Biomedical subjects

Philippe Marchetti

Publications and source records attributed to Philippe Marchetti.

At least 19 recordsLinked to original sources

PubMed2007

Annexin V detection of lipopolysaccharide-induced cardiac apoptosis.

Acute inflammatory response to lipopolysaccharide (LPS) exposure is typically associated with cardiac myocyte apoptosis, which is difficult to quantify because of heart tissue specificity. We report here that radioiodinated Annexin V (I-AnxV), a specific ligand of phosphatidylserine exposed by apoptotic cells, allows tissue detection of apoptosis in LPS-treated rat hearts. Heart I-AnxV uptake was significantly increased in all cardiac territories of LPS-treated rats. In contrast, I-human serum albumin myocardial uptake was only slightly increased in LPS-treated rat hearts, suggesting limited changes in vascular protein permeability. Autoradiography of endotoxin-treated rat heart sections with I-AnxV in association with deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling and caspase 3 staining allows identification of double positive cardiac myocytes. Inhibition of apoptosis by caspase inhibitors (i.e., ZVAD.fmk and DEVD.cmk) reduced I-AnxV myocardial uptake in LPS-treated rats. Eventually, endotoxin-treated rats displayed pathological uptake of Tc-annexin in the cardiac mediastinal region whereas zVAD.fmk reduced Tc-annexin mediastinal uptake. Our results show that radioactive I-AnxV signal emerging from LPS-treated rat hearts could be related to the activation of caspase-dependent apoptotic pathway in cardiac myocytes.

Animals↗

PubMed2006

The functionality of mitochondria differentiates human spermatozoa with high and low fertilizing capability.

By using flow-cytometric sorting, we compared some of the major factors related to fertility in sperm subpopulations with high and low mitochondrial membrane potential (DeltaPsi(m)). Results demonstrate that DeltaPsi(m)(high) spermatozoa represents a subpopulation of sperm with high fertility performance because they have normal morphology, high motility values, and calcium ionophore-induced acrosome reaction, suggesting the importance of mitochondrial functionality for fertilizing capacity of human spermatozoa.

Adult↗

PubMed2006

Inhibition of mitochondrial permeability transition prevents sepsis-induced myocardial dysfunction and mortality.

OBJECTIVES: The purpose of this study was to test whether mitochondrial dysfunction is causative of sepsis sequelae, a mouse model of peritonitis sepsis induced by cecal ligation and perforation. Inhibition of mitochondrial permeability transition was achieved by means of pharmacological drugs and overexpression of the antiapoptotic protein B-cell leukemia (Bcl)-2. BACKGROUND: Sepsis is the leading cause of death in critically ill patients and the predominant cause of multiple organ failure. Although precise mechanisms by which sepsis leads to multiple organ dysfunction are unknown, growing evidence suggests that perturbations of key mitochondrial functions, including adenosine triphosphate production, Ca2+ homeostasis, oxygen-derived free radical production, and permeability transition, might be involved in sepsis pathophysiology. METHODS: Heart and lung functions were evaluated respectively by means of isolated heart preparation, bronchoalveolar lavage fluid protein concentration, lung wet/dry weight ratio, lung homogenate myeloperoxidase activity, and histopathologic grading. Respiratory fluxes, calcium uptake, and membrane potential were evaluated in isolated heart mitochondria. RESULTS: Peritonitis sepsis induced multiple organ dysfunction, mitochondrial abnormalities, and increased mortality rate, which were reduced by pharmacological inhibition of mitochondrial transition by cyclosporine derivatives and mitochondrial Bcl-2 overexpression. CONCLUSIONS: Our study provides strong evidence that mitochondrial permeability transition plays a critical role in septic organ dysfunction. These studies demonstrate that mitochondrial dysfunction in sepsis is causative rather than epiphenomenal and relevant in terms of vital organ function and outcome. Regarding the critical role of heart failure in the pathophysiology of septic shock, our study also indicates a potentially new therapeutic approach for treatment of sepsis syndrome.

Animals↗

PubMed2006

Sphingosine impairs mitochondrial function by opening permeability transition pore.

Growing evidence suggest that, in the heart, sphingosine participates to contractile dysfunction by altering calcium transients and mitochondria function. However, mechanisms underlying sphingosine-induced cardiac mitochondria dysfunction are poorly understood. Here, we studied the effects of sphingosine on isolated cardiac mitochondria of either wild-type or Bcl-2 overexpressing transgenic mice. Sphingosine induced reductions in ADP-coupled respiration, membrane potential, mitochondrial cytochrome c content and ATP production, which were partially prevented by cyclosporine A and mitochondrial Bcl-2 overexpression. These data suggest that sphingosine promotes mitochondrial permeability transition pore opening, which may result in uncoupled respiration and participate in cardiac contractile dysfunction.

Animals↗

PubMed2006

Cancer cell mitochondria are direct proapoptotic targets for the marine antitumor drug lamellarin D.

Lamellarin D is a marine alkaloid with a pronounced cytotoxicity against a large panel of cancer cell lines and is a potent inhibitor of topoisomerase I. However, lamellarin D maintains a marked cytotoxicity toward cell lines resistant to the reference topoisomerase I poison camptothecin. We therefore hypothesized that topoisomerase I is not the only cellular target for the drug. Using complementary cell-based assays, we provide evidence that lamellarin D acts on cancer cell mitochondria to induce apoptosis. Lamellarin D, unlike camptothecin, induces early disruption of the inner mitochondrial transmembrane potential (Deltapsi(m)) in the P388 leukemia cell line. The functional alterations are largely prevented by cyclosporin A, an inhibitor of the mitochondrial permeability transition (MPT), but not by the inhibitor of caspases, benzyloxycarbonyl-Val-Ala-Asp(Ome)-fluoromethylketone. Deltapsi(m) disruption is associated with mitochondrial swelling and cytochrome c leakage. Using a reliable real-time flow cytometric monitoring of Deltapsi(m) and swelling of mitochondria isolated from leukemia cells, we show that lamellarin D has a direct MPT-inducing effect. Furthermore, mitochondria are required in a cell-free system to mediate lamellarin D-induced nuclear apoptosis. The direct mitochondrial effect of lamellarin D accounts for the sensitivity of topoisomerase I-mutated P388CPT5 cells resistant to camptothecin. Interestingly, a tumor-active analogue of lamellarin D, designated PM031379, also exerts a direct proapoptotic action on mitochondria, with a more pronounced activity toward mitochondria of tumor cell lines compared with nontumor cell lines. Altogether, this work reinforces the pharmacologic interest of the lamellarins and defines lamellarin D as a lead in the search for treatments against chemoresistant cancer cells.

Adenocarcinoma↗

PubMed2005

Ventricular myocyte caspases are directly responsible for endotoxin-induced cardiac dysfunction.

BACKGROUND: Although most of the deleterious effects of sepsis-induced apoptosis have been attributed to increased lymphocyte cell death, caspase activation may directly alter cell function of different organ systems. We postulated that left ventricular (LV) cardiomyocyte caspase activation is directly involved in sepsis-induced heart contractile dysfunction. METHODS AND RESULTS: LV cardiomyocytes isolated 4 hours after rat treatment with endotoxin injection (10 mg/kg) displayed major reductions in contractile reserve and myofilament response to Ca2+. Concomitantly, endotoxin also induced increases in LV cardiomyocyte caspase-3, -8, and -9-like activities, which were associated with sarcomeric structure destruction and cleavage of components of the cardiac myofilament. Interestingly, zVAD.fmk treatment of septic rat prevented LV cardiomyocyte contractile dysfunction, reductions in myofilament response to calcium, troponin T cleavage, and sarcomere destruction. Serum (10%) of endotoxin-treated rats induced contractile dysfunction, caspase-3-like activity increase, and troponin T cleavage of naive LV cardiomyocytes. The effects of septic serum were prevented in LV cardiomyocytes isolated from zVAD.fmk- or zDEVD.cmk-treated rats or LV cardiomyocytes preincubated with zVAD.fmk or zDEVD.cmk. CONCLUSIONS: The results show an important relationship between endotoxin-induced caspase activation and reduced contractile reserve and sarcomere disarray at the level of single LV cardiomyocytes.

Actin Cytoskeleton↗

PubMed2005

Expression of apoptosis regulatory factors during myocardial dysfunction in endotoxemic rats.

OBJECTIVES: To document the time course of apoptosis pathway activation in sepsis and to determine whether Bcl-2 overexpression would improve endotoxin-induced myocardial dysfunction and mortality rate. DESIGN: Randomized, controlled trial. SETTING: Experimental laboratory. SUBJECTS: Male Sprague Dawley rats, wild-type C57BL/6 female mice, C57BL/6 female mice overexpressing Bcl-2. INTERVENTIONS: Hearts were isolated from rats treated with endotoxin (10 mg/kg, intravenously) to perform heart function, immunohistochemistry (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick 3'-end labeling, caspase 3), RNase protection assay, reverse transcriptase polymerase chain reaction, Western blotting (caspase 3), and radiolabeled annexin V studies. Twenty-four hours before endotoxin challenge (10 mg/kg, intravenously), rats were pretreated with saline or endotoxin (0.5 mg/kg, intraperitoneally), with or without parthenolide (1 mg/kg, intraperitoneally). Isolated hearts were used to test myocardial function. Mortality induced by endotoxin (10 mg/kg, intraperitoneally) was tested on wild-type or mice overexpressing Bcl-2. MEASUREMENTS AND MAIN RESULTS: Endotoxin-induced heart dysfunction was maximal at 4 and 8 hrs postinjection, started to improve, and was fully restored at 24 hrs after endotoxin treatment. Endotoxin also induced phosphatidylserine outer leaflet membrane exposure, caspase 3 activation, nuclear apoptosis, and changes in apoptosis gene expression. Bcl-2 overexpression induced by endotoxin pretreatment prevented endotoxin-induced myocardial dysfunction. Mice overexpressing Bcl-2 had dramatic improvement in survival rate compared with wild-type mice. CONCLUSIONS: These observations suggest that both death receptor and caspase-mediated apoptosis processes are activated in this sepsis model. Bcl-2 overexpression before endotoxin challenge prevents myocardial dysfunction in rats and improves survival rate in mice.

Animals↗

PubMed2005

Induction of apoptosis-like mitochondrial impairment triggers antioxidant and Bcl-2-dependent keratinocyte differentiation.

Terminally differentiated keratinocytes are dead enucleated squams. We showed previously that the mitochondria-dependent cell death pathway might be gradually activated as differentiation progresses. In this study, we demonstrated that protoporphyrin IX, staurosporine, and rotenone induced apoptotic-like changes in the mitochondria, and early differentiation of keratinocytes without inducing apoptosis. Kinetics studies established that differentiation-related changes, including growth arrest, flattened morphology, stratification, and keratin 10 (K10) expression, were downstream of mitochondrial depolarization and proliferation, reactive oxygen species (ROS) production, and release of cytochrome c and apoptosis-inducing factor. When these changes were prevented by overexpressing Bcl-2 or pharmacologically decreasing the ROS level, K10 upregulation was inhibited, implying that the differentiated phenotype and K10 expression require apoptotic mitochondria, ROS being the most likely differentiation-mediating factor. Our data also suggest that the same mitochondria-affecting stimuli can induce either differentiation or apoptosis, depending on the keratinocyte's competency to undergo differentiation, a competency that may be controlled by Bcl-2.

Antioxidants↗

PubMed2004

Mitochondrial proliferation during apoptosis induced by anticancer agents: effects of doxorubicin and mitoxantrone on cancer and cardiac cells.

Doxorubicin is one of the most largely prescribed antitumor drug for the treatment of breast, liver and colon cancers as well as leukemia, but the cardiotoxicity of this anthracycline derivative limits its clinical use. Although doxorubicin is toxic to both cancer and cardiac cells, there are evidences suggesting that the mechanism of cell death is different for the two cell types. To investigate further this issue, we have compared the proapoptotic effects of doxorubicin and the functionally related anthracenedione compound mitoxantrone, which is also used in the clinic for the treatment of cancer. After evaluating the toxicity of the two drugs to mammary adenocarcinoma MTLn3 cells and H9C2 cardiomyocytes, we dissected the drug-induced apoptotic machinery by measuring the effects on the cell cycle progression, DNA condensation and fragmentation, production of endogenous peroxides and caspase activation. Both doxorubicin and mitoxantrone are potent inducers of apoptosis in H9C2 cardiomyocytes and MTLn3 breast cancer cells, but there are significant differences between the two cell types in terms of kinetics and order of the events. In particular, flow cytometry measurements of drug-induced changes in mitochondrial transmembrane potential and mitochondrial mass with different fluorescent probes suggested that the two drugs induced a progressive increase in mitochondrial mass in the cancer cells but not in the cardiac cells. The hypothesis was validated by means of electron microscopy, which revealed a significant increase in the number of mitochondria in drug-treated MTLn3 but not in H9C2 cells. The mitochondrial proliferation precedes the nuclear apoptosis in doxorubicin-treated MTLn3 cells. The changes in the architecture and number of mitochondria are linked to the drug-induced perturbation of the cell cycle progression and apoptosis. The proliferation of mitochondria could explain the higher toxicity of doxorubicin to cancer cells compared to cardiac cells and this suggests novel therapeutic opportunities to better control the cardiotoxicity of anthracyclines.

Adenocarcinoma↗

PubMed2004

Apoptosis-inducing factor determines the chemoresistance of non-small-cell lung carcinomas.

Non-small-cell lung carcinomas (NSCLCs) are resistant to the induction of apoptosis by conventional anticancer treatment. However, NSCLC cell lines are sensitive to the action of the broad protein kinase inhibitor, staurosporine (STS). In the NSCLC cell line U1810, STS induced the mitochondrial release of apoptosis-inducing factor (AIF) and cytochrome c (Cyt c) followed by activation of caspases, nuclear condensation, DNA fragmentation and finally cell death. Although preincubation of U1810 cells with the broad-spectrum caspase inhibitor z-VAD.fmk delayed the occurrence of nuclear apoptosis induced by STS, it did not impede mitochondrial alterations (such as the release of Cyt c and AIF) and cell death to occur. Moreover, the microinjection of neither Cyt c nor recombinant active caspase-3 into the cytoplasm promoted nuclear apoptosis-related changes in U1810 cells. Evaluation of the role of the caspase-independent factor AIF in STS-mediated death revealed that, upon immunodepletion of AIF, cytosols from STS-treated U1810 lost their capacity to induce nuclear condensation when incubated with isolated nuclei. In addition, microinjection of an anti-AIF antibody prevented AIF from translocating to the nuclei of STS-treated U1810 cells and reduced STS-induced cell death. Finally, although the transfection-enforced overexpression of AIF was not sufficient to induce cell death, it did enhance STS-mediated cell killing. Altogether, these results indicate that activation of caspases is not sufficient to kill U1810 cells and rather suggests an important role for the AIF-mediated mitochondrial-mediated death pathway.

Apoptosis Inducing Factor↗

PubMed2004

Comparison of four fluorochromes for the detection of the inner mitochondrial membrane potential in human spermatozoa and their correlation with sperm motility.

BACKGROUND: Sperm motility evaluation is associated with fertility in IVF programmes. The visual estimation of sperm motility is extremely subjective. Hence, alternative methods are required. Among them, determination of mitochondrial membrane potential (Deltapsi(m)) changes of spermatozoa using potentiometric dyes may be a reliable test to determine sperm quality. However, the use of the potentiometric dyes in sperm samples has not been compared. METHODS: We have studied sperm samples from 28 infertile patients enrolled in an IVF programme in flow cytometry after staining of spermatozoa with four commonly used potentiometric dyes. Sperm motility was evaluated visually. RESULTS: As expected, JC-1 seems to detect specifically Deltapsi(m) changes, CMX-Ros, DiOC(6)(3) and TMRE fluorescence is easily analysed and the latter three fluorochromes are particularly suitable for multiparametric staining. Irrespective of the Deltapsi(m)-dependent fluorochromes used to stain spermatozoa, a positive correlation was found between the percentage of Deltapsi(m)(high) cells and forward motility and also with high fertilization rates after IVF. CONCLUSION: The four fluorochromes may be useful for evaluation of sperm samples from infertile patients. The choice of the potentiometric dyes will depend on their fluorescence characteristics in order to use them in combination with other fluorescent markers.

Adult↗

PubMed2004

Staining of human sperm with fluorochrome-labeled inhibitor of caspases to detect activated caspases: correlation with apoptosis and sperm parameters.

BACKGROUND: Detection of apoptosis in sperm samples may help evaluate sperm quality. Recently, it has been suggested that in some ejaculated sperm populations, apoptosis is caspase dependent. The aim of this study was to investigate the presence of activated caspases and examine possible correlations with apoptosis and sperm parameters in semen samples prepared for IVF. METHODS: To detect activated caspases, neat semen from infertile patients and sperm prepared by PureSperm gradient were stained with the fluorescein isothyocyanate-Val-Ala-Asp-fluoromethylketone (FITC-VAD-fmk) and analysed by flow cytometry. Cell death was determined by DNA fragmentation (TUNEL) and mitochondrial membrane potential. Sperm parameters were studied by conventional microscopy. RESULTS: FITC-VAD-fmk stained sperm cells in situ and the subcellular labeling pattern was compatible with the known localization of caspases. A significant correlation was found between the frequency of FITC-VAD-fmk stained cells and cell death markers. In both prepared sperm and neat semen a negative correlation was found between the percentage of FITC-VAD-fmk positive cells and standard parameters (concentration/motility). FITC-VAD-fmk positive cells negatively correlated with high fertilization rates after IVF. CONCLUSIONS: Labelling of sperm cells with the activated caspases-reacting fluorochrome provides a sensitive assay for detection of sperm apoptosis. This cytometric assay can be helpful to test sperm before IVF.

Amino Acid Chloromethyl Ketones↗

PubMed2004

Apoptosis inhibition in P. aeruginosa-induced lung injury influences lung fluid balance.

OBJECTIVE: Pseudomonas aeruginosa-induced lung injury is characterized not only by the alteration in lung fluid movement but also by apoptosis of lung epithelial and endothelial cells. We studied whether inhibition of apoptosis using a broad spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (Z-VAD.fmk), would affect lung fluid balance in rat P. aeruginosa pneumonia. METHODS: Z-VAD.fmk (3 mg/kg) was administered intravenously simultaneously with P. aeruginosa intratracheal instillation (0.5 ml/kg, 2 x 10(9) CFU/ml). Apoptosis was evaluated with the TUNEL technique, cytoplasmic oligonucleosome assay, and caspase 3 activation. To evaluate lung permeability, extravascular plasma equivalent (EPE) and lung wet to dry weight ratio (W/D) were measured 4 h after intratracheal instillation of P. aeruginosa. RESULTS: We found an increase of lung apoptosis 4 h after P. aeruginosa instillation: cytoplasmic oligonucleosome assay increased from 3.17+/-0.78 to 26.82+/-4.67 ODx1000/mg of proteins/ml, Z-VAD.fmk administration decreased this parameter to 10.3+/-2.98 ODx1000/mg of proteins/ml. Caspase 3 levels followed the same pattern. Apoptosis involved both epithelial cells and endothelial cells. Endothelial permeability was increased after Pseudomonas instillation: W/D increased from 3.75+/-0.28 in the Co group to 4.42+/-0.23 in the Pn group; EPE was also higher in the Pn group compared with the Co group (0.125+/-0.04 and 0.002+/-0.01 ml, respectively). Both of these parameters were improved after Z-VAD.fmk administration; W/D decreased to 3.36+/-0.25 and EPE to 0.02+/-0.02 ml. CONCLUSION: Apoptosis occurs in the early phase of P. aeruginosa pneumonia. Administration of Z-VAD.fmk significantly decreases DNA fragmentation and caspase 3 levels. This is associated with an improvement of endothelial permeability and lung fluid balance.

Amino Acid Chloromethyl Ketones↗

PubMed2004

Endotoxin-induced myocardial dysfunction: evidence for a role of sphingosine production.

OBJECTIVE: To determine whether sphingomyelinase pathway activation would participate in myocardial depression induced by endotoxin. DESIGN: Randomized, controlled trial. SETTING: Experimental laboratory. SUBJECTS: Male Sprague-Dawley rats, isolated rat heart, and cardiac myocytes. INTERVENTIONS: Cardiovascular function was evaluated in rats injected with saline, endotoxin (10 mg/kg, intravenously), and N-oleoylethanolamine (NOE; 10 mg/kg, intravenously). In ex vivo experiments, isolated rat hearts were perfused with endotoxin (5 microg/mL). For pharmacologic intervention, NOE (1 micromol/L) was admixed to the perfusate 20 mins before endotoxin. In in vitro experiments, ventricular myocytes were incubated with sphingosine (20 microM). Myocyte cell shortening and calcium transient were measured. Mitochondrial membrane potential was measured using the cationic dye tetramethylrhodamine methylester fluorescence technique. MEASUREMENTS AND MAIN RESULTS: Endotoxin treatment at 4 hrs did not alter mean arterial pressure and abdominal blood flow compared with control rats. Left ventricle developed pressure (LVDP) and its first derivatives (i.e., maximal and minimal change in pressure over time [dP/dtmax and dP/dtmin]) were decreased after 4 hrs in endotoxin-treated rats compared with control rats. NOE (10 mg/kg) treatment largely prevented left ventricular systolic function alterations of endotoxin-treated hearts (n = 6 in each group). In isolated rat heart, endotoxin (5 microg/mL) caused increases in tumor necrosis factor-alpha perfusate concentration and delayed depression of LVDP, dP/dtmax, and dP/dtmin after 60 mins, which was partially abrogated in the presence of the ceramidase inhibitor NOE (1 micromol/L). Sphingosine (20 microM) caused decreases in cell fractional shortening, calcium transient, and mitochondrial membrane potential of cardiac myocytes. CONCLUSION: These observations suggest that the sphingomyelinase pathway participates in endotoxin-induced myocardial depression.

Animals↗

PubMed2003

The novel retinoid AHPN/CD437 induces a rapid but incomplete apoptotic response in human myeloma cells.

The synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN/CD437) appears to possess an apoptotic activity superior to classical retinoids in vitro as in vivo. Numerous studies have shown that CD437-induced apoptosis is independent of its nuclear receptor activity, suggesting that CD437 might have a unique mechanism of action. The purpose of this study was to compare CD437- and all-trans retinoic acid (atRA)-induced cell death. CD437 provoked a rapid apoptotic phenotype immediately followed by secondary necrosis in RPMI 8226, U266 and L363 human myeloma cell lines. Nuclear apoptotic features were observed upon both CD437 and atRA treatments. In contrast, membrane blebbing and the subsequent formation of apoptotic bodies, a classical apoptotic event, was only observed upon atRA treatment. In addition, CD437, contrary to atRA, was unable to induce tissue transglutaminase (tTG), an intracellular enzyme involved in the formation of cross-linked protein polymers contributing to apoptotic morphological changes. Taken together, these data suggest that CD437 induces rapid but incomplete apoptotic phenotype in human myeloma cells.

Antineoplastic Agents↗

PubMed2002

Protective effects of cyclosporin A from endotoxin-induced myocardial dysfunction and apoptosis in rats.

Myocardial depression can be demonstrated following administration of endotoxin. Proposed mechanisms of endotoxin-induced myocardial dysfunction include the release of proinflammatory mediators, focal myocardial ischemia, and the presence of activated leukocytes within the myocardium. Recently, myocardial caspase activation and mitochondria-related apoptotic events (i.e., release of cytochrome c) were demonstrated in the failing septic heart. Here, we tested the hypothesis that immunosuppressors, cyclosporin A and tacrolimus (FK 506), would improve inflammation, heart nuclear apoptosis, and myocardial dysfunction in endotoxin-treated rats. Myocardial contractility was assessed using an isolated heart preparation. Heart leukocyte infiltration was assessed by measurement of heart myeloperoxidase activity. Leukocyte activation was studied using the intravital microscopy of the mesenteric venule. Apoptosis was detected as myocardial DNA fragmentation, downstream caspase activation, and mitochondrial cytochrome c release. Both cyclosporin A and FK 506 reduced heart leukocyte sequestration and venular adhesion in endotoxin-treated rats. Cyclosporin A, which blocks mitochondrial cytochrome c release, was able to reduce endotoxin-induced myocardial end-stage nuclear apoptosis and heart dysfunction, whereas tacrolimus had no such effects. These effects could be related to the unique properties of cyclosporin A to act on mitochondria.

Analysis of Variance↗

PubMed2002

Mitochondrial dysfunction is an essential step for killing of non-small cell lung carcinomas resistant to conventional treatment.

Apoptosis, a tightly controlled multi-step mechanism of cell death, is important for anti-cancer therapy-based elimination of tumor cells. However, this process is not always efficient. Small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) cells display different susceptibility to undergo apoptosis induced by anticancer treatment. In contrast to SCLC, NSCLC cells are cross-resistant to a broad spectrum of apoptotic stimuli, including receptor stimulation, cytotoxic drugs and gamma-radiation. Since resistance of tumor cells to treatment often accounts for the failure of traditional forms of cancer therapy, in the present study attempts to find a potent broad-range apoptosis inductor, which can kill therapy-resistant NSCLC cells were undertaken and the mechanism of apoptosis induction by this drug was investigated in detail. We found that staurosporine (STS) had cell killing effect on both types of lung carcinomas. Release of cytochrome c, activation of apical and effector caspases followed by cleavage of their nuclear substrates and morphological changes specific for apoptosis were observed in STS-treated cells. In contrast to treatment with radiation or chemotherapy drugs, STS induces mitochondrial dysfunction followed by translocation of AIF into the nuclei. These events preceded the activation of nuclear apoptosis. Thus, in lung carcinomas two cell death pathways, caspase-dependent and caspase-independent, coexist. In NSCLC cells, where the caspase-dependent pathway is less efficient, the triggering of an AIF-mediated caspase-independent mechanism circumvents the resistance of these cells to treatment.

Amino Acid Chloromethyl Ketones↗

PubMed2002

Study of mitochondrial membrane potential, reactive oxygen species, DNA fragmentation and cell viability by flow cytometry in human sperm.

BACKGROUND: Sperm cell death appears to be a cause of male infertility. The objective of this study was to determine the most reliable method for the evaluation of sperm quality in semen samples during sperm preparation for IVF. METHODS: Conventional analysis of semen samples was compared with several cytofluorometric methods detecting death-associated changes. Neat semen from infertile patients and sperm prepared by PureSperm gradient were studied by conventional microscopy and analysed for mitochondrial membrane potential (Delta Psi(m)), generation of reactive oxygen species, DNA fragmentation and cell viability. RESULTS: In neat semen, a positive correlation was found between the percentage of Delta Psi(m)(high) sperm cells and standard semen parameters (concentration/motility). Sperm cells depicting Delta Psi(m)(high) and cells with low DNA fragmentation displayed high fertilization rate after IVF. The only changes that could be detected in prepared sperm were changes in Delta Psi(m), with Delta Psi(m)(high) sperm positively correlated with forward motility and also with high fertilization rates after IVF. CONCLUSION: Analysis of mitochondrial membrane potential is the most sensitive test by which to determine sperm quality. These findings promise development of a test that may help to predict successful IVF.

Adult↗