. Functional characterization reveals that ZY-312 inhibited C. sakazakii invasion, restoring epithelial barrier dysfunction, decreasing the expression of inflammatory cytokines, and reducing dual cell death (pyroptosis and apoptosis). Furthermore, the presence of ZY-132 was sufficient to hinder the adverse reaction seen with C. sakazakii in a C. sakazakii-induced NEC model. Taking the results together, our study demonstrated the utility of ZY-312 as a promising probiotic agent for the prevention of NEC.

Activation of necroptosis to overcome drug resistance in leukemia

The understanding of cell death mechanisms is crucial for the development and application of novel anti-cancer therapies to avoid or circumvent drug-resistance in refractory malignancies. Impairment of apoptotic cell death plays a major role in therapy resistance and relapse of acute lymphoblastic leukemia (ALL) patients. Therefore, efforts are being directed at new agents reactivating apoptosis or inducing alternative cell death pathways such as necroptosis, a regulated form of necrosis. In a recent study published in Science Translational Medicine we show that the IAP (inhibitor of apoptosis proteins) inhibitor birinapant potently induces cell death in patient-derived ALL cells in vitro and in vivo through a receptor-interacting protein kinase 1- (RIP1) dependent mechanism. To define the cell death modality induced downstream of RIP1, we used a multicolor lentiCRISPR approach that allows simultaneous knockout of multiple genes. We observed that apoptosis and necroptosis are induced simultaneously as the inhibition of both pathways is required to restore cell viability upon birinapant treatment. This induction of dual cell death makes birinapant and other IAP inhibitors interesting agents for the treatment of refractory or drug resistant malignancies.

A novel microtubule depolymerizing colchicine analogue triggers apoptosis and autophagy in HCT-116 colon cancer cells.

Colchicine is a tubulin-binding natural product isolated from Colchicum autumnale. Here we report the in vitro anticancer activity of C-ring modified semi-synthetic derivative of colchicine; N-[(7S)-1,2,3-trimethoxy-9-oxo-10-(4-phenyl-piperidin-1-yl)-5,6,7,9 tetrahydrobenzo[a]heptalen-7-yl]acetamide (4h) on colon cancer HCT-116 cell line. The compound 4h was screened for anti-proliferative activity against different human cancer cell lines and was found to exhibit higher cytotoxicity against colon cancer cell lines HCT-116 and Colo-205 with IC50 of 1 and 0.8 μM respectively. Cytotoxicity of the compound to the normal fR2 breast epithelial cells and normal HEK293 human embryonic kidney cells was evaluated in concentration and time-dependent manner to estimate its selectivity for cancer cells which showed much better selectivity than that of colchicine. Compound 4h induced cell death in HCT-116 cells by activating apoptosis and autophagy pathways. Autophagy inhibitor 3-MA blocked the production of LC3-II and reduced the cytotoxicity in response to 4h, but did not affect apoptosis, suggesting thereby that these two were independent events. Reactive oxygen species scavenger ascorbic acid pretreatment not only decreased the reactive oxygen species level but also reversed 4h induced cytotoxicity. Treatment with compound 4h depolymerized microtubules and the majority of cells arrested at the G2/M transition. Together, these data suggest that 4h has better selectivity and is a microtubule depolymerizer, which activates dual cell-death machineries, and thus, it could be a potential novel therapeutic agent in cancer therapy.

Methyl 3-hydroxyimino-11-oxoolean-12-en-28-oate (HIMOXOL), a synthetic oleanolic acid derivative, induces both apoptosis and autophagy in MDA-MB-231 breast cancer cells

HIMOXOL (methyl 3-hydroxyimino-11-oxoolean-12-en-28-oate) is a synthetic derivative of oleanolic acid (OA). HIMOXOL revealed the highest cytotoxic effect among tested synthetic OA analogs. In this study we focused on elucidating the cytotoxic mechanism of HIMOXOL in MDA-MB-231 breast cancer cells. HIMOXOL reduced MDA-MB-231 cell viability with an IC50 value of 21.08±0.24μM. In contrast to OA, the tested compound induced cell death by activating apoptosis and the autophagy pathways. More specifically, we found that HIMOXOL was able to activate the extrinsic apoptotic pathway, which was proven by observation of caspase-8, caspase-3 and PARP-1 protein activation in Western blot analysis. An increase in the ratio of Bax/Bcl-2 protein levels was also detected. Moreover, HIMOXOL triggered microtubule-associated protein LC3-II expression and upregulated beclin 1. This observed compound activity was modulated by mitogen-activated protein kinases and NFκB/p53 signaling pathways.Together, these data suggest that HIMOXOL, a synthetic oleanolic acid derivative which activates dual cell death machineries, could be a potential and novel chemotherapeutic agent.

The microtubule depolymerizing agent naphthazarin induces both apoptosis and autophagy in A549 lung cancer cells

Naphthazarin (DHNQ, 5,8-dihydroxy-l,4-naphthoquinone) is a naturally available 1,4-naphthoquinone derivatives. In this study, we focused on elucidating the cytotoxic mechanism of naphthazarin in A549 non-small cell lung carcinoma cells. Naphthazarin reduced the A549 cell viability considerably with an IC(50) of 16.4±1.6μM. Naphthazarin induced cell death in a dose- and time-dependent manner by activating apoptosis and autophagy pathways. Specifically, we found naphthazarin inhibited the PI3K/Akt cell survival signalling pathway, measured by p53 and caspase-3 activation, and PARP cleavage. It also resulted in an increase in the ratio of Bax/Bcl2 protein levels, indicating activation of the mitochondrial apoptotic pathway. Similarly naphthazarin triggered LC3II expression and induced autophagic flux in A549 cells. We demonstrated further that naphthazarin is a microtubule inhibitor in cell-free system and in A549 cells. Naphthazarin treatment depolymerized interphase microtubules and disorganised spindle microtubules and the majority of cells arrested at the G(2)/M transition. Together, these data suggest that naphthazarin, a microtubule depolymerizer which activates dual cell death machineries, could be a potential novel chemotherapeutic agent.

Sanguinarine induces bimodal cell death in K562 but not in high Bcl-2-expressing JM1 cells.

Our previous studies with low Bcl-2-expressing K562 cells have shown that, when treated with the putative anti-cancer drug sanguinarine, concentrations of 1.5 microg/ml induced the morphology of apoptosis or programmed cell death (PCD), while concentrations of 12.5 microg/ml induced a morphology of blister formation or blister cell death (BCD). To elucidate the possible role of Bcl-2 in this dual cell death modality induced by sanguinarine, K562 and the high Bcl-2-expressing JM1 cells were treated with sanguinarine concentrations of 1.5 microg/ml and 12.5 microg/ml respectively, and multiple parameters of their effects were studied using light and electron microscopy, terminal deoxynucleotidyl transferase (TdT) end-labeling, 51Cr release, trypan blue exclusion, propidium iodide exclusion, and annexin-V binding. In general, we found that, while K562 cells underwent PCD and BCD when treated with sanguinarine, JM1 cells failed to undergo either PCD or BCD under the same experimental conditions. Thus, the over-expression of anti-apoptotic Bcl-2 may have prevented sanguinarine from inducing PCD and BCD in JM1 cells. These results indicate that the resistance of JM1 cells to the alkaloid sanguinarine may have been due to an anti-BCD role played by Bcl-2, in addition to its widely reported anti-apoptotic role.

Mitochondrial DNA Damage as a Mechanism of Cell Loss in Alzheimer's Disease

Aging is associated with impaired mitochondrial function caused by accumulation of oxygen free radical-induced mitochondrial (Mt) DNA mutations. One prevailing theory is that age-associated diseases, including Alzheimer's disease (AD), may be precipitated, propagated, or caused by impaired mitochondrial function. To investigate the role of MtDNA relative to genomic (Gn) DNA damage in AD, temporal lobe samples from postmortem AD (n = 37) and control (n = 25) brains were analyzed for MtDNA and GnDNA fragmentation, mitochondrial protein and cytochrome oxidase expression, MitoTracker Green fluorescence (to assess mitochondrial mass/abundance), and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-OHdG) immunoreactivity. Brains with AD had more extensive nicking and fragmentation of both MtDNA and GnDNA as demonstrated by agarose gel electrophoresis, end-labeling, and the in situ terminal deoxynucleotide transferase end-labeling (TUNEL) assay, and only the brains with AD had detectable 8-OHdG immunoreactivity in cortical neurons. Increased MtDNA damage in AD was associated with reduced MtDNA content, as demonstrated by semiquantitative PCR analysis and reduced levels of Mt protein and cytochrome oxidase expression by Western blot analysis or immunohistochemical staining with image analysis. The finding of reduced MitoTracker Green fluorescence in AD brains provided additional evidence that reduced Mt mass/abundance occurs with AD neurodegeneration. The presence of increased MtDNA and GnDNA damage in AD suggest dual cell death cascades in AD. Impaired mitochondrial function caused by MtDNA damage may render brain cells in AD more susceptible to oxidative injury and thereby provide a mechanism by which systemic or environmental factors could influence the course of disease.