Intrinsic Mitochondrial Apoptotic Pathway Research Articles

Resistance to butyrate impairs bile acid-induced apoptosis in human colon adenocarcinoma cells via up-regulation of Bcl-2 and inactivation of Bax

A critical risk factor in colorectal carcinogenesis and tumor therapy is the resistance to the apoptotic effects of different compounds from the intestinal lumen, among them butyrate (main regulator of colonic epithelium homeostasis). Insensitivity to butyrate-induced apoptosis yields resistance to other agents, as bile acids or chemotherapy drugs, allowing the selective growth of malignant cell subpopulations. Here we analyze bile acid-induced apoptosis in a butyrate-resistant human colon adenocarcinoma cell line (BCS-TC2.BR2) to determine the mechanisms that underlay the resistance to these agents in comparison with their parental butyrate-sensitive BCS-TC2 cells. This study demonstrates that DCA and CDCA still induce apoptosis in butyrate-resistant cells through increased ROS production by activation of membrane-associated enzymes and subsequent triggering of the intrinsic mitochondrial apoptotic pathway. Although this mechanism is similar to that described in butyrate-sensitive cells, cell viability is significantly higher in resistant cells. Moreover, butyrate-resistant cells show higher Bcl‐2 levels that confer resistance to bile acid-induced apoptosis sequestering Bax and avoiding Bax-dependent pore formation in the mitochondria. We have confirmed that this resistance is reverted using the Bcl-2 inhibitor ABT-263, thus demonstrating that the lower sensitivity of butyrate-resistant cells to the apoptotic effects of bile acids is mainly due to increased Bcl-2 levels.

Effects of 2-Chlorodeoxyadenosine (Cladribine) on Primary Rat Microglia

2-chlorodeoxyadenosine (CdA, Cladribine) is an immunosuppressant that has recently been shown to be effective in the treatment of multiple sclerosis (MS). There is extensive clinical experience with CdA for the treatment of neoplastic diseases, especially hematologic malignancies, due to its apoptotic effects on leukemic and several other neoplastic cells. Furthermore, CdA crosses the blood-brain-barrier and thus may also exert its effects directly on cells of the central nervous system (CNS). Therefore, we have studied the effects of CdA on cultured primary rat microglia, the resident macrophage in the CNS, which is also thought to be involved in the pathogenesis of MS. Treatment of microglia with CdA inhibited their proliferation and induced apoptosis. Phosphorylation of CdA to CdATP was required for both effects and was inhibited by deoxycytidine. Furthermore, activation of caspase-3 and -9 revealed the involvement of the intrinsic mitochondrial mediated apoptotic pathway. However, the absence of caspase-8 activation specified independency from the extrinsic death receptor mediated apoptosis. The mitochondrial membrane potential was significantly reduced after CdA exposure and was not conserved with Bax or caspase-3 inhibition. Assessment of DNA fragmentation by TUNEL and DNA-release-assay showed microglia with fragmented nuclei. Other functions of microglia like phagocytosis and LPS-induced NO and TNF-α release were not affected by CdA. These data suggest a potential of CdA treatment to induce not only leukopenia but also apoptosis in microglia in the CNS. These results help to understand the mechanism of action of CdA in CNS diseases and may open the possibility to target microglia.

Artepillin C (3,5-diprenyl-4-hydroxycinnamic acid) sensitizes LNCaP prostate cancer cells to TRAIL-induced apoptosis

Naturally occurring phenolic compounds have been shown to sensitize prostate cancer cells to tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. TRAIL is a potent stimulator of apoptosis in cancer cells and an important immune effector molecule in the surveillance and elimination of developing tumours. However, many cancer cells are resistant to TRAIL-mediated death. In this study, we aimed to determine the mechanisms by which TRAIL resistance can be overcome in prostate cancer cells by 3,5-diprenyl-4-hydroxycinnamic acid (artepillin C). Artepillin C is a bioactive component of Brazilian green propolis that possesses antitumour and chemopreventive activities. TRAIL-resistant LNCaP prostate cancer cells were treated with TRAIL and artepillin C. Cytotoxicity was measured by MTT and lactate dehydrogenase (LDH) assays. Apoptosis was detected using Annexin V-FITC staining by flow cytometry and fluorescence microscopy. Death receptor (DR) (TRAIL-R1/DR4 and TRAIL-R2/DR5) expression was analyzed using flow cytometry. Mitochondrial membrane potential (ΔΨm) was evaluated using DePsipher staining by fluorescence micro scopy. The inhibition of NF-κB (p65) activation was confirmed with the ELISA-based TransAM NF-κB kit. Caspase-8 and caspase-3 activities were determined by colorimetric protease assays. The results showed that artepillin C sensitized the TRAIL-resistant LNCaP cells by engaging the extrinsic (receptor-mediated) and intrinsic (mitochondrial) apoptotic pathways. Artepillin C increased the expression of TRAIL-R2 and decreased the activity of NF-κB. Co-treatment with TRAIL and artepillin C induced the significant activation of caspase-8 and caspase-3, as well as the disruption of ΔΨm. These findings show that prostate cancer cells can be sensitized to TRAIL-mediated immunoprevention by artepillin C and confirm the role of phenolic compounds in prostate cancer immunochemoprevention.

Activation of Wnt/β-Catenin Protein Signaling Induces Mitochondria-mediated Apoptosis in Hematopoietic Progenitor Cells

The canonical Wnt/β-catenin signaling is activated during development, tumorigenesis, and in adult homeostasis, yet its role in maintenance of hematopoietic stem/progenitor cells is not firmly established. Here, we demonstrate that conditional expression of an active form of β-catenin in vivo induces a marked increase in the frequency of apoptosis in hematopoietic stem/progenitor cells (HSCs/HPCs). Activation of Wnt/β-catenin signaling in HPCs in vitro elevates the activity of caspases 3 and 9 and leads to a loss of mitochondrial membrane potential (ΔΨ(m)), indicating that it induces the intrinsic mitochondrial apoptotic pathway. In vivo, expression of activated β-catenin in HPCs is associated with down-regulation of Bcl2 and expression of Casp3. Bone marrow transplantation assays reveal that enhanced cell survival by a Bcl2 transgene re-establishes the reconstitution capacity of HSCs/HPCs that express activated β-catenin. In addition, a Bcl2 transgene prevents exhaustion of these HSCs/HPCs in vivo. Our data suggest that activation of the Wnt/β-catenin pathway contributes to the defective function of HPCs in part by deregulating their survival.

PGE2 promotes apoptosis induced by cytokine deprivation through EP3 receptor on mast cells (177.1)

Abstract Increased mast cells numbers are observed at sites of allergic inflammation. Although a critical factor in the resolution of these inflammatory responses is the restoration of normal mast cell numbers. Early studies showed that cytokines protect mast cells from programmed cells death, suggesting a simple model in which diminished cytokines levels during resolution leads to cell death. The report that prostaglandins (PGs) can contribute to the resolution of inflammation together with the demonstration that mast cells express all four PGE2 receptors raises the possibility that PGE2 act in part by augmenting mast cell apoptosis. We report here that, while exposure of mast cells to PGE2 alone does not lead to apoptosis, PGE2 through the EP3 receptor promotes cell death initiated by cytokine withdrawal. Apoptosis is accompanied by higher dissipation of mitochondrial potential (ΔΨm), increased caspase-3 activation, and chromatin condensation with low molecular weight cleavage. PGE2 augmented cell death is dependent on increase in intracellular calcium release, calmodulin dependent kinase II and MAPK activation. Synergy between the EP3 pathway and the intrinsic mitochondrial apoptotic pathway results in increased Bim expression and higher sensitivity of mast cells to cytokine deprivation. This supports a model in which PGE2 contributes to the resolution of inflammatory response in part by augmenting the removal of inflammatory cells particularly mast cells.

A new way of measuring apoptosis by absolute quantitation of inter-nucleosomally fragmented genomic DNA

Several critical events of apoptosis occur in the cell nucleus, including inter-nucleosomal DNA fragmentation (apoptotic DNA) and eventual chromatin condensation. The generation of apoptotic DNA has become a biochemical hallmark of apoptosis because it is a late ‘point of no return’ step in both the extrinsic (cell-death receptor) and intrinsic (mitochondrial) apoptotic pathways. Despite investigators observing apoptotic DNA and understanding its decisive role as a marker of apoptosis for over 20 years, measuring it has proved elusive. We have integrated ligation-mediated PCR and qPCR to design a new way of measuring apoptosis, termed ApoqPCR, which generates an absolute value for the amount (picogram) of apoptotic DNA per cell population. ApoqPCR’s advances over current methods include a 1000-fold linear dynamic range yet sensitivity to distinguish subtle low-level changes, measurement with a 3- to 4-log improvement in sample economy, and capacity for archival or longitudinal studies combined with high-throughput capability. We demonstrate ApoqPCR’s utility in both in vitro and in vivo contexts. Considering the fundamental role apoptosis has in vertebrate and invertebrate health, growth and disease, the reliable measurement of apoptotic nucleic acid by ApoqPCR will be of value in cell biology studies in basic and applied science.

Is ARE/poly(U)-binding factor 1 (AUF1) a new player in cytokine-mediated beta cell apoptosis?

Type 1 diabetes is a chronic autoimmune disease involving the progressive loss of beta cell mass. Cytokines released by immune cells are early contributors to beta cell apoptosis. Thus, an understanding of the signal transduction mechanisms induced by cytokines in beta cells is necessary for the rational design of novel therapies to prevent or to cure this disease. Cytokine-mediated beta cell apoptosis is a complex phenomenon that includes activation of the transcription factors signal transducer and activator of transcription 1 and nuclear factor κB (NFκB), c-Jun N-terminal kinase, endoplasmic reticulum (ER) stress and the intrinsic mitochondrial apoptotic pathway. NFκB has both a pro-inflammatory and a pro-apoptotic role in beta cells. One of the mechanisms by which NFκB contributes to beta cell apoptosis is via activation of ER stress. The role for ER stress in beta cell apoptosis is not completely clarified but involves production of C/EBP homologous protein and activation of the intrinsic mitochondrial apoptotic pathway. In this issue of Diabetologia, Roggli et al (DOI 10.1007/s00125-011-2399-7) report on a new player in this elaborate response, the RNA-binding protein ARE/poly(U)-binding factor 1. This commentary discusses these findings and their relevance to the field.

Abstract 4671: Assessment of molecular processes relative to DNA damage response using primary human hematopoietic cells exposed to high-dose chemotherapy

Abstract A major adverse side effect in many cancer treatments is irreversible damage to primitive and mature hematopoietic cells. Depending of the level and duration of therapy, this can subsequently lead to myelosuppression or life-threatening myeloablation. Understanding molecular processes that regulate how hematopoietic cells respond to and repair chemotherapy-mediated DNA damage could provide key information towards development of cancer treatments with acceptable hematotoxicity. However, model systems designed to study the response of human hematopoietic cells to cytotoxic therapy are lacking. An in vitro culture system that allows for robust expansion of primary human hematopoietic cells was developed. Primary human CD34+ cells were incubated in granulocyte-colony stimulating factor and stem-cell factor for 10 days. This yielded large numbers of human myeloid CD33+ cells so that activation of DNA damage response mechanisms induced by high-dose chemotherapy could be interrogated. A combination therapy of O6-benzylguanine (O6BG) and temozolomide (TMZ)-a regimen previously shown to cause myelosuppression in clinical trials-was used. Exposure of myeloid cells to O6BG/TMZ led to expected decreases in O6-methylguanine DNA methyltransferase and increases in p53, p21, H2AX, and cell cycle arrest. TMZ exposure also led to increased mitochondrial DNA damage which was further pronounced when MGMT activity was inhibited pharmacologically by O6BG, suggesting that MGMT may play a role in repairing mitochondrial DNA damage in human myeloid cells. The extrinsic versus intrinsic apoptotic pathways were also analyzed to determine if a predominant mode of cell death was evident in the myeloid cells following exposure to O6BG/TMZ. Increases in mitochondrial membrane depolarization as well as caspase-9 and caspase-3 activities were observed in cells treated with O6BG/TMZ. Additionally, while ∼50% of the myeloid cells expressed the Fas receptor, expression increased to 80-100% following exposure to 6BG/TMZ. Therefore, to what extent Fas-mediated signaling was operative following 6BG/TMZ exposure was investigated. Experiments using agonistic and neutralizing anti-Fas antibodies indicated that Fas-mediated signaling was not operative in vehicle or myeloid cells exposed to O6BG/TMZ. These data indicate that 6BG/TMZ-mediated apoptosis is initiated predominantly via the intrinsic-mitochondrial apoptotic pathway- and that while Fas is expressed on human myeloid cells, it does not contribute to cell death in myeloid cells. This model system provides a feasible approach to probe further how hematopoietic cells respond to therapy and to search for new strategies that will maintain genome stability in primary hematopoietic cells during anti-cancer therapy. 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 4671. doi:1538-7445.AM2012-4671

Abstract 1827: Developing chemotherapeutics which selectively disable the actin cytoskeleton of tumor cells

Abstract The actin cytoskeleton is an important regulator of a variety of cellular functions making it an ideal chemotherapeutic target. Despite this promise no anti-actin compounds are used in current chemotherapy, primarily due to the inability of existing anti-actin agents to discriminate between the actin cytoskeleton of tumor cells and that of the muscle sarcomere. We have previously shown that tropomyosin, an integral component of the actin cytoskeleton, defines functionally distinct actin filament populations. We have designed a new class of compounds which target a specific cancer associated tropomyosin isoform, Tm5NM1. Anti-tropomyosin (Tm) compounds were selected based on ability to target the actin cytoskeleton and efficacy against a panel of neuroblastoma and melanoma cell lines. The lead compound, TR100, was shown to be effective against a panel of tumor cell lines (average EC50 3µM). Preliminary studies demonstrate that TR100 induces cell death via the intrinsic (mitochondrial) apoptotic pathway and the mechanism appears to be independent of the pro apoptotic factors BAX and BAK. In 3D melanoma spheroid models, which more accurately mimic the tumor microenvironment, TR100 inhibited cell growth and motility. This translated to a reduction in tumor cell growth in neuroblastoma and melanoma xenograft models. Analysis of drug treated animals showed no evidence of cardiac damage as measured by blood Troponin I levels and the intraventricular septum thickness of isolated hearts. These results demonstrate the possibility of targeting distinct actin filament populations based on tropomyosin composition. Next generation anti-Tm compounds with improved efficacy and specificity have now been developed. Preliminary data demonstrate that these compounds exhibit increased selectivity for transformed cells. Taken together, our findings suggest that the anti-Tm compounds show a significant improvement in the therapeutic window compared to existing anti-actin agents. This novel approach and the development of the new class of anti-Tm compounds may be the key for disabling a long sought after target, the actin cytoskeleton, and may lead to a new class of chemotherapeutics active against a broad range of cancer types. 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 1827. doi:1538-7445.AM2012-1827

Development of a small anti‐cancer molecule targeting both the intrinsic and extrinsic pathways of apoptosis

Our chemical genetic screen for small molecules that increase lifespan in yeast cellular models of aging by targeting mitochondria‐controlled apoptosis identified lithocholic acid (LCA) as one of such molecules. Aging is one of the major risk factors of cancer, an age‐related disease whose onset can be delayed and incidence reduced by certain pharmacological antiaging interventions. We therefore sought to examine if LCA exhibits an anti‐tumor effect in cultured human cancer cells by activating certain anti‐cancer processes that may play an essential role in cellular aging. We found that LCA, at concentrations that are not cytotoxic to primary cultures of human neurons, kills the neuroblastoma (NB) cell lines BE(2)‐m17, SK‐n‐SH, SK‐n‐ MCIXC and Lan‐1. In the case of BE(2)‐m17, SK‐n‐SH and SK‐n‐ MCIXC cells, the LCA anti‐tumor effect is due to apoptotic cell death. In contrast, the LCA‐triggered death of Lan‐1 cells is not caused by apoptosis. Our findings imply that LCA kills BE(2)‐m17 and SK‐n‐MCIXC cell lines by triggering not only the intrinsic (mitochondrial) apoptotic cell death pathway driven by mitochondrial outer membrane permeabilization and initiator caspase‐9 activation, but also the extrinsic (death receptor) pathway of apoptosis involving activation of the initiator caspase‐ 8. Our data suggest a molecular mechanism underlying a potent and selective anti‐tumor effect of LCA in cultured human NB cells. Importantly, we found that a similar mechanism underlies a broad anti‐tumor effect of LCA in cultured mammalian cancer cells derived from different tissues and organisms. Supported by NSERC of Canada.

Predominant requirement of Bax for apoptosis in HCT116 cells is determined by Mcl-1's inhibitory effect on Bak

The intrinsic mitochondrial apoptotic pathway acts through two core pro-apoptotic proteins Bax and Bak. While Bax and Bak seem to play redundant roles in apoptosis, accumulating evidence also suggests that they might not be interchangeable under certain conditions, at least in some human cell lines. Here we report the generation of Bak knockout as well as BaxBak double knockout HCT116 human colon carcinoma cells. We show that Bak is dispensable for apoptosis induced by a variety of stimuli including ABT-737 but not for 5-FU-induced apoptosis. In addition, Bax deficiency only provides partial protection against camptothecin and cisplatin-induced apoptosis and no protection against killing by Puma or ABT-737 plus Noxa overexpression. Moreover, Bak is activated normally in response to many chemotherapeutic drugs in the presence of Bax but remains kept in check by Mcl-1 in the absence of Bax. Our data suggest that Bax and Bak are functionally redundant but they are counteracted by distinct anti-apopotic Bcl-2 family proteins in different species.

The cytotoxicity of methacryloxylethyl cetyl ammonium chloride, a cationic antibacterial monomer, is related to oxidative stress and the intrinsic mitochondrial apoptotic pathway

Antibacterial monomers incorporated in dentin bonding systems may have toxic effects on the pulp. Thus, the cytotoxicity of antibacterial monomers and its underlying mechanisms must be elucidated to improve the safety of antibacterial monomer application. The influence of an antibacterial monomer, methacryloxylethyl cetyl ammonium chloride (DMAE-CB), on the vitality of L929 mouse fibroblasts was tested using MTT assay. Cell cycle progression was studied using flow cytometry. Production of intracellular reactive oxygen species (ROS) after DMAE-CB treatment was measured using 2,7-dichlorodihydrofluorescein diacetate staining and flow cytometry analysis. Loss of mitochondrial membrane potential, disturbance of Bcl-2 and Bax expression, as well as release of cytochrome C were also measured using flow cytometry analysis or Western blot to explore the possible involvement of the mitochondrial-related apoptotic pathway. DMAE-CB elicited cell death in a dose-dependent manner and more than 50% of cells were killed after treatment with 30 µM of the monomer. Both necrosis and apoptosis were observed. DMAE-CB also induced G1- and G2-phase arrest. Increased levels of intracellular ROS were observed after 1 h and this overproduction was further enhanced by 6-h treatment with the monomer. DMAE-CB may cause apoptosis by disturbing the expression of Bcl-2 and Bax, reducing the mitochondrial potential and inducing release of cytochrome C. Taken together, these findings suggest that the toxicity of the antibacterial monomer DMAE-CB is associated with ROS production, mitochondrial dysfunction, cell cycle disturbance, and cell apoptosis/necrosis.

Lithocholic bile acid selectively kills neuroblastoma cells, while sparing normal neuronal cells

Aging is one of the major risk factors of cancer. The onset of cancer can be postponed by pharmacological and dietary anti-aging interventions. We recently found in yeast cellular models of aging that lithocholic acid (LCA) extends longevity. Here we show that, at concentrations that are not cytotoxic to primary cultures of human neurons, LCA kills the neuroblastoma (NB) cell lines BE(2)-m17, SK-n-SH, SK-n-MCIXC and Lan-1. In BE(2)-m17, SK-n-SH and SK-n-MCIXC cells, the LCA anti-tumor effect is due to apoptotic cell death. In contrast, the LCA-triggered death of Lan-1 cells is not caused by apoptosis. While low concentrations of LCA sensitize BE(2)-m17 and SK-n-MCIXC cells to hydrogen peroxide-induced apoptotic cell death controlled by mitochondria, these LCA concentrations make primary cultures of human neurons resistant to such a form of cell death. LCA kills BE(2)-m17 and SK-n-MCIXC cell lines by triggering not only the intrinsic (mitochondrial) apoptotic cell death pathway driven by mitochondrial outer membrane permeabilization and initiator caspase-9 activation, but also the extrinsic (death receptor) pathway of apoptosis involving activation of the initiator caspase-8. Based on these data, we propose a mechanism underlying a potent and selective anti-tumor effect of LCA in cultured human NB cells. Moreover, our finding that LCA kills cultured human breast cancer and rat glioma cells implies that it has a broad anti-tumor effect on cancer cells derived from different tissues and organisms.

The dietary flavonol fisetin enhances the apoptosis-inducing potential of TRAIL in prostate cancer cells

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is an endogenous agent that induces apoptosis selectively in cancer cells. Soluble or expressed in immune cells, TRAIL plays an important role in the defense against tumour cells. The resistance of cancer cells to TRAIL immune surveillance is implicated in tumour development. Naturally occurring flavonoids can sensitize TRAIL-resistant cancer cells and augment their apoptotic activity. Fisetin, a dietary flavonol has cancer preventive properties. This study was designed to investigate the effect of fisetin on the TRAIL-induced apoptosis potential in prostate cancer cells. Prostate cancer cell lines represent an ideal model for research in chemoprevention. Cytotoxicity was measured by MTT and LDH assays. Apoptosis was detected using ΑnnexinV-FITC by flow cytometry and fluorescence microscopy. Mito-chondrial membrane potential (ΔΨm) was evaluated using DePsipher staining by fluorescence microscopy. Death receptor (TRAIL-R1 and TRAIL-R2) expression was analysed by flow cytometry. Inhibition of NF-κB (p65) activation was confirmed with an ELISA-based TransAM NF-κB kit. Caspase-8 and caspase-3 activities were determined by colorimetric protease assays. Our study demonstrates that fisetin sensitizes the TRAIL-resistant androgen-dependent LNCaP and the androgen-independent DU145 and PC3 prostate cancer cells to TRAIL-induced death. Fisetin augmented TRAIL-mediated cytotoxicity and apoptosis in prostate cancer LNCaP cells by engaging the extrinsic (receptor-mediated) and intrinsic (mitochondrial) apoptotic pathways. Fisetin increased the expression of TRAIL-R1 and decreased the activity of NF-κB. Co-treatment of cancer cells with TRAIL and fisetin caused significant activation of caspase-8 and caspase-3 and disruption of ΔΨm. Our data indicate the usefulness of fisetin in prostate cancer chemoprevention through enhancement of TRAIL-mediated apoptosis.

The natural triterpene maslinic acid induces apoptosis in HT29 colon cancer cells by a JNK-p53-dependent mechanism

BackgroundMaslinic acid, a pentacyclic triterpene found in the protective wax-like coating of the leaves and fruit of Olea europaea L., is a promising agent for the prevention of colon cancer. We have shown elsewhere that maslinic acid inhibits cell proliferation to a significant extent and activates mitochondrial apoptosis in colon cancer cells. In our latest work we have investigated further this compound's apoptotic molecular mechanism.MethodsWe used HT29 adenocarcinoma cells. Changes genotoxicity were analyzed by single-cell gel electrophoresis (comet assay). The cell cycle was determined by flow cytometry. Finally, changes in protein expression were examined by western blotting. Student's t-test was used for statistical comparison.ResultsHT29 cells treated with maslinic acid showed significant increases in genotoxicity and cell-cycle arrest during the G0/G1 phase after 72 hours' treatment and an apoptotic sub-G0/G1 peak after 96 hours. Nevertheless, the molecular mechanism for this cytotoxic effect of maslinic acid has never been properly explored. We show here that the anti-tumoral activity of maslinic acid might proceed via p53-mediated apoptosis by acting upon the main signaling components that lead to an increase in p53 activity and the induction of the rest of the factors that participate in the apoptotic pathway. We found that in HT29 cells maslinic acid activated the expression of c-Jun NH2-terminal kinase (JNK), thus inducing p53. Treatment of tumor cells with maslinic acid also resulted in an increase in the expression of Bid and Bax, repression of Bcl-2, release of cytochrome-c and an increase in the expression of caspases -9, -3, and -7. Moreover, maslinic acid produced belated caspase-8 activity, thus amplifying the initial mitochondrial apoptotic signaling.ConclusionAll these results suggest that maslinic acid induces apoptosis in human HT29 colon-cancer cells through the JNK-Bid-mediated mitochondrial apoptotic pathway via the activation of p53. Thus we propose a plausible sequential molecular mechanism for the expression of the different proteins responsible for the intrinsic mitochondrial apoptotic pathway. Further studies with other cell lines will be needed to confirm the general nature of these findings.

Oncogenic role of p53 is suppressed by si-RNA bicistronic construct of uPA, uPAR and cathepsin-B in meningiomas both in vitro and in vivo

Meningiomas are the most commonly occurring intracranial tumors and account for approximately 15-20% of central nervous system tumors. Patients whose tumors recur after surgery and radiation therapy have limited therapeutic options. It has also been reported recently that radiation triggers DNA repair, cell survival and cell proliferation, and reduces apoptosis via the induction of cellular protective mechanisms. Earlier studies have reported that proteases such as uPA, uPAR and cathepsin B play important roles in tumor progression. In the present study, we attempted to determine the effectiveness of two bicistronic siRNA constructs pUC (uPAR/cathepsin B) and pU2 (uPA/uPAR) either alone or in combination with radiation, both in in vitro and in vivo models. Transfection of a plasmid vector expressing double-stranded RNA for uPA, uPAR and cathepsin B significantly induced the sub-G0-G1 cell population by the mitochondrial intrinsic apoptotic pathway. Results showed that pUC efficiently enhanced sub-G0-G1 phases compared to pU2 and was more effective. Interestingly, we observed that in IOMM-Lee cell lines, combined treatment of radiation with pUC and pU2 is more effective in comparison to SF-3061 and MN cell lines. We showed that apoptosis caused by these bicistronic constructs involves Bcl-2, Bcl-xL, p53 inactivation, cytochrome c release from mitochondria and caspase-9 activation, followed by the activation of caspase-3. We also determined that apoptosis caused by pUC and pU2 involves a mechanism which includes inactivation of p53 by its translocation from nucleus to cytoplasm as confirmed by immunofluorescence, which shows the oncogenic potential of p53 in meningiomas. However, the simultaneous RNAi-mediated targeting of uPAR and cathepsin B (pUC), in combination with irradiation, has greater potential application for the treatment of human meningioma in comparison to pU2 by decreasing p53 expression both in vitro and in vivo.

Corneal Endothelial Cells Are Protected from Apoptosis by Gene Therapy

Corneal grafting is the most prevalent form of transplantation. Corneal endothelial cells (ECs), which form a monolayer of the cornea with minimal proliferative potential, are pivotal for maintenance of corneal clarity. Loss of EC viability and apoptosis leads to graft failure posttransplantation and reduces the quality of donor corneas in storage, such that up to 30% do not meet selection criteria and must be discarded. The current study investigates antiapoptotic effects of transduced mammalian Bcl-x(L) and baculoviral p35 on human ECs. Multiple apoptotic cell features are observed while inducing apoptosis either via the extrinsic (death receptor) or intrinsic (mitochondrial) apoptotic pathway. Human ECs were studied under three experimental conditions: (1) as an immortalized cell line, (2) as primary cells, and (3) in an intact cornea. Interestingly, in primary EC suspensions, Bcl-x(L) was protective against apoptosis mediated via both pathways. However, p35 was significantly more protective against apoptosis mediated via the intrinsic pathway compared with Bcl-x(L). Our results provide critical insight into the role of apoptotic pathways in the maintenance of EC viability and the efficacy with which these protective proteins exert their effect. These observations could form the basis for future applications of antiapoptotic gene therapy to corneal preservation aiming to reduce both graft failure after transplantation as well as donor corneal damage during storage.

Study on potential antitumor mechanism of a novel Schiff Base copper(II) complex: Synthesis, crystal structure, DNA binding, cytotoxicity and apoptosis induction activity

A new cytotoxic copper(II) complex with Schiff base ligand [CuII(5-Cl-pap)(OAc)(H2O)]·2H2O (1) (5-Cl-pap=N-2-pyridiylmethylidene-2-hydroxy-5-chloro-phenylamine), was synthesized and structurally characterized by X-ray diffraction. Single-crystal analysis revealed that the copper atom shows a 4+1 pyramidal coordination, a water oxygen appears in the apical position, and three of the basal positions are occupied by the NNO tridentate ligand and the fourth by an acetate oxygen. The interaction of Schiff base copper(II) complex 1 with DNA was investigated by UV–visible spectra, fluorescence spectra and agarose gel electrophoresis. The apparent binding constant (Kapp) value of 6.40×105M−1 for 1 with DNA suggests moderate intercalative binding mode. This copper(II) complex displayed efficient oxidative cleavage of supercoiled DNA, which might indicate that the underlying mechanism involve hydroxyl radical, singlet oxygen-like species, and hydrogen peroxide as reactive oxygen species. In addition, our present work showed the antitumor effect of 1 on cell cycle and apoptosis. Flow cytometric analysis revealed that HeLa cells were arrested in the S phase after treatment with 1. Fluorescence microscopic observation indicated that complex 1 can induce apoptosis of HeLa cells, whose process was mediated by intrinsic mitochondrial apoptotic pathway owing to the activation of caspase-9 and caspase-3.

Impact of Polyunsaturated Fatty Acids on Oxidized Low Density Lipoprotein-Induced U937 Cell Apoptosis

Dietary supplements in polyunsaturated fatty acids (PUFA), particularly omega-3, are well known for their beneficial effects in preventing cardiovascular diseases (CVD). The aim of this study was to determine the role of PUFA on the modulation of apoptosis induced by hypochlorous acidoxidized LDL (HOCl-oxLDL) in U937 cells. We tested the effect of monocyte cell line U937 supplementation with eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (ARA) or oleic acid (OA) on the modulation of HOCl-oxLDL-induced apoptosis. First, we showed the incorporation of fatty acids in the cellular membrane in U937 cells. Then, we showed that both EPA and ARA exerted a pro-apoptotic effect through the intrinsic mitochondrial apoptotic pathway including the dissipation of mitochondrial membrane potential followed by cardiolipin depletion, the downstream activation of caspase-3 and the increase in DNA fragmentation. The pro-apoptotic effect of EPA or ARA was completely blocked in U937/Bcl-2 cells. A new mechanism of dietary supplements in PUFA with likely consequences in apoptosis could be suggested through the mitochondrial pathway in monocytes.

Mitochondrial dysfunction and oxidative stress in seizure-induced neuronal cell death.

Epilepsy is considered one of the most common neurological disorders worldwide. The burst firing associated with prolonged epileptic discharges could lead to a large number of changes and cascades of events at the cellular level. From its role as the cellular powerhouse, the mitochondrion is emerging as a key participant in cell death because of its association with an ever-growing list of apoptosis-related proteins. Prolonged seizures may result in the mitochondrial dysfunction and increased production of reactive oxygen species and nitric oxide (NO) precede neuronal cell death and cause subsequent epileptogenesis. Emerging evidences also showed that intrinsic mitochondrial apoptotic pathway may contribute to the neuropathology of human epilepsy, particularly in the hippocampus. Subsequent laboratory studies in the animal model of status epilepticus provide credence to the notion that activation of nuclear factor-κB upregulates NO synthase (NOS) II gene expression with temporal correlation of NOS II derived NO-, superoxide anion- and peroxynitrite-dependent reduction in mitochondrial Complex I activity, leading to apoptotic neuronal cell death in the hippocampus. These results will broaden our understanding on the intimate link between mitochondrial function, oxidative stress and mitochondria-dependent apoptotic signaling triggered by epileptic seizures. It will open a new vista in the development of more effective neuroprotective strategies against seizure-induced brain damage by modification of bioenergetic failure in the mitochondria and in the design of novel treatment perspectives for therapy-resistant forms of epilepsy.