Role Of Mitochondria In Apoptosis Research Articles

SIRT6 suppresses colon cancer growth by inducing apoptosis and autophagy through transcriptionally down-regulating Survivin

SIRT6, an evolutionarily conserved histone deacetylase, has been identified as a novel direct downstream target of Akt/FoxO3a and a tumor suppressor in colon cancer in our previous research. Nevertheless, the precise mechanisms through which SIRT6 hinders tumor development remain unclear. To ascertain whether SIRT6 directly impacts Survivin transcription, a ChIP assay was conducted using an anti-SIRT6 antibody to isolate DNA. YM155 was synthesized to explore Survivin’s role in mitochondrial apoptosis, autophagy and tumor progression. Our investigation into the regulation of Survivin involved real-time fluorescence imaging in living cells, real-time PCR, immunohistochemistry, flow cytometry, and xenograft mouse assays. In this current study, we delved into the role of SIRT6 in colon cancer and established that activated SIRT6 triggers mitochondrial apoptosis by reducing Survivin expression. Subsequent examinations revealed that SIRT6 directly binds to the Survivin promoter, impeding its transcription. Notably, direct inhibition of Survivin significantly impeded colon cancer proliferation by inducing mitochondrial apoptosis and autophagy both in vitro and in vivo. More interestingly, Survivin inhibition reactivated the Akt/FoxO3a pathway and elevated SIRT6 levels, establishing a positive feedback loop. Our results identify Survivin as a novel downstream transcriptional target of SIRT6 that fosters tumor growth and holds promise as a prospective target for colon cancer therapy.

IL-22 relieves hepatic ischemia-reperfusion injury by inhibiting mitochondrial apoptosis based on the activation of STAT3

IntroductionInterleukin-22 (IL-22) has been proven to exhibit a protective role in hepatic ischemia-reperfusion injury (HIRI). This study aimed to explore the change of IL-22 and IL-22 receptor 1 (IL-22R1) axis in HIRI and its role in mitochondrial apoptosis associated with STAT3 activation. Materials and MethodsI/R mice were examined for the expression of IL-22, IL-22R1 and IL-22BP. The roles of IL-22 in hepatic histopathology and oxidative stress injuries (ALT, MDA and SOD) were determined. Oxidative stress damages of AML-12 cells were induced by H2O2, and were indicated by apoptosis, Ca2+ concentration, and mitochondrial function. The effects of IL-22 on p-STAT3Try705 were analyzed. ResultsWe found that the expression of IL-22, IL-22R1, and IL-22BP was elevated 24 h after I/R induction, while decreased 48 h after I/R induction. Furthermore, we also discovered that IL-22 rescued the morphological damages and dysfunction of hepatocytes induced by H2O2, which were antagonized by IL-22BP, an endogenous antagonist of IL-22. Additionally, increased levels of Ca2+ concentration, MDA, ROS, apoptosis and mitochondrial dysfunction were noticed in H2O2-treated hepatocytes. However, IL-22 ameliorated the effects of I/R or H2O2. The protective effects of IL-22 were reversed by AG490, a specific antagonist of STAT3. ConclusionsIn conclusion, our results indicated that IL-22 inhibited I/R-induced oxidative stress injury, Ca2+ overload, and mitochondrial apoptosis via STAT3 activation.

Chinese giant salamander Bcl-w: An inhibitory role in iridovirus-induced mitochondrial apoptosis and virus replication

B-cell lymphoma-2 (BCL-2) superfamily molecules play crucial roles in mitochondrial apoptosis induced by Chinese giant salamander iridovirus (GSIV). As an anti-apoptotic molecule in the BCL-2 family, the molecular mechanism of Bcl-w during GSIV infection remains unknown. In this study, we characterized for the first time an amphibian Bcl-w from Chinese giant salamander Andrias davidianus (AdBcl-w), and its function and regulatory mechanism during GSIV infection were investigated. AdBcl-w possesses the conserved structural features of Bcl-w and shares 35–54% sequence identities with other Bcl-w. mRNA expression of AdBcl-w was most abundant in liver and muscle. The AdBcl-w mRNA expression was regulated during GSIV infection. Western blotting assays revealed that the level of Bcl-w protein was downregulated markedly as the infection progresses. Confocal microscopy showed that overexpressed AdBcl-w was translocated to the mitochondria after infection with GSIV. Flow cytometry analysis demonstrated that compared with control, the apoptotic progress in cells transfected with AdBcl-w was reduced while that in cells transfected with AdBcl-w siRNA was enhanced. The number of virus major capsid protein gene copies was lower and protein synthesis was reduced in AdBcl-w overexpressing cells. In addition, AdBcl-w could bind directly to the pro-apoptotic molecule AdBak, while this interaction was weakened with GSIV infection. Moreover, p53 level was reduced and the mRNA expression levels of crucial regulatory molecules in the p53 pathway were regulated in AdBcl-w overexpressing cells during GSIV infection. These results suggested that AdBcl-w inhibit GSIV replication by regulating the virus induced mitochondrial apoptosis.

An Emergent Role for Mitochondrial Bioenergetics in the Action of Snake Venom Toxins on Cancer Cells.

Beyond the role of mitochondria in apoptosis initiation/execution, some mitochondrial adaptations support the metastasis and chemoresistance of cancer cells. This highlights mitochondria as a promising target for new anticancer strategies. Emergent evidence suggests that some snake venom toxins, both proteins with enzymatic and non-enzymatic activities, act on the mitochondrial metabolism of cancer cells, exhibiting unique and novel mechanisms that are not yet fully understood. Currently, six toxin classes (L-amino acid oxidases, thrombin-like enzymes, secreted phospholipases A2, three-finger toxins, cysteine-rich secreted proteins, and snake C-type lectin) that alter the mitochondrial bioenergetics have been described. These toxins act through Complex IV activity inhibition, OXPHOS uncoupling, ROS-mediated permeabilization of inner mitochondrial membrane (IMM), IMM reorganization by cardiolipin interaction, and mitochondrial fragmentation with selective migrastatic and cytotoxic effects on cancer cells. Notably, selective internalization and direct action of snake venom toxins on tumor mitochondria can be mediated by cell surface proteins overexpressed in cancer cells (e.g. nucleolin and heparan sulfate proteoglycans) or facilitated by the elevated Δψm of cancer cells compared to that non-tumor cells. In this latter case, selective mitochondrial accumulation, in a Δψm-dependent manner, of compounds linked to cationic snake peptides may be explored as a new anti-cancer drug delivery system. This review analyzes the effect of snake venom toxins on mitochondrial bioenergetics of cancer cells, whose mechanisms of action may offer the opportunity to develop new anticancer drugs based on toxin scaffolds.

The Effects of P53 in the Globular Heads of the C1q Receptor in Gastric Carcinoma Cell Apoptosis Are Exerted via a Mitochondrial-Dependent Pathway.

The globular heads of the C1q receptor (gC1qR), located in the B cell cytoplasm, perform important roles in many cellular processes. A recent studies reported a major role of mitochondrial apoptosis in several cancers, but there has been no report on gastric carcinoma (GC). In this study, the mechanism by which cell apoptosis is induced by gC1qR in GC was explored. Western blot showed that gC1qR and P53 protein levels were lower in GC tissues than in normal tissues. Cytotoxicity was dynamically increased in gC1qR-overexpressing GC cells compared to the control. CCK8 assay indicated that overexpression of gC1qR induced GC cell apoptosis, increased reactive oxygen species (ROS) production, decreased the mitochondrial transmembrane potential and promoted mitochondrial apoptosis. Moreover, the P53 level increased in response to gC1qR. The viability, migration, and mitochondrial transmembrane potential of GC cells increased in association with decreased levels of ROS and mitochondrial apoptosis in the P53-silenced group. Collectively, our findings indicate that apoptosis of GC cells is enhanced when gC1qR overexpression is induced by P53-mediated mitochondrial apoptosis.

A Putative Prohibitin-Calcium Nexus in β-Cell Mitochondria and Diabetes.

The role of mitochondria in apoptosis is well known; however, the mechanisms linking mitochondria to the proapoptotic effects of proinflammatory cytokines, hyperglycemia, and glucolipotoxicity are not completely understood. Complex Ca2+ signaling has emerged as a critical contributor to these proapoptotic effects and has gained significant attention in regulating the signaling processes of mitochondria. In pancreatic β-cells, Ca2+ plays an active role in β-cell function and survival. Prohibitin (PHB), a mitochondrial chaperone, is actively involved in maintaining the architecture of mitochondria. However, its possible interaction with Ca2+-activated signaling pathways has not been explored. The present review aims to examine potential crosstalk between Ca2+ signaling and PHB function in pancreatic β-cells. Moreover, this review will focus on the effects of cytokines and glucolipotoxicity on Ca2+ signaling and its possible interaction with PHB. Improved understanding of this important mitochondrial protein may aid in the design of more targeted drugs to identify specific pathways involved with stress-induced dysfunction in the β-cell.

Heavy-Metals-Mediated Phospholipids Scrambling by Human Phospholipid Scramblase 3: A Probable Role in Mitochondrial Apoptosis.

Human phospholipid scramblases are a family of four homologous transmembrane proteins (hPLSCR1-4) mediating phospholipids (PLs) translocation in plasma membrane upon Ca2+ activation. hPLSCR3, the only homologue localized to mitochondria, plays a vital role in mitochondrial structure, function, maintenance, and apoptosis. Upon Ca2+ activation, hPLSCR3 mediates PL translocation at the mitochondrial membrane enhancing t-bid-induced cytochrome c release and apoptosis. Mitochondria are important target organelles for heavy-metals-induced apoptotic signaling cascade and are the central executioner of apoptosis to trigger. Pb2+ and Hg2+ toxicity mediates apoptosis by increased reactive oxygen species (ROS) and cytochrome c release from mitochondria. To discover the role of hPLSCR3 in heavy metal toxicity, hPLSCR3 was overexpressed as a recombinant protein in Escherichia coli Rosetta (DE3) and purified by affinity chromatography. The biochemical assay using synthetic proteoliposomes demonstrated that hPLSCR3 translocated aminophospholipids in the presence of micromolar concentrations of Pb2+ and Hg2+. A point mutation in the Ca2+-binding motif (F258V) led to a ∼60% loss in the functional activity and decreased binding affinities for Pb2+ and Hg2+ implying that the divalent heavy metal ions bind to the Ca2+-binding motif. This was further affirmed by the characteristic spectra observed with stains-all dye. The conformational changes upon heavy metal binding were monitored by circular dichroism, intrinsic tryptophan fluorescence, and light-scattering studies. Our results revealed that Pb2+ and Hg2+ bind to hPLSCR3 with higher affinity than Ca2+ thus mediating scramblase activity. To summarize, this is the first biochemical evidence for heavy metals binding to the mitochondrial membrane protein leading to bidirectional translocation of PLs specifically toward phosphatidylethanolamine.

Transcutaneous carbon dioxide enhances the antitumor effect of radiotherapy on oral squamous cell carcinoma.

Radiotherapy (RT) is one of the main treatment modalities for oral squamous cell carcinoma (OSCC), however, radioresistance is a major impediment to its clinical success and poses as a concern that needs to be addressed. Tumor hypoxia is known to be significantly associated with radioresistance in various malignancies, hence, resolving the hypoxic state of a tumor may improve the antitumor effect of RT on OSCC. We have previously revealed that transcutaneous CO2 induced mitochondrial apoptosis and suppressed tumor growth in OSCC by resolving hypoxia. Considering the previous study, we hypothesized that transcutaneous CO2 may enhance the antitumor effect of RT on OSCC by improving intratumoral hypoxia, thereby overcoming radioresistance. In the present study, the combination of transcutaneous CO2 and RT significantly inhibited tumor growth compared with other treatments. This combination therapy also led to decreased expression of HIF-1α in parallel with increased expression of the cleaved forms of caspase-3-8-9 and PARP, which play essential roles in mitochondrial apoptosis. Additionally, the combination therapy increased the expression of ROS modulator1 and subsequent mitochondrial ROS production, compared to RT alone. These results indicated that transcutaneous CO2 could potentially improve the antitumor effect of RT by decreasing the intratumoral hypoxia and increasing the mitochondrial apoptosis. Our findings indicated that CO2 therapy may be a novel adjuvant therapy in combination with RT for OSCC.

Alterations of the MT-ATP8 Gene and 9-bp Deletion in Vietnamese Patients with Breast Cancer

Alterations of the MT-ATP8 Gene and 9-bp Deletion in Vietnamese Patients with Breast Cancer

Mitochondrial levels determine variability in cell death by modulating apoptotic gene expression

Fractional killing is the main cause of tumour resistance to chemotherapy. This phenomenon is observed even in genetically identical cancer cells in homogeneous microenvironments. To understand this variable resistance, here we investigate the individual responses to TRAIL in a clonal population of HeLa cells using live-cell microscopy and computational modelling. We show that the cellular mitochondrial content determines the apoptotic fate and modulates the time to death, cells with higher mitochondrial content are more prone to die. We find that all apoptotic protein levels are modulated by the mitochondrial content. Modelling the apoptotic network, we demonstrate that these correlations, and especially the differential control of anti- and pro-apoptotic protein pairs, confer mitochondria a powerful discriminatory capacity of apoptotic fate. We find a similar correlation between the mitochondria and apoptotic proteins in colon cancer biopsies. Our results reveal a different role of mitochondria in apoptosis as the global regulator of apoptotic protein expression.

Eupatorin and Salvigenin Potentiate Doxorubicin-Induced Apoptosis and Cell Cycle Arrest in HT-29 and SW948 Human Colon Cancer Cells

Background:Cancer persists as one of the world’s most pressing maladies. Notable points about chemotherapy are drug side effects which are almost universally encountered. Emerging knowledge focusing on mechanisms of toxicity due to chemotherapy has led to characterization of novel methods, including the exploitation of natural compounds, in combination therapies. Flavonoids are natural polyphenolic compounds that play protective roles against tumor cell development. The focus of this study was apoptotic effects of two flavonoids, eupatorin and salvigenin, in combination with doxorubicin on a cellular model of colon cancer.Method:Upon establishing a non-effective dose of doxorubicin, and effective doses of eupatorin (100µM) and salvigenin (150µM) via MTT, morphological features of apoptosis were distinguished using DAPI staining and cell cycle blockage in the sub-G1 phase. Apoptosis was determined by annexin/PI and western blotting. ROS levels and MMP were measured to show any role of mitochondria in apoptosis.Results:Co-administration of flavonoids with doxorubicin induced apoptosis via the mitochondrial pathway as mitochondrial membrane potential and ROS production were changed. Annexin/PI analysis demonstrated that apoptosis frequency was increased with the combination treatments in colon cancer cells. Finally, the combination of these flavonoids with doxorubicin increased the Bax/Bcl-2 ratio, caspase-3 expression and PARP cleavage.Conclusion:Combination of flavonoids with doxorubicin induces apoptosis and enhances effect on cancer cells which might allow amelioration of side effects by dose lowering.

KDM4B plays an important role in mitochondrial apoptosis by upregulating HAX1 expression in colorectal cancer.

Histone methyltransferases and demethylases regulate transcription by altering the epigenetic marks on histones in tumorigenesis. Members of the histone lysine(K)-specific demethylase 4 (KDM4) family are dysregulated in several types of cancer. Here, we report a novel role for KDM4B in mitochondrial apoptosis. In this study, we demonstrate that KDM4B is overexpressed in colorectal cancer (CRC) tissues. Decreased expression of KDM4B significantly promoted apoptosis of CRC cell lines. Moreover, our data indicate that HAX1 is required for KDM4B-mediated mitochondrial apoptosis. The transcription of HAX1 was directly activated by KDM4B. We also show that HAX1 is overexpressed in CRC tissues and is positively correlated with KMD4B expression. Collectively, we demonstrate that KDM4B may play an important role in mitochondrial apoptosis and represent a potential therapeutic cancer target in CRC.

Elevated caspase 3 activity and cytosolic cytochrome c in NT2 cybrids containing amyotrophic lateral sclerosis subject mtDNA

Apoptosis of motor neurons is an important feature in amyotrophic lateral sclerosis (ALS). A vital role of mitochondria in apoptosis and cell survival is well documented. Eventually mitochondria have shown to be an early target in the pathogenesis of ALS. On account of these facts, we investigated the involvement of mitochondrial-dependent apoptosis in ALS and control (CTR) cybrids, generated fusing human platelets with mitochondrial DNA-depleted NT2-neuroteratocarcinoma cells. After a 6 week selection process during which transferred subject mtDNA repopulated the NT2 cells and restored mitochondrial oxygen consumption, we assessed cell viability and two programmed cell death parameters, caspase 3 activity and cytosolic cytochrome c levels. Compared to the control cybrid lines (n = 5), the ALS cybrid lines (n = 10) showed 45% less XTT reduction and higher caspase 3 activity ( p < 0.05, two-way Student's t test) exhibiting lesser cell viability and execution of apoptosis. Elevated cytosolic cytochrome c levels in ALS cybrid lines (n = 8) than in CTR (n = 4) ( p < 0.05, two-way Student's t-test) indicating its mitochondrial release and initiation of apoptosis. This indicates apoptosis as one of the possible mechanisms of cell death in ALS. Our findings support the view that in ALS, subject's mitochondria are altered in non-degenerating tissues in such a way that intrinsic apoptotic pathway activity is relatively increased.

Mitochondrial-Mediated Apoptosis Pathway in Alveolar Epithelial Cells Exposed to the Metals in Combustion-Generated Particulate Matter

Previously a significant mitochondrial impairment was identified in alveolar epithelial cells exposed to metals adsorbed to combustion-generated particulate matter (PM). Due to the critical role of mitochondria in apoptosis, the aim of this study was to investigate the pro-apoptotic potential of metals present in oil fly ash (OFA). A549 cells were exposed to water-soluble components of an OFA sample, containing vanadium [V(IV)], iron [Fe(III)], and nickel [Ni(II)] (68.8, 110.4, and 18 μM, respectively). Experiments were also performed using individual metal solutions. Apoptosis was detected and the mitochondrial role was assessed by a caspase-9 inhibitor (Z-LEHD-FMK). To determine whether the presence of impaired mitochondria in unexposed daughter cells increased apoptosis, an in vitro model was developed that allowed determination of effects until the third cell generation. To specifically examine the toxicity of vanadium (V), that characterize the airborne pollutant examined in this study, p53involvement and metabolic impairment through changes in HIF-1α and Glut-1 expression were determined. OFA and individual metal solutions produced significant apoptosis in the progeny of exposed cells, triggering the intrinsic apoptosis pathway. In apoptosis induced by poorly genotoxic metal V, p53 did not play a significant role. However, V exposure increased nuclear translocation of HIF-1α and expression of the Glut-1 receptor, indicating metabolic impairment due to metal-induced mitochondrial dysfunction. Overall, these results improve our knowledge of the pathogenic role that airborne metals and in particular V exerted in respiratory epithelium.

Animal Mitochondria: Evolution, Function, and Disease

Mitochondria are sub-cellular organelles responsible for producing the majority of cellular energy through the process of oxidative phosphorylation (OXPHOS), and are found in nearly all eukaryotic cells. Mitochondria have a unique genetic system, mitochondrial DNA (mtDNA), which is a small, self-replicating and diverse genome. In the past 30 years, mtDNA has made significant contribution to molecular ecology and phylogeography. Mitochondria also represent a unique system of mitochondrial-nuclear genomic cooperation. Additionally, mitochondrial dysfunction can be fatal. In this paper, we review several aspects of mitochondria, including evolution and the origin of mitochondria, energy supply and the central role of mitochondria in apoptosis, and mitochondrial dysfunction. It is shown that mitochondria play a critical role in many aspects of life.

Mitochondrial ion channels as oncological targets

Mitochondria, the key bioenergetic intracellular organelles, harbor a number of proteins with proven or hypothetical ion channel functions. Growing evidence points to the important contribution of these channels to the regulation of mitochondrial function, such as ion homeostasis imbalances profoundly affecting energy transducing processes, reactive oxygen species production and mitochondrial integrity. Given the central role of mitochondria in apoptosis, their ion channels with the potential to compromise mitochondrial function have become promising targets for the treatment of malignancies. Importantly, in vivo evidence demonstrates the involvement of the proton-transporting uncoupling protein, a mitochondrial potassium channel, the outer membrane located porin and the permeability transition pore in tumor progression/control. In this review, we focus on mitochondrial channels that have been assigned a definite role in cell death regulation and possess clear oncological relevance. Overall, based on in vivo and in vitro genetic and pharmacological evidence, mitochondrial ion channels are emerging as promising targets for cancer treatment.

The retinoblastoma protein induces apoptosis directly at the mitochondria

The retinoblastoma protein gene RB-1 is mutated in one-third of human tumors. Its protein product, pRB (retinoblastoma protein), functions as a transcriptional coregulator in many fundamental cellular processes. Here, we report a nonnuclear role for pRB in apoptosis induction via pRB's direct participation in mitochondrial apoptosis. We uncovered this activity by finding that pRB potentiated TNFα-induced apoptosis even when translation was blocked. This proapoptotic function was highly BAX-dependent, suggesting a role in mitochondrial apoptosis, and accordingly, a fraction of endogenous pRB constitutively associated with mitochondria. Remarkably, we found that recombinant pRB was sufficient to trigger the BAX-dependent permeabilization of mitochondria or liposomes in vitro. Moreover, pRB interacted with BAX in vivo and could directly bind and conformationally activate BAX in vitro. Finally, by targeting pRB specifically to mitochondria, we generated a mutant that lacked pRB's classic nuclear roles. This mito-tagged pRB retained the ability to promote apoptosis in response to TNFα and also additional apoptotic stimuli. Most importantly, induced expression of mito-tagged pRB in Rb(-/-);p53(-/-) tumors was sufficient to block further tumor development. Together, these data establish a nontranscriptional role for pRB in direct activation of BAX and mitochondrial apoptosis in response to diverse stimuli, which is profoundly tumor-suppressive.

Abstract 1119: The oxidation of proline by proline oxidase provides a regulated source of ROS for mitochondria derived cellular signaling

Abstract A role for reactive oxygen species (ROS) in intracellular signaling has been generally accepted, but the organellar localization, the enzymatic source and the regulation of ROS generation are not well understood. Recent work by others has focused on mitochondria as a source of ROS and specifically on the reduction of oxygen by electrons from complex III to form superoxide. The accessibility of complex III to the mitochondrial intermembrane space and cytosol provides an attractive hypothesis for mitochondrial ROS as regulators of cellular redox signaling. We now report that the regulated metabolism of proline makes it a special substrate for this process. Proline oxidase (POX) is an inner mitochondrial membrane protein that oxidizes proline to generate superoxide as well as ATP. Work in our laboratory has documented that POX activity is highly regulated during genotoxic and metabolic stress. Inducers of POX include p53, and proline-dependent ROS play a role in mitochondrial apoptosis. PPAR-γ and its ligands also induce POX but depending on the metabolic context, POX initiates autophagy as well as apoptosis. Mechanistic models for ROS generation by POX have come from microorganisms. PutA, the prokaryotic homolog of POX can generate ROS by electrons reducing oxygen directly from its catalytic center. On the other hand, in S. cerevisiae, the POX homolog, Put1, has been shown to directly interact with ubiquinone, thereby contributing electrons derived from proline oxidation into the electron transport chain. In this work, we now report that human POX interacts directly with ubiquinone and that POX activity is markedly increased by the presence of ubiquinone. In addition, by using selective inhibitors of electron transport, we demonstrate that electrons derived from proline can be converted to superoxide by complex III of the electron transport chain. These radicals are directed into both the mitochondrial matrix as well as into the intermembrane space and the cytosol. Thus, POX is not only specifically regulated but also specially located and coupled to the mitochondrial electron transport chain to mediate ROS signaling. These POX- mediated mechanisms provide important insights into its novel apoptotic and metabolic tumor suppressive functions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1119. doi:1538-7445.AM2012-1119

Abstract 3278: Mitochondria derived from breast cancer MDA-MB-231 cells confer oncogenic properties in the normal breast epithelial nuclear background

Abstract Previous reports showing somatic mitochondrial DNA alterations, the reduction of respiratory chain function, and the essential roles of mitochondria in apoptosis and autophagy, have implicated that mitochondria are vital in cancer development. To study the contribution of cancer mitochondria to tumor properties, the major effects from the nucleus must be removed. We developed an in vitro transmitochondrial cybrid system, in which, mitochondria derived from normal breast epithelial cells (MCF-10A) or breast cancer cells (MDA-MB-231) were fused with ρ0 nuclear donor cells derived from immortalized normal breast epithelial A1N4 cells. Studies of these cybrids showed reduced respiratory chain activities, ATP synthesis rates, and oxygen consumption, in 231/A1N4 cybrids compared to 10A/A1N4 cybrids. In addition, higher production of ROS and lactate were also noted. These results suggested that cancer mitochondria had reduced oxidative energy production function. With the same normal breast epithelial nuclear background, cybrids containing cancer mitochondria exhibited higher expression of oncogenes and growth factors than cybrids containing mitochondria derived from non-cancer epithelial cells (MCF-10A). The 231/A1N4 cybrid cells lost growth contact inhibition, had higher ability to form colonies, and were more resistant to serum starvation compared to 10A/A1N4 cybrids, supporting the hypothesis that mitochondria derived from cancer cells contribute to tumor properties even in the normal nuclear background. These studies provide useful insights to the cross-talk between the mitochondrial and nuclear genomes of altered mitochondria to normal cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3278.

Essential role of mitochondria in apoptosis of cancer cells induced by the marine alkaloid Lamellarin D

Lamellarin D, a potent cytotoxic marine alkaloid, exerts its antitumor action through two complementary pathways: a nuclear route via topoisomerase I inhibition and a mitochondrial targeting. The present study was designed to investigate the contribution of these two pathways for apoptosis in cancer cells. Lamellarin D promoted nuclear apoptosis in leukemia cells without prominent cell cycle arrest. Signals transmitted by lamellarin D initiated apoptosis via the intrinsic apoptotic pathway. The drug induced conformational activation of Bax and decreased the expression levels of antiapoptotic proteins Bcl-2 and cIAP2 in association with activation of caspase-9 and caspase-3. Upon lamellarin D exposure, Fas and Fas-L expression was not modified in leukemia cells. Moreover, leukemia cells deficient in caspase-8 or Fas-associated protein with death domain underwent apoptosis through the typical mitochondrial apoptotic cascade, indicating that cell death induced by lamellarin D was independent of the extrinsic apoptotic pathway. Lamellarin D also exerted a topoisomerase I-mediated DNA damage response resulting in H2AX phosphorylation, and the upregulation of the DNA repair protein Rad51 and of p53, as well as the phosphorylation of p53 at serine 15. However, lamellarin D killed efficiently mutated p53 or p53 null cancer cells, and sensitivity to lamellarin D was abrogated neither by cycloheximide nor in enucleated cells. Lamellarin D-induced cytochrome c release occurs independently of nuclear factors in a cell-free system. These results suggest that lamellarin D exerts its cytotoxic effects primarily by inducing mitochondrial apoptosis independently of nuclear signaling. Thus, lamellarin D constitutes a new proapoptotic agent that may bypass certain forms of apoptosis resistance that occur in tumor cells.