Collapse Of Mitochondrial Transmembrane Potential Research Articles

Induction of apoptosis by Polygonatum odoratum lectin and its molecular mechanisms in murine fibrosarcoma L929 cells

Background The Galanthus nivalis agglutinin (GNA)-related lectins have been reported to bear antiproliferative and apoptosis-inducing activities in cancer cells; however, the precise mechanisms by which GNA-related lectins induce cell death are still only rudimentarily understood. Methods In the present study, Polygonatum odoratum lectin (designated POL), a mannose-binding specific GNA-related lectin, possessed a remarkable antiproliferative activity toward murine fibrosarcoma L929 cells. And, this lectin induced L929 cell apoptosis in a caspase-dependent manner. In addition, POL treatment increased the levels of FasL and Fas-Associated protein with Death Domain (FADD) proteins and resulted in caspase-8 activation. Also, POL treatment caused mitochondrial transmembrane potential collapse and cytochrome c release, leading to activations of caspase-9 and caspase-3. Moreover, POL treatment enhanced tumor necrosis factor α (TNFα)-induced L929 cell apoptosis. Results Our data demonstrate for the first time that this lectin induces apoptosis through both death-receptor and mitochondrial pathways, as well as amplifies TNFα-induced L929 cell apoptosis. General significance These inspiring findings would provide new molecular basis for further understanding cell death mechanisms of the Galanthus nivalis agglutinin (GNA)-related lectins in future cancer investigations.

A Novel Combination Therapy With Arsenic Trioxide and Parthenolide Against Pancreatic Cancer Cells

To investigate the anticancer effect and potential mechanism of combination treatment with arsenic trioxide (ATO) and parthenolide (PTL) in human pancreatic cancer cells. The effects of PTL/ATO on 2 pancreatic carcinoma cell lines, PANC-1 and BxPC-3, were determined by trypan blue exclusion, annexin V/propidium iodide, 4',6-diamidino-2-phenylindole HCl, terminal deoxynucleotidyltransferase-mediated nick-end labeling, flow cytometry, Western blot, and clonogenic assay. In vivo study was performed in PANC-1 tumor xenografts in nude mice. The combination of PTL and ATO inhibited the growth of pancreatic tumor cell lines much greater than each agent alone. The PTL/ATO treatment induced apoptosis and reactive oxygen species generation. Both of them were inhibited by L-N-acetylcysteine and diphenylene iodonium chloride. During ATO/PTL-mediated apoptosis, the collapse of mitochondrial transmembrane potential occurred with cytochrome c release, which was reversed by L-N-acetylcysteine. The combination treatment significantly reduced tumor growth rates of PANC-1 xenografts compared with those treated with either PTL or ATO alone. Combination therapy with ATO and PTL has an augmented anticancer effect on pancreatic cancer in vitro and in vivo, which provides a novel promising approach in the treatment of pancreatic cancer. The mechanism of growth-suppressive effect of combination therapy was correlated with its ability to induce reactive oxygen species generation and apoptosis via the mitochondrial pathway.

Aspirin‐triggered lipoxins override the apoptosis‐delaying action of myeloperoxidase in human neutrophils

Myeloperoxidase (MPO), a heme protein abundantly expressed in the azurophilic granules in neutrophils generates cytotoxic oxidants and signals through the adhesion molecule CD11b/CD18 (Mac‐1) to activate and rescue neutrophils from apoptosis. Since aspirin‐triggered 15‐epi‐lipoxin A4 (15‐epi‐LXA4) inhibits Mac‐1‐mediated neutrophil adhesion, we studied its impact on MPO suppression of neutrophil apoptosis. Pretreatment of neutrophils with 15‐epi‐LXA4 or its metabolically stable analog 15‐epi‐16‐p‐fluorophenoxy‐LXA4 through downregulation of surface expression of Mac‐1 and inhibition of MPO release overcame the powerful anti‐apoptosis signal from MPO. 15‐epi‐LXA4 promoted apoptosis by attenuating MPO‐induced concurrent activation of ERK and phosphatidylinositol 3‐kinase/Akt‐mediated phosphorylation of Bad and reducing the expression of the anti‐apoptotic protein Mcl‐1. These led to collapse of mitochondrial transmembrane potential, cytochrome c release, resulting in increased caspase‐3 activity. The pro‐apoptotic action of 15‐epi‐LXA4 was predominant over MPO‐mediated effects even when 15‐epi‐LXA4 was added at 4‐hour post‐MPO. 15‐epi‐LXA4 did not inhibit the catalytic activity of MPO. Our results indicate that through overriding the MPO survival signal, aspirin‐triggered lipoxins may prevent neutrophil‐mediated tissue injury. (Grant support: CIHR MOP‐64283).

Dihydroartemisinin (DHA) induces caspase-3-dependent apoptosis in human lung adenocarcinoma ASTC-a-1 cells

BackgroundDihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, isolated from the traditional Chinese herb Artemisia annua, is recommended as the first-line anti-malarial drug with low toxicity. DHA has been shown to possess promising anticancer activities and induce cancer cell death through apoptotic pathways, although the molecular mechanisms are not well understood.MethodsIn this study, cell counting kit (CCK-8) assay was employed to evaluate the survival of DHA-treated ASTC-a-1 cells. The induction of apoptosis was detected by Hoechst 33258 and PI staining as well as flow cytometry analysis. Collapse of mitochondrial transmembrane potential (ΔΨm) was measured by dynamic detection under a laser scanning confocal microscope and flow cytometry analysis using Rhodamine123. Caspase-3 activities measured with or without Z-VAD-fmk (a broad spectrum caspase inhibitor) pretreatment by FRET techniques, caspase-3 activity measurement, and western blotting analysis.ResultsOur results indicated that DHA induced apoptotic cell death in a dose- and time-dependent manner, which was accompanied by mitochondrial morphology changes, the loss of ΔΨm and the activation of caspase-3.ConclusionThese results show for the first time that DHA can inhibit proliferation and induce apoptosis via caspase-3-dependent mitochondrial death pathway in ASTC-a-1 cells. Our work may provide evidence for further studies of DHA as a possible anticancer drug in the clinical treatment of lung adenocarcinoma.

Inhibition of p66ShcA redox activity in cardiac muscle cells attenuates hyperglycemia-induced oxidative stress and apoptosis

Apoptotic myocyte cell death, diastolic dysfunction, and progressive deterioration in left ventricular pump function characterize the clinical course of diabetic cardiomyopathy. A key question concerns the mechanism(s) by which hyperglycemia (HG) transmits danger signals in cardiac muscle cells. The growth factor adapter protein p66ShcA is a genetic determinant of longevity, which controls mitochondrial metabolism and cellular responses to oxidative stress. Here we demonstrate that interventions which attenuate or prevent HG-induced phosphorylation at critical position 36 Ser residue (phospho-Ser36) inhibit the redox function of p66ShcA and promote the survival phenotype. Adult rat ventricular myocytes obtained by enzymatic dissociation were transduced with mutant-36 p66ShcA (mu-36) dominant-negative expression vector and plated in serum-free media containing 5 or 25 mM glucose. At HG, adult rat ventricular myocytes exhibit a marked increase in reactive oxygen species production, upregulation of phospho-Ser36, collapse of mitochondrial transmembrane potential, and increased formation of p66ShcA/cytochrome-c complexes. These indexes of oxidative stress were accompanied by a 40% increase in apoptosis and the upregulation of cleaved caspase-3 and the apoptosis-related proteins p53 and Bax. To test whether p66ShcA functions as a redox-sensitive molecular switch in vivo, we examined the hearts of male Akita diabetic nonobese (C57BL/6J) mice. Western blot analysis detected the upregulation of phospho-Ser36, the translocation of p66ShcA to mitochondria, and the formation of p66ShcA/cytochrome-c complexes. Conversely, the correction of HG by recombinant adeno-associated viral delivery of leptin reversed these alterations. We conclude that p66ShcA is a molecular switch whose redox function is turned on by phospho-Ser36 and turned off by interventions that prevent this modification.

Angiopoietin-1 inhibits intrinsic apoptotic signaling and vascular hyperpermeability following hemorrhagic shock

Studies from our laboratory demonstrated the involvement of intrinsic apoptotic signaling in hyperpermeability following hemorrhagic shock (HS). Angiopoietin 1 (Ang-1), a potent inhibitor of hyperpermeability, was recently shown to inhibit apoptosis. The purpose of our study was to determine the effectiveness of Ang-1 in attenuating HS-induced hyperpermeability and its relationship to apoptotic signaling. HS was induced in rats by withdrawing blood to reduce the mean arterial pressure to 40 mmHg for 1 h, followed by reperfusion. Mesenteric postcapillary venules were examined for changes in hyperpermeability by intravital microscopy. Mitochondrial release of second mitochondrial derived activator of caspases (smac) and cytochrome c were determined by Western blot and ELISA, respectively. Caspase-3 activity was determined by fluorometric assay. Parallel studies were performed in rat lung microvascular endothelial cell (RLMEC) monolayers, utilizing HS serum and the proapoptotic Bcl-2 homologous antagonist/killer [BAK (BH3)] peptide as inducers of hyperpermeability. In rats, Ang-1 (200 ng/ml) attenuated HS-induced hyperpermeability versus the HS group (P < 0.05). Ang-1 prevented HS-induced collapse of mitochondrial transmembrane potential (DeltaPsi(m)), smac and cytochrome c release, and caspase-3 activity (P < 0.05). In RLMEC monolayers, HS serum and BAK (BH3) peptide both induced hyperpermeability that was inhibited by Ang-1 (P < 0.05). Ang-1 attenuated HS and BAK (BH3) peptide-induced collapse of DeltaPsi(m), smac release, cytochrome c release, activation of caspase-3, and vascular hyperpermeability. In vivo, BAK (BH3) induced vascular hyperpermeability that was attenuated by Ang-1 (P < 0.05). These findings suggest that Ang-1's role in maintaining microvascular endothelial barrier integrity involves the intrinsic apoptotic signaling cascade.

Myeloperoxidase suppresses apoptosis of human neutrophils

Myeloperoxidase (MPO), a heme protein abundantly expressed in the azurophilic granules in neutrophils mediates killing of bacteria and oxidative tissue injury. Since elevated plasma MPO levels and prolonged survival of neutrophils are characteristic features of inflammatory diseases, we studied the impact of MPO on neutrophil apoptosis. Culture of neutrophils with MPO at clinically relevant concentrations (10–160 nM) markedly enhanced neutrophil viability by suppressing apoptosis. These actions were prevented by an anti‐CD11b antibody, but not by 4‐aminobenzoic acid hydrazide, an inhibitor of the enzymatic activity of MPO. MPO evoked concurrent activation of the ERK and phosphatidylinositol 3‐kinase/Akt signaling pathways, leading to phosphorylation of Bad at Ser112 and Ser136, respectively. These led to prevention of collapse of mitochondrial transmembrane potential and cytochrome c release, resulting in decreased caspase‐3 activity. Consistently, pharmacological inhibition of either ERK or phosphatidylinositol 3‐kinase almost completely blocked the responses to MPO. MPO and the pan‐caspase inhibitor z‐VAD‐fmk (20 μM) did not produce additive suppression of neutrophil apoptosis. Our results identify MPO as a survival signal for neutrophils by delaying intrinsic neutrophil apoptosis and thus may contribute to amplification of the inflammatory response. (Supported by CIHR MOP‐64283).

Novel substituted 1,4-anthracenediones with antitumor activity directly induce permeability transition in isolated mitochondria

Synthetic analogs of 1,4-anthraquinone (AQ code number), which block nucleoside transport, decrease DNA, RNA and protein syntheses, trigger cytochrome c release without caspase activation, induce apoptotic DNA fragmentation and inhibit the proliferation of wild-type and multidrug resistant tumor cells in the nM range in vitro, rapidly cause the collapse of mitochondrial transmembrane potential in cell and cell-free systems. Because mitochondrial permeability transition (MPT) requires more than depolarization to occur, antitumor AQs were tested for their ability to directly trigger specific markers of MPT in isolated mitochondria. In contrast to a spectrum of conventional anticancer drugs that are inactive, various AQs interact with isolated mitochondria in a concentration- and time-dependent manner to rapidly cause large amplitude swelling and Ca2+ release in relation with their effectiveness against L1210, HL-60 and LL/2 tumor cells in vitro. Indeed, the lead antitumor AQ8, AQ9 and AQ17 are also the most effective inducers of MPT in isolated mitochondria, whereas all AQ derivatives devoid of anti-proliferative activity also fail to trigger mitochondrial swelling and Ca2+ release. Moreover, the ability of 4 microM AQ17 to maximally induce mitochondrial swelling and Ca2+ release within 15 min is similar to that of classic MPT-inducing agents, such as 5 microg/ml alamethicin, 200 microM atractyloside, 5 microM phenylarsine oxide, 100 microM arsenic trioxide and a 100 microM Ca2+ overload. Interestingly, AQ17 requires a priming concentration of 20 microM Ca2+ to trigger mitochondrial swelling and Ca2+ release and these 0.1 microM ruthenium red-sensitive MPT events are abolished by 1 microM cyclosporin A, 2 mM ADP and 20 microM bongkrekic acid, which block components of the permeability transition pore (PTP), and also inhibited by 50-100 microM of various ubiquinones, which interact with the quinone binding site of the PTP and raise the Ca2+ load required for PTP opening. Hence, antitumor AQs that target isolated mitochondria and trigger MPT might directly interact with components of the PTP to induce conformational changes that increase its Ca2+ sensitivity and transition from the closed to the open state.

Aspirin-Triggered Lipoxins Override the Apoptosis-Delaying Action of Serum Amyloid A in Human Neutrophils: A Novel Mechanism for Resolution of Inflammation

Elevated plasma levels of the acute-phase reactant serum amyloid A (SAA) have been used as a marker and predictor of inflammatory diseases. SAA regulates leukocyte activation; however, it is not known whether it also modulates neutrophil apoptosis, which is critical to the optimal expression and resolution of inflammation. Culture of human neutrophils with SAA (0.1-20 microg/ml) markedly prolonged neutrophil longevity by delaying constitutive apoptosis. SAA evoked concurrent activation of the ERK and PI3K/Akt signaling pathways, leading to phosphorylation of BAD at Ser(112) and Ser(136), respectively, and to prevention of collapse of mitochondrial transmembrane potential, cytochrome c release, and caspase-3 activation. These actions were abrogated by pharmacological inhibition of the formyl peptide receptor, ERK or PI3K. Furthermore, aspirin-triggered 15-epi-lipoxin A(4) (15-epi-LXA(4)) and its stable analog 15-epi-16-p-fluorophenoxy-LXA(4), which binds to the same receptor as SAA, effectively overrode the antiapoptosis signal from SAA even when neutrophils were treated with 15-epi-LXA(4) at either 1 or 4 h postculture with SAA. 15-Epi-LXA(4) itself did not affect neutrophil survival and apoptosis. Our results indicate that SAA at clinically relevant concentrations promotes neutrophil survival by suppressing the apoptotic machinery, an effect that can be opposed by 15-epi-LXA(4). The opposing actions of SAA and aspirin-triggered 15-epi-LXA(4) may contribute to the local regulation of exacerbation and resolution of inflammation, respectively.

Protective effect of Phyllanthus virgatus extract on Cd 2+ ‐induced loss of mitochondrial membrane potential

Phyllanthus virgatus, family Euphorbiaceae, is a plant traditionally employed for the treatment of several diseases in Thailand, especially for liver ailments. The mechanisms responsible for the hepatoprotective action of P. virgatus, however, are not well defined. In this study, the protection of P. virgatus extract on mitochondrial dysfunction induced by Cd2+ was examined with isolated rat liver mitochondria. It was found that the extract of P. virgatus at 80 to 240 μg/ml blocked the collapse of mitochondrial transmembrane potential induced by 5 μM CdCl2 in a dose-dependent manner. Similar results were also obtained with Ca2+-induced loss of mitochondrial membrane potential. Increasing the cation concentrations did not diminish this protective effect of the extract. The extract also inhibited Cd2+-induced cytochrome c release from mitochondria. Cd2+ has been suggested to cause mitochondrial damage by opening the permeability transition pore (PTP) after entering the mitochondria via Ca2+ uniporter. We therefore postulated that P. virgatus extract may act by blocking Ca2+ uniporter to impede Cd2+ influx into the mitochondria and/or by inhibiting the Cd2+-induced PTP opening.

Functional characterization and target validation of alternative complex I of Plasmodium falciparum mitochondria.

This study reports on the first characterization of the alternative NADH:dehydrogenase (also known as alternative complex I or type II NADH:dehydrogenase) of the human malaria parasite Plasmodium falciparum, known as PfNDH2. PfNDH2 was shown to actively oxidize NADH in the presence of quinone electron acceptors CoQ(1) and decylubiquinone with an apparent K(m) for NADH of approximately 17 and 5 muM, respectively. The inhibitory profile of PfNDH2 revealed that the enzyme activity was insensitive to rotenone, consistent with recent genomic data indicating the absence of the canonical NADH:dehydrogenase enzyme. PfNDH2 activity was sensitive to diphenylene iodonium chloride and diphenyl iodonium chloride, known inhibitors of alternative NADH:dehydrogenases. Spatiotemporal confocal imaging of parasite mitochondria revealed that loss of PfNDH2 function provoked a collapse of mitochondrial transmembrane potential (Psi(m)), leading to parasite death. As with other alternative NADH:dehydrogenases, PfNDH2 lacks transmembrane domains in its protein structure, and therefore, it is proposed that this enzyme is not directly involved in mitochondrial transmembrane proton pumping. Rather, the enzyme provides reducing equivalents for downstream proton-pumping enzyme complexes. As inhibition of PfNDH2 leads to a depolarization of mitochondrial Psi(m), this enzyme is likely to be a critical component of the electron transport chain (ETC). This notion is further supported by proof-of-concept experiments revealing that targeting the ETC's Q-cycle by inhibition of both PfNDH2 and the bc(1) complex is highly synergistic. The potential of targeting PfNDH2 as a chemotherapeutic strategy for drug development is discussed.

Sequential events of apoptosis involving docetaxel, a microtubule-interfering agent: A cytometric study

BackgroundDespite the great advances in the understanding of programmed cell death, little attention has been paid to the sequence of the events that characterise it. In particular, the course of apoptotic events induced by microtubule-interfering agents such as taxanes is poorly understood. In order to increase such knowledge, we studied a number of independent biochemical and cytological modifications using cytometric methods in a bladder cancer cell line treated with the second generation taxane, docetaxel.ResultsWithin a few hours, drug treatment had induced mitochondrial membrane transition, cell shrinkage and a decrease in granularity. Cell cycle was almost completely blocked in G2/M phase within 24 hours. The hypodiploid peak started to become prominent 48 hours after the treatment. At the same time, the appearance of a DNA ladder demonstrated caspase-dependent chromatin fragmentation. Concurrently, specific cell surface modifications took place, involving at first glycoprotein syalilation and later phospholipid asymmetry. DNA fragmentation was subsequently detected by TUNEL assay. Over time, cell membranes became permeable to propidium iodide. A very similar time-course of apoptotic events was found after treatment of a myelomonocytic cell line with the same drug.ConclusionAfter discussing some characteristics of the methods employed and their limitations, a succession of apoptotic events over time is suggested, in which the collapse of mitochondrial transmembrane potential (Δψm) is the earliest sign of apoptosis.

Antitumor triptycene analogs induce a rapid collapse of mitochondrial transmembrane potential in HL-60 cells and isolated mitochondria

Since synthetic analogs of triptycene (TT code number), such as bisquinones TT2 and TT13, can trigger cytochrome c release without caspase activation and retain their ability to induce apoptosis in multidrug-resistant (MDR) tumor cells, fluorescent probes of transmembrane potential have been used to determine whether these antitumor compounds might directly target mitochondria in cell and cell-free systems to cause the collapse of mitochondrial membrane potential ( downward arrow Deltapsim) that is linked to permeability transition pore (PTP) opening. Using JC-1 dye, the abilities of various TT analogs to induce the downward arrow Deltapsim in wild-type and MDR HL-60 cells are rapid (within 5-20 min), irreversible after drug removal, concentration dependent in the 0.64-25 microM range, and generally related to their antitumor activities in vitro. The downward arrow Deltapsim caused by TT2 and TT13, which are more potent than mitoxantrone, staurosporine and the reference depolarizing agent, carbonyl cyanide m-chlorophenylhydrazone (CCCP), in HL-60 cells, are not prevented by caspase-2 or -8 inhibitors, suggesting that activation of these apical caspases upstream of mitochondria is not involved in this process. Antitumor TT analogs (0.64-25 microM) also mimic the abilities of the known depolarizing agents, CCCP, alamethicin, gramicidin A and 100 microM CaCl(2), to directly induce within 20 min the downward arrow Deltapsim in isolated mitochondria prepared from mouse liver and loaded with rhodamine 123 dye. The fact that 20 microM Ca(2+), which is insufficient to trigger depolarization on its own, is required to reveal the depolarizing effect of TT2 in isolated mitochondria suggests that antitumor TT analogs might interact with the PTP to alter its conformation and increase its Ca(2+) sensitivity. Indeed, such Ca(2+)-dependent downward arrowDeltapsim of isolated mitochondria treated with 25 microM TT2 or 100 microM Ca(2+) are blocked by ruthenium red. Daunorubicin (DAU) is unable to mimic the rapid downward arrowDeltapsim caused by antitumor TT bisquinones within 5-40 min of treatment in HL-60 cells or isolated mitochondria. Moreover, the downward arrowDeltapsim caused by 25 microM TT2 or 100 microM Ca(2+) in isolated mitochondria are similarly blocked by cyclosporin A (CsA), bongkrekic acid and decylubiquinone, which prevent PTP opening, suggesting that, in contrast to DAU, antitumor TT analogs that directly target mitochondria to trigger the Ca(2+)-dependent and CsA-sensitive downward arrowDeltapsim, might induce PTP opening and the mitochondrial pathway of apoptosis even in the absence of nuclear signals.

Anti-CD20 and Anti-CD52 Monoclonal Antibodies Enhance Cytotoxicity of Bortezomib Against Chronic Lymphocytic Leukemia Cells.

Background: Inhibitor of proteasome, bortezomib (BOR), has high in vitro cytotoxic activity in chronic lymphocytic leukemia (CLL). However, first clinical trials showed low efficacy of BOR used alone in CLL patients. One way to increase the efficacy of BOR in vivo may be its combinatory use with other agents active in CLL, what can bring also benefit from lowering their doses. Indeed, there is some evidence, that efficacy of BOR may be increased by its combination with other agents active in CLL.Aim: To assess in vitro cytotoxic effects exerted by BOR in concomitant treatment with monoclonal antibodies anti-CD20 (rituximab, RIT) or anti-CD52 (alemtuzumab, ALT), agents with confirmed activity in CLL. Additionally, we investigated mechanisms responsible for observed interactions.Methods: The study was performed on cells isolated from 61 untreated CLL patients. Cell viability was evaluated by propydium iodide and MTT assays. Proapoptotic effects were measured by detection of active caspase-3, collapse of mitochondrial transmembrane potential (Mitotracker Red dye) and annexin V flow cytometry assays. Additionally, expression of several promoters of apoptosis (Bid, Bax, Bak, Bcl-w) and anti-apoptotic proteins (Bcl-2, Mcl-1, XIAP and FLIP) was measured. BOR concentration varied in different experiments between 1,25nM and 5nM. Based on preliminarily performed experiments RIT and ALT were used at final doses 10 μg/ml and 20 μg/ml, respectively. Cells were cultured for 24 hours in following sets of samples: 1/untreated control, 2/BOR alone, 3/RIT alone, 4/ALT alone, 5/BOR+RIT and 6/BOR+ALT. Moreover, all those sets of cultures were either done in RPMI 1640 medium supplemented with 5% human serum as a source of complement or in the presence of 20 μg/ml anti-human IgG crosslinking antibodies. Finally, in additional series of experiments cells were pretreated for 24 hours with RIT or ALT, and then BOR was added for the next 24 hours of incubation.Results: Combinations of BOR+RIT and BOR+ALT showed additive cytotoxicity, especially when 24-hour incubation with the antibodies crosslinked with anti-IgG preceded BOR administration (combination index 1.07 and 0.83, respectively). In these settings both combinations, BOR+RIT and BOR+ALT, significantly increased caspase-3 activation. Interestingly, addition of complement enhanced significantly only cell death mediated by ALT. Caspase-3 activation correlated with collapse of mitochondrial potential in samples treated with BOR+RIT (R= 0.71; p<0.02). Upregulation of Bax and downregulation of Bcl-2 or Mcl-1 were found after BOR+RIT treatment (p<0.005 and p<0.01, respectively). ALT+BOR triggered significant downregulation of Bcl-2 (p<0.005) and XIAP (p<0.01) proteins in CLL cells. For both combinations, significant increase in Bax/Bcl-2 ratio was shown (p<0.001).Conclusions: We found additive cytotoxic effects of both combinations, BOR+RIT and BOR+ALT. Upregulation of Bax and downregulation of Bcl-2 and Mcl-1 proteins were main mechanisms of additive interaction of BOR+RIT. The BOR+ALT combination significantly increased Bid and p53 expression and downregulated Bcl-2 and XIAP proteins. These data indicate feasibility of treatment combining proteasome inhibition with the use of RIT or CAM monoclonal antibodies in CLL.

Rapid collapse of mitochondrial transmembrane potential in HL-60 cells and isolated mitochondria treated with anti-tumor 1,4-anthracenediones

Since synthetic analogs of 1,4-anthraquinone (AQ code number), such as AQ8, AQ9 and AQ10, can trigger cytochrome c release without caspase activation and retain their ability to induce apoptosis in multidrug-resistant (MDR) tumor cells, fluorescent probes of transmembrane potential have been used to determine whether these anti-tumor compounds might directly target mitochondria in cell and cell-free systems to cause the collapse of mitochondrial membrane potential (/Deltapsim) that is linked to permeability transition pore (PTP) opening. Using JC-1 dye, the abilities of various AQ analogs to induce the /Deltapsim in wild-type and MDR HL-60 cells are rapid (within 2.5-10 min), irreversible after drug removal, concentration dependent in the 0.256-10 micromol/l range and generally related to their anti-tumor activities in vitro. The /Deltapsim caused by AQ9 and AQ10, which are more potent than mitoxantrone, staurosporine and the reference depolarizing agent carbonyl cyanide m-chlorophenylhydrazone (CCCP) in HL-60 cells, are not prevented by caspase-2 or -8 inhibitors, suggesting that activations of these apical caspases upstream of mitochondria are not involved in this process. Antitumor AQ analogs (0.256-10 micromol/l) also mimic the abilities of the known depolarizing agents CCCP, alamethicin, gramicidin A and 100 micromol/l CaCl2 to directly induce within 15 min the /Deltapsim in isolated mitochondria prepared from mouse liver and loaded with rhodamine 123 dye. The fact that 20 micromol/l Ca2+, which is insufficient to trigger depolarization on its own, is required to reveal the depolarizing effect of AQ9 in isolated mitochondria suggests that anti-tumor AQ analogs might interact with the PTP to alter its conformation and increase its Ca2+ sensitivity. Indeed, such Ca2+-dependent /Deltapsim of isolated mitochondria treated with 1.6 micromol/l AQ9 or 100 micromol/l Ca2+ are blocked by ruthenium red. Daunorubicin (DAU) is unable to mimic the rapid /Deltapsim caused by anti-tumor AQ analogs within 2.5-40 min of treatment in HL-60 cells or isolated mitochondria. Moreover, the /Deltapsim caused by 1.6 micromol/l AQ9 or 100 micromol/l Ca2+ in isolated mitochondria are similarly blocked by cyclosporin A (CsA), bongkrekic acid and decylubiquinone, which prevent PTP opening, suggesting that, in contrast to DAU, anti-tumor AQ analogs that directly target mitochondria to trigger the Ca2+-dependent and CsA-sensitive /Deltapsim, might induce PTP opening and the mitochondrial pathway of apoptosis even in the absence of nuclear signals.

A rapid and transient ROS generation by cadmium triggers apoptosis via caspase-dependent pathway in HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulation

Although reactive oxygen species (ROS) have been implicated in cadmium (Cd)-induced hepatotoxicity, the role of ROS in this pathway remains unclear. Therefore, we attempted to determine the molecular mechanisms relevant to Cd-induced cell death in HepG2 cells. Cd was found to induce apoptosis in the HepG2 cells in a time- and dose-dependent fashion, as confirmed by DNA fragmentation analysis and TUNEL staining. In the early stages, both rapid and transient ROS generation triggered apoptosis via Fas activation and subsequent caspase-8-dependent Bid cleavage, as well as by calpain-mediated mitochondrial Bax cleavage. The timing of Bid activation was coincided with the timing at which the mitochondrial transmembrane potential (MMP) collapsed as well as the cytochrome c (Cyt c) released into the cytosol. Furthermore, mitochondrial permeability transition (MPT) pore inhibitors, such as cyclosporin A (CsA) and bongkrekic acid (BA), did not block Cd-induced ROS generation, MMP collapse and Cyt c release. N-acetylcysteine (NAC) pretreatment resulted in the complete inhibition of the Cd-induced apoptosis via catalase upregulation and subsequent Fas downregulation. NAC treatment also completely blocked the Cd-induced intracellular ROS generation, MMP collapse and Cyt c release, indicating that Cd-induced mitochondrial dysfunction may be regulated indirectly by ROS-mediated signaling pathway. Taken together, a rapid and transient ROS generation by Cd triggers apoptosis via caspase-dependent pathway and subsequent mitochondrial pathway. NAC inhibits Cd-induced apoptosis through the blocking of ROS generation as well as the catalase upregulation.

Mitochondria are the primary target in isothiocyanate-induced apoptosis in human bladder cancer cells

Many isothiocysanates (ITC) are promising cancer-preventive agents, and induction of apoptosis is one of their underlying mechanisms of action. We recently found that caspase-9 was preferentially activated over other initiator caspases in human bladder cancer UM-UC-3 cells. We report here that caspase-9 activation is the major step leading to ITC-induced apoptosis in this cell line. More importantly, our results show that caspase-9 activation by the ITCs may result primarily from mitochondrial damage. Four common naturally occurring ITCs were studied, including allyl ITC, benzyl ITC (BITC), phenethyl ITC (PEITC), and sulforaphane. BITC and PEITC showed more potent mitochondria-damaging ability than the other two ITCs, correlating well with their stronger apoptosis-inducing potentials. Furthermore, BITC and PEITC damaged both the outer and inner mitochondrial membranes. Use of isolated mitochondria allowed us to establish that ITCs, and more importantly their major intracellular derivatives (glutathione conjugates) at concentrations that are readily achievable in cells, damage mitochondria, leading to the collapse of mitochondrial trans-membrane potential and release of cytochrome c. The mitochondria-damaging potencies of the ITCs correlate well with their lipophilicities. Bcl-2 family members are known to influence the stability of mitochondrial membrane. Our results show that the ITCs caused phosphorylation of Bcl-2, induced mitochondrial translocation of Bak, and disrupted the association of Bcl-xl with both Bak and Bax in mitochondrial membrane, indicating that ITC-induced mitochondrial damage results at least in part from modulation of select Bcl-2 family members.

Cell apoptosis induced by a synthetic carbazole compound LCY-2-CHO is mediated through activation of caspase and mitochondrial pathways

The mechanisms involved in the apoptotic effect of LCY-2-CHO [9-(2-chlorobenzyl)-9 H-carbazole-3-carbaldehyde], a synthetic carbazole derivative identified as an anti-inflammatory compound, were studied. Cell cycle analysis by propidium iodide staining in human THP-1 monocytic leukemia cells showed the ability of LCY-2-CHO to increase cell population in sub-G1 stage with time- and concentration-dependent manners. LCY-2-CHO-mediated cell death was also demonstrated by DNA laddering and was not related to the release of lactate dehydrogenase. Apoptosis in THP-1 cells induced by LCY-2-CHO was accompanied by the Bid cleavage, collapse of mitochondrial transmembrane potential, the release of cytochrome c and the activation of caspase-3. The apoptotic effect of LCY-2-CHO was diminished by the presence of zVEID-fmk (caspase-6 inhibitor), zIETD-fmk (caspase-8 inhibitor), and zVAD-fmk (non-selective caspase inhibitor), but was not altered by several antioxidants, and cathepsin inhibitor. The Bid cleavage and loss of mitochondrial transmembrane potential, but not the cytochrome c release, were reversed by zIETD-fmk. Comparing the cell selectivity of LCY-2-CHO, we found T-cell acute lymphoblastic CEM leukemia cells were sensitive to 1 μM LCY-2-CHO, acute myeloid leukemia HL-60 cells underwent apoptosis at 10 μM, while adherent cancer cells, such as PC3, HT29 and MCF-7, were resistant to 30 μM LCY-2-CHO within 24-h incubation. Taken together in the present study, we demonstrated LCY-2-CHO might be apoptotic for malignant hematopoietic cells but not anchorage-dependent cells. This action is mediated by an intrinsic caspase-dependent apoptotic event involving mitochondria.

Interactions of fluorochrome-labeled caspase inhibitors with apoptotic cells: a caution in data interpretation.

Fluorochrome-labeled inhibitors of caspases (FLICA, e.g., FAM-VAD-FMK, FITC-VAD-FMK) have been designed as affinity labels of the enzyme active center of caspases Their binding by apoptotic cells was interpreted as reflecting activation of caspases. We have recently observed, however, that their binding is more complex and may involve additional mechanisms. Our goal in this study was to clarify the ongoing utility of these probes. Apoptosis of HL-60, Jurkat, MCF-7 and T-24 cells was induced by the DNA topoisomerase I inhibitor, topotecan, or by oxidative stress (H(2)O(2)). Lymphocytes were induced by their mitogenic activation. Using multiparameter laser scanning and flow cytometry analysis, the correlation between FLICA binding and the number of known apoptotic indicators was examined. These included: collapse of the mitochondrial transmembrane potential; activation of caspase-3 (detected immunocytochemically); binding of annexin V; chromatin condensation; the presence of DNA strand breaks; and loss of plasma membrane capability to exclude propidium iodide (PI). FLICA binding specificity was tested by pretreatment with z-VAD-FMK or z-DEVD-FMK. FLICA binding was subsequent to the collapse of mitochondrial transmembrane potential, nearly concurrent with caspase-3 activation, and preceded annexin V binding, chromatin condensation, DNA fragmentation and loss of plasma membrane integrity. The predominant portion of FAM-VAD-FMK, FITC-VAD-FMK or FAM-DEVD-FMK binding to apoptotic cells could not be inhibited by z-VAD-FMK or z-DEVD-FMK, respectively, when the unlabeled inhibitors were added post-induction of apoptosis. FLICA are specific and convenient to use markers of apoptotic cells and they detect very early events of apoptosis associated with caspases activation. Assays that combine their binding with either the loss of mitochondrial potential or with exclusion of PI as a probe of plasma membrane integrity, distinguish sequential stages of apoptosis and are particularly useful to differentiate between apoptosis and necrosis. Our results conform with the published data that unlabeled caspase inhibitors, when added after induction of apoptosis, cannot prevent activation of caspases detected by binding of biotinylated inhibitors or by cleavage of fluorogenic substrates. While FLICA binding by apoptotic cells most likely is a consequence of caspase activation, these binding events may also involve other or additional mechanisms than simply their specific attachment to the active enzyme centers of caspases.

Arsenic trioxide-induced mitotic arrest and apoptosis in acute promyelocytic leukemia cells.

Arsenic trioxide (As(2)O(3)), an effective drug for the treatment of acute promyelocytic leukemia (APL), can induce apoptosis and partial differentiation in APL cells in vitro and in vivo. However, As(2)O(3) also induces apoptosis in cancer cells other than APL with complex mechanisms, which seem to be cell type dependent. In this study, we report that APL cells (NB4 cell line) are arrested at early mitotic phase before the collapse of mitochondrial transmembrane potential (Deltavarphi(m)) and apoptosis after treatment with pharmacological concentrations (1.0-2.0 micro M) of As(2)O(3). We have also made the following new discoveries: (1) 0.5 micro M As(2)O(3) that fails to induce apoptosis has no effects on cell cycle distribution. (2) With inhibition of As(2)O(3)-induced Deltavarphi(m) collapse and apoptosis, dithiothreitol also effectively inhibits As(2)O(3)-induced mitotic arrest, suggesting that both As(2)O(3)-induced apoptosis and mitotic arrest involve proteins with thiol groups. (3) 1.5 mM caffeine that relieves cells from G(2)/M arrest also inhibits As(2)O(3)-induced Deltavarphi(m) collapse and apoptosis, (4) 1.0 micro M As(2)O(3) increases the expression of both cyclin B(1) and hCDC20 whereas it inhibits Tyr15 phosphorylation of p34(cdc2). In conclusion, our results strongly support that there is a tight link between As(2)O(3)-induced apoptosis and mitotic arrest, the latter being one of common mechanisms for As(2)O(3)-induced apoptosis in cancer cells.