A rapid and strong apoptotic process is triggered by hyperosmotic stress in cultured rat cardiac myocytes

In all cell types, the maintenance of normal cell volume is an essential homeostatic function. Relatively little is known about the induction of apoptosis by hyperosmotic stress and its molecular mechanism in terminally differentiated cardiac myocytes. We compared the apoptotic response of cultured neonatal rat cardiomyoctes to hyperosmotic stress by sorbitol (SOR) with those induced by doxorubicin (Doxo) or angiotensin II (Ang II). We also examined the apoptotic-signaling pathway stimulated by the hyperosmotic stress. Apoptosis was assessed by the observation of: (1) cell viability, (2) DNA fragmentation detected by the TUNEL method and by agarose gel electrophoresis, and (3) poly(ADP-ribose)polymerase (PARP) degradation, and Bcl-XS and Bcl-XL levels by Western blot analysis. Exposure of cardiomyocytes to 0.3 M SOR for 24 h resulted in decreased cell viability and increased generation of oligosomal DNA fragments (2.5-fold of controls). At this time, 83 +/- 5% of SOR-treated myocytes were TUNEL-positive (vs 23.7 +/- 6.8% in controls; P<0.01). PARP levels also decreased by approximately 42% when cardiac myocytes were exposed to SOR. Hyperosmotic stress induced a more rapid and stronger apoptotic response in cardiomyocytes than Doxo or Ang II. In addition, SOR increased 3.2-fold Bcl-XS proapoptotic protein without changes in Bcl-XL antiapoptotic protein levels and in the p53-transactivating activity. Taken together, these results strongly suggest that hyperosmotic stress triggers cardiac myocyte apoptosis in a p53-independent manner, being earlier and stronger than apoptosis induced by Doxo and Ang II.     

Poly(ADP-ribose) polymerase-1-deficient mice are protected from angiotensin II-induced cardiac hypertrophy

Poly(ADP-ribose) polymerase-1 (PARP), a chromatin-bound enzyme, is activated by cell oxidative stress. Because oxidative stress is also considered a main component of angiotensin II-mediated cell signaling, it was postulated that PARP could be a downstream target of angiotensin II-induced signaling leading to cardiac hypertrophy. To determine a role of PARP in angiotensin II-induced hypertrophy, we infused angiotensin II into wild-type (PARP(+/+)) and PARP-deficient mice. Angiotensin II infusion significantly increased heart weight-to-tibia length ratio, myocyte cross-sectional area, and interstitial fibrosis in PARP(+/+) but not in PARP(-/-) mice. To confirm these results, we analyzed the effect of angiotensin II in primary cultures of cardiomyocytes. When compared with PARP(-/-) cardiomyocytes, angiotensin II (1 microM) treatment significantly increased protein synthesis in PARP(+/+) myocytes, as measured by (3)H-leucine incorporation into total cell protein. Angiotensin II-mediated hypertrophy of myocytes was accompanied with increased poly-ADP-ribosylation of nuclear proteins and depletion of cellular NAD content. When cells were treated with cell death-inducing doses of angiotensin II (10-20 microM), robust myocyte cell death was observed in PARP(+/+) but not in PARP(-/-) myocytes. This type of cell death was blocked by repletion of cellular NAD levels as well as by activation of the longevity factor Sir2alpha deacetylase, indicating that PARP induction and subsequent depletion of NAD levels are the sequence of events causing angiotensin II-mediated cardiomyocyte cell death. In conclusion, these results demonstrate that PARP is a nuclear integrator of angiotensin II-mediated cell signaling contributing to cardiac hypertrophy and suggest that this could be a novel therapeutic target for the management of heart failure.     

Aldose reductase induced by hyperosmotic stress mediates cardiomyocyte apoptosis: differential effects of sorbitol and mannitol

Cells adapt to hyperosmotic conditions by several mechanisms, including accumulation of sorbitol via induction of the polyol pathway. Failure to adapt to osmotic stress can result in apoptotic cell death. In the present study, we assessed the role of aldose reductase, the key enzyme of the polyol pathway, in cardiac myocyte apoptosis. Hyperosmotic stress, elicited by exposure of cultured rat cardiac myocytes to the nonpermeant solutes sorbitol and mannitol, caused identical cell shrinkage and adaptive hexose uptake stimulation. In contrast, only sorbitol induced the polyol pathway and triggered stress pathways as well as apoptosis-related signaling events. Sorbitol resulted in activation of the extracellular signal-regulated kinase (ERK), p54 c-Jun N-terminal kinase (JNK), and protein kinase B. Furthermore, sorbitol treatment resulting in induction and activation of aldose reductase, decreased expression of the antiapoptotic protein Bcl-xL, increased DNA fragmentation, and glutathione depletion. Apoptosis was attenuated by aldose reductase inhibition with zopolrestat and also by glutathione replenishment with N-acetylcysteine. In conclusion, our data show that hypertonic shrinkage of cardiac myocytes alone is not sufficient to induce cardiac myocyte apoptosis. Hyperosmolarity-induced cell death is sensitive to the nature of the osmolyte and requires induction of aldose reductase as well as a decrease in intracellular glutathione levels.     

Herpes simplex virus 1 blocks caspase-3-independent and caspase-dependent pathways to cell death

Earlier reports have shown that herpes simplex virus 1 (HSV-1) mutants induce programmed cell death and that wild-type HSV blocks the execution of the cell death program triggered by viral gene products, by the effectors of the immune system such as the Fas and tumor necrosis factor pathways, or by nonspecific stress agents such as either osmotic shock induced by sorbitol or thermal shock. A report from this laboratory showed that caspase inhibitors do not block DNA fragmentation induced by infection with the HSV-1 d120 mutant. To identify the events in programmed cell death induced and blocked by HSV-1, we examined cells infected with wild-type virus or the d120 mutant or cells infected and exposed to sorbitol. We report that: (i) the HSV-1 d120 mutant induced apoptosis by a caspase-3-independent pathway inasmuch as caspase 3 was not activated and DNA fragmentation was not blocked by caspase inhibitors even though the virus caused cytochrome c release and depolarization of the inner mitochondrial membrane. (ii) Cells infected with wild-type HSV-1 exhibited none of the manifestations associated with programmed cell death assayed in these studies. (iii) Uninfected cells exposed to osmotic shock succumbed to caspase-dependent apoptosis inasmuch as cytochrome c was released, the inner mitochondrial potential was lost, caspase-3 was activated, and chromosomal DNA was fragmented. (iv) Although caspase-3 was activated in cells infected with wild-type HSV-1 and exposed to sorbitol, cytochrome c outflow, depolarization of the inner mitochondrial membrane, and DNA fragmentation were blocked. We conclude that although d120 induces apoptosis by a caspase-3-independent pathway, the wild-type virus blocks apoptosis induced by this pathway and also blocks the caspase-dependent pathway induced by osmotic shock. The block in the caspase-dependent pathway may occur downstream of caspase-3 activation.     

L-carnitine prevents doxorubicin-induced apoptosis of cardiac myocytes: role of inhibition of ceramide generation.

Besides the well-documented effect of the chemotherapeutic drug doxorubicin on free radical generation, the exact signaling mechanisms by which it causes cardiac damage remain largely unknown and are of fundamental importance in understanding anthracycline cardiotoxicity. In this study, we describe that a 1 h treatment of isolated adult rat cardiac myocytes with doxorubicin (0.5 microM) induced DNA fragmentation associated with the classical morphological features of apoptosis observed after 7 days of culture. The doxorubicin toxicity was preceded by an increase in intracellular ceramide levels with a concurrent decrease in sphingomyelin. Anthracycline-induced ceramide accumulation resulted from the activation of a sphingomyelinase assayed under acidic conditions, an effect related to an increase in V(max). Pretreatment of cardiac myocytes with L-carnitine (200 microgram/ml), a compound known for its protective effect on cardiac metabolic injuries, was found to dose-dependently inhibit the doxorubicin-induced sphingomyelin hydrolysis and ceramide generation as well as subsequent cell death. However, L-carnitine did not protect cardiac myocytes from apoptosis induced by exogenous cell-permeant ceramide. L-carnitine pretreatment did not affect the sphingomyelinase basal activity but abolished the doxorubicin-induced increase in V(max). Moreover, in vitro studies conducted on cell extracts or with purified acid sphingomyelinase demonstrated that L-carnitine exerted a dose-dependent, sphingomyelinase inhibitory effect (through V(max) reduction). Taken together, these findings show that by inhibiting a (perhaps novel) drug-activated acid sphingomyelinase and ceramide generation, L-carnitine can prevent doxorubicin-induced apoptosis of cardiac myocytes.     

Elevation of IGF-2 receptor and the possible underlying implications in end-stage heart failure patients before and after heart transplantation

Up-regulation of insulin-like growth factor 2 receptor (IGF-2R) involved in angiotensin II-induced cell apoptosis in cardiomyoblasts, and correlated with cardiomyocyte apoptosis in hypertensive rat hearts. Here, we detected IGF-2R levels and explored the possible underlying implications in end-stage heart failure (HF) patients before and after heart transplantation. Western blot and immunohistochemistry were used to measure cardiac IGF-2R levels. ELISA was used to detect serum IGF-2R and CD8 levels. Labelling of DNA strand breaks and dihydroethidium detection were used to determine cellular apoptosis and reactive oxygen species, respectively. Cardiac IGF-2R levels increased in end-stage HF patients (n = 11) compared with non-failing control subjects. Leu27-IGF-2, an IGF-2 analogue to activate specially the IGF-2R, could induce apoptosis and reactive oxygen species production in neonatal rat ventricular myocytes. The serum IGF-2R levels were significantly higher in HF patients than those in non-failing control subjects. An unexpected observation is that the serum IGF-2R levels further increased after heart transplantation, peaked at the first month, and gradually reduced close to the levels before heart transplantation at the 6th months after heart transplantation. Serum CD8, a marker of acute rejection, had no change after heart transplantation, but IGF-2R and Granzyme B, as a ligand for the IGF-2R and a marker for CD8 T lymphocyte activation, coexisted in the transplanted hearts. Our preliminary studies suggest that elevation of IGF-2R may participate in pathological process of end-stage HF and involved in the acute cellular rejection after heart transplantation.     

The essential role of MEKK3 signaling in angiotensin II-induced calcineurin/nuclear factor of activated T-cells activation

Calcineurin is a serine/threonine protein phosphatase that plays a critical role in many physiologic processes, such as T-cell activation, apoptosis, skeletal myocyte differentiation, and cardiac hypertrophy. We determined that active MEKK3 was capable of activating calcineurin/nuclear factor of activated T-cells (NFAT) signaling in cardiac myocytes and reprogramming cardiac gene expression. In contrast, small interference RNA directed against MEKK3 and a dominant negative form of MEKK3 caused the reduction of NFAT activation in response to angiotensin II in cardiac myocytes. Genetic studies showed that MEKK3-deficient mouse embryo fibroblasts failed to activate calcineurin/NFAT in response to angiotensin II, a potent NFAT activator. Conversely, restoring MEKK3 to the MEKK3-deficient cells restored angiotensin II-mediated calcineurin/NFAT activation. We determined that angiotensin II induced MEKK3 phosphorylation. Thus, MEKK3 functions downstream of the AT1 receptor and is essential for calcineurin/NFAT activation. Finally, we determined that MEKK3-mediated activation of calcineurin/NFAT signaling was associated with the phosphorylation of modulatory calcineurin-interacting protein 1 at Ser(108) and Ser(112). Taken together, our studies reveal a previously unrecognized novel essential regulatory role of MEKK3 signaling in calcineurin/NFAT activation.     

Effects of subcultivation and culture medium on differentiation of human fetal cardiac myocytes

Summary Previous work has suggested that subcultivated human fetal heart muscle cell cultures contain immature cardiac muscle cells capable only of limited differentiation after mitogen withdrawal. We studied several human fetal heart cultures (14–15 wk gestation) at several passage levels using immunocytochemistry, autoradiography, and Northern blot analysis. Characteristics in high-mitogen (growth) medium were compared with those after serum withdrawal. Cultured cells from one heart, expanded through 2 passages in growth medium, did not beat; however, 75% of cells did beat after subsequent culture for 24 days in low-serum (differentiation) medium containing insulin. In confluent cultures after 1 passage, there was no detectable difference in the number of cardiac myocytes present in growth medium compared with that 7 days after serum withdrawal. After 4 passages, however, serum withdrawal increased the number of cells expressing immunoreactive sarcomeric myosin heavy chain by 100-fold; expression of immunoreactive sarcomeric actin andα-cardiac actin mRNA also increased in the same cultures. Similar results were obtained in cultures kept in differentiation medium for 20 days before passage and expansion in growth medium. Using isopycinc centrifugation, a high-density cell fraction was isolated which contained no immunostained myocytes in growth medium but numerous myocytes after serum withdrawal. Combined immunocytochemistry/autoradiography showed that myocytes synthesize DNA in growth medium and in serum-free medium containing fibroblast growth factor, but not in serum-free medium alone. The results indicate that a) human fetal cardiac muscle cells proliferate in vitro and can maintain a phenotype characteristic of fetal myocytes after multiple subcultivations followed by serum withdrawal; b) after subcultivation in growth medium, some myocytes modulate their phenotype into one in which detectable levels of cardiac contractile proteins are expressed only after mitogen withdrawal, and c) the phenotype attained after serum withdrawal is in part dependent on passage level. Cultured human fetal myocardial cells my provide a useful experimental system for the study of human cardiac muscle cell biology.     

Angiotensin II does not induce apoptosis but rather prevents apoptosis in cardiomyocytes

The ability of angiotensin II (ang II) to produce apoptosis is controversial. Cardiomyocytes, isolated from 7-day embryonic chick hearts and maintained in culture for 72 h, were treated with ang II. There was no evidence of ang II-induced apoptosis consistently demonstrated by six different techniques: electrophoretic separation of fragmented DNA, staining with TUNEL, enzyme-linked immunosorbent assay specific for fragmented DNA, dual staining of cells with fluorescein diacetate and propidium iodide with analysis by flow cytometry, staining of nuclei with propidium iodide and cell microscopy. In contrast, apoptosis was readily induced by camptothecin or staurosporine or serum deprivation. The absence of ang II-induced cell death was also demonstrated in neonatal mouse cardiomyocytes in culture. We further sought to answer the question whether ang II Type 1 receptor blockade by antagonizing the potential beneficial effects mediated through this receptor and producing more ang II binding to the ang II Type 2 receptors, would lead to cardiac apoptosis. There was no evidence of ang II-induced apoptosis in the presence of the ang II Type 1 receptor antagonist losartan in embryonic chick cardiomyocytes. Rather ang II prevented serum deprivation-induced apoptosis. In summary, in these cardiomyocytes ang II does not induce but rather prevents apoptosis.     

DNA damage is an early event in doxorubicin-induced cardiac myocyte death

Anthracyclines are antitumor agents the main clinical limitation of which is cardiac toxicity. The mechanism of this cardiotoxicity is thought to be related to generation of oxidative stress, causing lethal injury to cardiac myocytes. Although protein and lipid oxidation have been documented in anthracycline-treated cardiac myocytes, DNA damage has not been directly demonstrated. This study was undertaken to determine whether anthracyclines induce cardiac myocyte DNA damage and whether this damage is linked to a signaling pathway culminating in cell death. H9c2 cardiac myocytes were treated with the anthracycline doxorubicin at clinically relevant concentrations, and DNA damage was assessed using the alkaline comet assay. Doxorubicin induced DNA damage, as shown by a significant increase in the mean tail moment above control, an effect ameliorated by inclusion of a free radical scavenger. Repair of DNA damage was incomplete after doxorubicin treatment in contrast to the complete repair observed in H2O2-treated myocytes after removal of the agent. Immunoblot analysis revealed that p53 activation occurred subsequent in time to DNA damage. By a fluorescent assay, doxorubicin induced loss of mitochondrial membrane potential after p53 activation. Chemical inhibition of p53 prevented doxorubicin-induced cell death and loss of mitochondrial membrane potential without preventing DNA damage, indicating that DNA damage was proximal in the events leading from doxorubicin treatment to cardiac myocyte death. Specific doxorubicin-induced DNA lesions included oxidized pyrimidines and 8-hydroxyguanine. DNA damage therefore appears to play an important early role in anthracycline-induced lethal cardiac myocyte injury through a pathway involving p53 and the mitochondria.     

Photosystem I shows a higher tolerance to sorbitol‐induced osmotic stress than photosystem II in the intertidal macro‐algae Ulva prolifera (Chlorophyta)

The photosynthetic performance of the desiccation‐tolerant, intertidal macro‐algae Ulva prolifera was significantly affected by sorbitol‐induced osmotic stress. Our results showed that photosynthetic activity decreased significantly with increases in sorbitol concentration. Although the partial activity of both photosystem I (PS I) and photosystem II (PS II) was able to recover after 30 min of rehydration, the activity of PS II decreased more rapidly than PS I. At 4 M sorbitol concentration, the activity of PS II was almost 0 while that of PS I was still at about one third of normal levels. Following prolonged treatment with 1 and 2 M sorbitol, the activity of PS I and PS II decreased slowly, suggesting that the effects of moderate concentrations of sorbitol on PS I and PS II were gradual. Interestingly, an increase in non‐photochemical quenching occurred under these conditions in response to moderate osmotic stress, whereas it declined significantly under severe osmotic stress. These results suggest that photoprotection in U. prolifera could also be induced by moderate osmotic stress. In addition, the oxidation of PS I was significantly affected by osmotic stress. P700⁺ in the thalli treated with high concentrations of sorbitol could still be reduced, as PS II was inhibited by 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU), but it could not be fully oxidized. This observation may be caused by the higher quantum yield of non‐photochemical energy dissipation in PS I due to acceptor‐side limitation (Y(NA)) during rehydration in seawater containing DCMU.     

The IGF-1-IGF-1 Receptor System Modulates Myocyte Proliferation but Not Myocyte Cellular Hypertrophy in Vitro

In preliminary experiments it was established that the hypertrophic and hyperplastic responses of neonatal cardiac myocytes in culture were associated with enhanced expression of IGF-1 and IGF-1 receptors in these cells. Therefore, to determine the role of IGF-1 receptors on myocyte growth, cells were exposed to antisense oligodeoxynucleotides to IGF-1 receptor mRNA and the effects of this intervention on DNA synthesis, nuclear mitotic division, and changes in the number of myocytes were measured. Moreover, the influence of this procedure on ANF induction and myocyte cell volume was examined. Inhibition of the formation of IGF-1 receptors on myocytes suppressed DNA replication, mitosis, and cell proliferation. In contrast, the antisense treatment did not alter the expression of ANF in myocytes or cellular hypertrophy. Finally, IGF-1 stimulated DNA synthesis in myocytes cultured in serum-free medium, without inducing cellular hypertrophy. In conclusion, ligand activation of IGF-1 receptors on myocytes appears to be coupled with cell proliferation, whereas myocyte cellular hypertrophy seems to be independent from this effector pathway.     

IGF-1 regulates cardiac fibroblast apoptosis induced by osmotic stress

In this study we have determined the ability of IGF-1 to protect cardiac fibroblasts against osmotic-induced apoptosis and investigated the potential mechanism(s) underlying this protection. Treatment with IGF-1 (1-100 ng/ml) promoted a dose dependent increase in cell survival against osmotic cell death. Both Akt and ERK1/2 were rapidly phosphorylated by IGF-1 and blocked by wortmannin and PD98059, inhibitors of their upstream activators respectively. However, IGF-1-induced protection was mediated via a wortmannin-dependent but PD98059-independent pathway as determined by cell survival assay suggesting a role of PI3-K/Akt. Furthermore, IGF-1 appeared to reduce the activation of a number of early components in the apoptotic pathway in a wortmannin dependent manner including the osmotic stress-induced perturbation in mitochondrial membrane potential, cleavage and activation of caspase-3 and DNA fragmentation. Thus, the results suggest that IGF-1 regulates osmotic stress-induced apoptosis via the activation of the PI3-K/Akt pathway at a point upstream of the mitochondria and caspase-3.     

Transforming growth factor beta-1 enhances cytotoxic effect of doxorubicin in human lung adenocarcinoma cells of A549 line

Transforming growth factor beta-1 (TGFbeta-1) is a regulator of cell proliferation, differentiation and apoptosis. Doxorubicin (adriamycin), an anthracycline drug causing double-strand DNA breaks, is widely used in anticancer chemotherapy. Here we demonstrated that TGFbeta-1 enhanced cytotoxic (proapoptotic) action of doxorubicin towards cultured human lung carcinoma A549 cells. Western-blot analysis and immunocytochemistry were used to show that doxorubicin induced PARP degradation in A549 cells, and TGFbeta-1 enhanced that action of the drug. The obtained results suggest a possibility of biomodulating effect of TGFbeta-1 on tumor cell treatment with doxorubicin.     

Double strand DNA breaks in C57B1 and mdx mice cardiomyocytes after dynamical stress

The survival of cardiac myocytes under different physiological and pathological conditions presents pressing problem. mdx mice cardiac myocytes are a promising model of cell survival under condition of oxidative stress. Our early results have shown that some part of mdx mice cardiomyocytes is in early stage of apoptosis (Kazakov, Mikhailov, 2001). But the development of cell death with loss of apoptotical cardiac myocytes occurs only after dynamical stress (bathing during 5 min) (Mikhailov et al., 2001). DNA endonuclease activity in the myocardium and low level of cardiac myocytes death during usual being of mdx mice allowed us to suggest DNA repair to be involved in the survival of mdx mice cardiac myocytes (Mikhailov et al., 2003). To confirm the suggestion we have studied the dynamics of formation and elimination of double strand DNA breaks in mdx myocardium cells after 5 min bathing at 12 degrees C. To visualise double strand DNA breaks formation cell nuclei were stained by monoclonal antibodies to phosphorylated H2Ax histone and to mouse PAP. Double staining with monoclonal anti-H2Ax antibodies and monoclonal anti-a-actin antibodies were used to separate cardiac myocytes from other myocardial cell types. The results showed that during 40 min after stress the deal of H2Ax-positive nuclei in mdx myocardium cells grew up to 41.7 +/- 11.4 % as compared with the initial control level of 6.7 +/- 0.2 %. The number of H2Ax-positive nuclei in these cells decreased after 24 h to 5.7 +/- 0.2 %. The quantity of tagged myocardium cell nuclei in C57B1/6 mice after stress was negligible and did not go beyond 0.01%. Dynamical stress also induced the increase in the rate of 3H-Thymidine incorporation by mdx mice cardiac myocytes from 0.3 +/- 0.3 up to 2.9 +/- 0.5 %. There was not change in the rate of 3H-Thymidine incorporation by cardiac myocytes in C57B1/6 mice. The numbers of labelled nuclei before and after stress were 0.2 and 0.3 %, correspondingly. The number of 3H-Thymidine labelled mdx cardiac myocytes fell down up to 0.4 +/- 0.2 % within 24 h after stress; the level of labelled C57B1/6 cardiac myocytes did not change. We have concluded that 3H-Thymidine incorporation into cardiac myocytes nuclei and staining of these nuclei by monoclonal antiboies phosphorylated H2Ax histone after stress demonstrate rather DNA repair than cardiomyocytes entry into the cell cycle.     

Anticancer drugs induce necrosis of human endothelial cells involving both oncosis and apoptosis

The endothelium is the first physiological barrier between blood and tissues and can be injured by physical or chemical stress, particularly by the drugs used in cancer therapy. We found that four anticancer agents: etoposide, doxorubicin, bleomycin and paclitaxel induced apoptosis in human umbilical vein endothelial cells (HUVECs) (as judged by DNA fragmentation) with a time- and concentration-dependent decrease in bcl-2 protein but without the involvement of p53. As revealed by immunoblotting, bax protein was expressed in HUVECs treated with 1 mg/ml etoposide whereas bcl-2 protein disappeared. Oncosis occurred parallel to apoptosis with the release of lactate dehydrogenase into the supernatant, and, for doxorubicin and etoposide with the inversion of the distribution of angiotensin I-converting enzyme between supernatant and cells. Among the four tested anticancer drugs, only doxorubicin induced an oxidative stress, with significative malondialdehyde production. Thus, human endothelial cells in confluent cultures seem to be in an equilibrium of resistance to apoptosis related to bcl-2 expression; this equilibrium can be disrupted by a chemical stress, such as the antiproliferative drugs known as pro-apoptotic for tumour cells. For doxorubicin and bleomycin, this cellular toxicity can be related to their unwanted effects in human cancer therapy. Low doses of doxorubicin, paclitaxel or etoposide, however, could induce apoptosis of endothelial cells of new vessels surrounding the tumour, thus leading to specific vessel regression with minimal toxic effects for the endothelium of the other vessels. These findings provide evidence of relationships between endothelial toxicity of anticancer drugs and the key role of bcl-2 for resistance of endothelium cells toward apoptosis; moreover lack of p53 and bax in quiescent cells contributes to resistance of endothelial cells to DNA-damaging agents.     

NADPH oxidase is involved in angiotensin II-induced apoptosis in H9C2 cardiac muscle cells: effects of apocynin

Angiotensin II stimulates NADPH oxidase activity in vascular cells. However, it is not fully understood whether angiotensin II, which plays an important role in heart failure, stimulates NADPH oxidase activation and expression in cardiac myocytes. Previous studies have shown that angiotensin II induces myocyte apoptosis, but whether the change is mediated via NADPH oxidase remains to be elucidated. In this study we proposed to determine whether angiotensin II stimulated NADPH oxidase activation and NADPH oxidase subunit p47-phox expression in H9C2 cardiac muscle cells. If so, we would determine whether the NADPH oxidase inhibitor apocynin prevented angiotensin II-induced apoptosis. The results showed that angiotensin II increased NADPH oxidase activity, p47-phox protein and mRNA expression, intracellular reactive oxygen species, and apoptosis in H9C2 cells. Angiotensin II elevated p38 mitogen-activated protein kinase (MAPK) activity, decreased Bcl-2 protein, and increased Bax protein and caspase-3 activity. Apocynin treatment inhibited angiotensin II-induced NADPH oxidase activation and increases in p47-phox expression, intracellular reactive oxygen species, and apoptosis. The effect of apocynin on apoptosis was associated with reduced p38 MAPK activity, increased Bcl-2 protein, and decreased Bax protein and caspase-3 activity. These results suggest that angiotensin II-induced apoptosis is mediated via NADPH oxidase activation probably through p38 MAPK activation, a decrease in Bcl-2 protein, and caspase activation.     

Mechanism of apoptosis induced by doxorubicin through the generation of hydrogen peroxide

The main anticancer action of doxorubicin (DOX) is believed to be due to topoisomerase II inhibition and free radical generation. Our previous study has demonstrated that TAS-103, a topoisomerase inhibitor, induces apoptosis through DNA cleavage and subsequent H(2)O(2) generation mediated by NAD(P)H oxidase activation [H. Mizutani et al. J. Biol. Chem. 277 (2002) 30684-30689]. Therefore, to clarify whether DOX functions as an anticancer drug through the same mechanism or not, we investigated the mechanism of apoptosis induced by DOX in the human leukemia cell line HL-60 and the H(2)O(2)-resistant sub-clone, HP100. DOX-induced DNA ladder formation could be detected in HL-60 cells after a 7 h incubation, whereas it could not be detected under the same condition in HP100 cells, suggesting the involvement of H(2)O(2)-mediated pathways in apoptosis. Flow cytometry revealed that H(2)O(2) formation preceded the increase in Delta Psi m and caspase-3 activation. Poly(ADP-ribose) polymerase (PARP) and NAD(P)H oxidase inhibitors prevented DOX-induced DNA ladder formation in HL-60 cells. Moreover, DOX significantly induced formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine, an indicator of oxidative DNA damage, in HL-60 cells at 1 h, but not in HP100 cells. DOX-induced apoptosis was mainly initiated by oxidative DNA damage in comparison with the ability of other topoisomerase inhibitors (TAS-103, amrubicin and amrubicinol) to cause DNA cleavage and apoptosis. These results suggest that the critical apoptotic trigger of DOX is considered to be oxidative DNA damage by the DOX-induced direct H(2)O(2) generation, although DOX-induced apoptosis may involve topoisomerase II inhibition. This oxidative DNA damage causes indirect H(2)O(2) generation through PARP and NAD(P)H oxidase activation, leading to the Delta Psi m increase and subsequent caspase-3 activation in DOX-induced apoptosis.