Curcumin induces crosstalk between autophagy and apoptosis mediated by calcium release from the endoplasmic reticulum, lysosomal destabilization and mitochondrial events.

Curcumin, a major active component of turmeric (Curcuma longa, L.), has anticancer effects. In vitro studies suggest that curcumin inhibits cancer cell growth by activating apoptosis, but the mechanism underlying these effects is still unclear. Here, we investigated the mechanisms leading to apoptosis in curcumin-treated cells. Curcumin induced endoplasmic reticulum stress causing calcium release, with a destabilization of the mitochondrial compartment resulting in apoptosis. These events were also associated with lysosomal membrane permeabilization and of caspase-8 activation, mediated by cathepsins and calpains, leading to Bid cleavage. Truncated tBid disrupts mitochondrial homeostasis and enhance apoptosis. We followed the induction of autophagy, marked by the formation of autophagosomes, by staining with acridine orange in cells exposed curcumin. At this concentration, only the early events of apoptosis (initial mitochondrial destabilization with any other manifestations) were detectable. Western blotting demonstrated the conversion of LC3-I to LC3-II (light chain 3), a marker of active autophagosome formation. We also found that the production of reactive oxygen species and formation of autophagosomes following curcumin treatment was almost completely blocked by N-acetylcystein, the mitochondrial specific antioxidants MitoQ10 and SKQ1, the calcium chelators, EGTA-AM or BAPTA-AM, and the mitochondrial calcium uniporter inhibitor, ruthenium red. Curcumin-induced autophagy failed to rescue all cells and most cells underwent type II cell death following the initial autophagic processes. All together, these data imply a fail-secure mechanism regulated by autophagy in the action of curcumin, suggesting a therapeutic potential for curcumin. Offering a novel and effective strategy for the treatment of malignant cells.

Non-Gaussian space-variant resolution modelling for list-mode reconstruction.

Partial volume effect is an important source of bias in PET images that can be lowered by accounting for the point spread function (PSF) of the scanner. We measured such a PSF in various points of a clinical PET scanner and modelled it as a product of matrices acting in image space, taking the asymmetrical, shift-varying and non-Gaussian character of the PSF into account (AMP modelling), and we integrated this accurate image space modelling into a conventional list-mode OSEM algorithm (EM-AMP reconstruction). We showed on the one hand that when a sufficiently high number of iterations are considered, the AMP modelling lead to better recovery coefficients at reduced background noise compared to reconstruction where no or only partial resolution modelling is performed, and on the other hand that for a small number of iterations, a Gaussian modelling gave the best recovery coefficients. Moreover, we have demonstrated that a deconvolution based on the AMP system response model leads to the same recovery coefficients as the corresponding EM-AMP reconstruction, but at the expense of an increased background noise.

Cellular uptake of phosphorothioate oligonucleotide facilitated by cationic porphyrin: a microfluorescence study.

Cationic 5,10,15,20-tetrakis (1-methyl-4-pyridyl) porphyrin was tested as a delivery agent for oligonucleotides. By using fluorescence microimaging, it has been shown that complexation of the porphyrin to the phosphorothioate analog of dT(15) labeled by rhodamine enabled its nonendocytic penetration into the cell and regular distribution in the cytoplasm and preferentially into the nucleus. Time-resolved microfluorescence spectroscopy revealed that the oligonucleotide integrity was kept. A small fraction of the porphyrin molecules seems to undergo change of the binding mode after internalization, probably due to duplex formation between the oligonucleotide and its cellular target sequences, or due to dissociation of the porphyrin from the oligonucleotide and subsequent interactions in the cellular environment.

On the evolutionary conservation of the cell death pathway: mitochondrial release of an apoptosis-inducing factor during Dictyostelium discoideum cell death.

Mitochondria play a pivotal role in apoptosis in multicellular organisms by releasing apoptogenic factors such as cytochrome c that activate the caspases effector pathway, and apoptosis-inducing factor (AIF) that is involved in a caspase-independent cell death pathway. Here we report that cell death in the single-celled organism Dictyostelium discoideum involves early disruption of mitochondrial transmembrane potential (DeltaPsim) that precedes the induction of several apoptosis-like features, including exposure of the phosphatidyl residues at the external surface of the plasma membrane, an intense vacuolization, a fragmentation of DNA into large fragments, an autophagy, and the release of apoptotic corpses that are engulfed by neighboring cells. We have cloned a Dictyostelium homolog of mammalian AIF that is localized into mitochondria and is translocated from the mitochondria to the cytoplasm and the nucleus after the onset of cell death. Cytoplasmic extracts from dying Dictyostelium cells trigger the breakdown of isolated mammalian and Dictyostelium nuclei in a cell-free system, and this process is inhibited by a polyclonal antibody specific for Dictyostelium discoideum apoptosis-inducing factor (DdAIF), suggesting that DdAIF is involved in DNA degradation during Dictyostelium cell death. Our findings indicate that the cell death pathway in Dictyostelium involves mitochondria and an AIF homolog, suggesting the evolutionary conservation of at least part of the cell death pathway in unicellular and multicellular organisms.

Oxidation of pyridine nucleotides during Fas- and ceramide-induced apoptosis in Jurkat cells: correlation with changes in mitochondria, glutathione depletion, intracellular acidification and caspase 3 activation.

Jurkat T cells showed a major, early decrease in blue autofluorescence in response to Fas/Apo-1/CD95 cross-linking or stimulation with cell-permeant ceramide. This indicates the oxidation/depletion of NADH or NADPH before the onset of apoptosis. Kinetic studies, cytofluorimetric multiparameter analyses and cell sorting experiments indicated a close temporal relationship between NAD(P)H oxidation/depletion and the dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)). In contrast, NAD(P)H depletion was detected well before several other changes associated with late apoptosis, including enhanced superoxide generation, phosphatidylserine exposure on the cell surface, loss of cytosolic K(+), decreased cytoplasmic pH, nuclear DNA fragmentation, cell shrinkage, loss of viability and the appearance of the mitochondrial antigen APO2.7. Full activation of caspase 9 and caspase 3 appeared to be correlated with the appearance of superoxide anions in the mitochondria, and followed the drop in NADPH. Overexpression of the apoptosis-inhibitory proto-oncogene Bcl-2, which encodes an inhibitor of the mitochondrial permeability transition (PT) pore, delayed both the DeltaPsi(m) disruption and the depletion of NAD(P)H. Similar effects were observed with the pharmacological PT pore inhibitors, bongkrekic acid and cyclosporin A. Thus there appears to be a close functional relationship between mitochondrial and cellular redox changes during early apoptosis; events that are inhibited by Bcl-2.

The effect of hypericin and hypocrellin-A on lipid membranes and membrane potential of 3T3 fibroblasts.

Hypericin (HY) and Hypocrellin-A (HA) photosensitization induce rapid depolarization of plasma membrane in 3T3 cells as revealed by confocal microspectrofluorimetry using diO-C5(3) fluorescent probe. HY and HA are also able to rigidify the lipid membrane of DMPC liposomes as indicated by the decrease of pyrene excimer fluorescence used as a marker of the lipid membrane fluidity. We have also observed a nonspecific inhibition of Na+,K+-ATPase activity due to the HY and HA photosensitization. The described effects are concentration- and light dose-dependent and generally more pronounced for HA than for HY. All these observations suggest that the lipid membranes can play an important role in the photosensitization process induced by HY and HA at the cellular level. It can be hypothesized that for HA and HY the secondary mechanism following type I or type II photosensitization process can be the peroxidation of membrane lipids as well, and thus intracellular membranes seem to be one of the most important targets of these photosensitizers.

Over-expression of Bcl-2 does not protect cells from hypericin photo-induced mitochondrial membrane depolarization, but delays subsequent events in the apoptotic pathway.

Hypericin (HY) is a powerful photo-inducer of apoptosis in Jurkat cells as measured by caspase-3 activation, cell shrinkage, phosphatidylserine (PS) exposure and the appearance of hypoploid DNA. These processes are preceded by rapid Bcl-2-independent mitochondrial transmembrane depolarization and a drop in cytoplasmic pH. Pre-incubation of cells with inhibitors of the mitochondrial permeability transition pore, such as cyclosporin A or bongkrekic acid, does not protect cells from mitochondrial membrane potential (deltapsim) decrease. However, monitoring of mitochondrial entrapped calcein by confocal fluorescence imaging gives clear evidence of HY photo-induced mitochondrial permeability. This should be considered as the result of a non-specific alteration of mitochondrial membrane integrity brought about by lipid peroxidation. Nevertheless, synthesis of the anti-apoptotic protein Bcl-2 appears to delay the subsequent time course of PS exposure and to reduce caspase-3 activation and the fraction of cells which become hypoploid. We interpret this partially protective effect as the consequence of a direct interaction of Bcl-2 with cytosolic cytochrome c previously released from mitochondria upon deltapsim decrease and/or of Bcl-2 inhibition of the deleterious retro-effect of caspase-3 on the mitochondrial permeability transition pore and/or the mitochondrial membrane components.

Light-induced photoactivation of hypericin affects the energy metabolism of human glioma cells by inhibiting hexokinase bound to mitochondria.

Glucose-dependent energy required for glioma metabolism depends on hexokinase, which is mainly bound to mitochondria. A decrease in intracellular pH leads to a release of hexokinase-binding, which in turn decreases glucose phosphorylation, ATP content, and cell proliferation. Thus, intracellular pH might be a target for therapy of gliomas, and a search for agents able to modulate intracellular pH was initiated. Hypericin, a natural photosensitizer, displays numerous biological activities when exposed to light. Its mechanism and site of action at the cellular level remain unclear, but it probably acts by a type II oxygen-dependent photosensitization mechanism producing singlet oxygen. Hypericin is also able to induce a photogenerated intracellular pH drop, which could constitute an alternative mechanism of hypericin action. In human glioma cells treated for 1 h with 2.5 microg/ml hypericin, light exposure induced a fall in intracellular pH. In these conditions, mitochondria-bound hexokinase was inhibited in a light- and dose-dependent manner, associated with a decreased ATP content, a decrease of mitochondrial transmembrane potential, and a depletion of intracellular glutathione. Hexokinase protein was effectively released from mitochondria, as measured by an ELISA using a specific anti-hexokinase antibody. In addition to decreased glutathione, a response to oxidative stress was confirmed by the concomitant increase in mRNA expression of gamma-glutamyl cysteine synthetase, which catalyzes the rate-limiting step in overall glutathione biosynthesis, and is subject to feedback regulation by glutathione. Hypericin also induced a dose- and light-dependent inhibition of [3H]thymidine uptake and induced apoptosis, as demonstrated by annexin V-FITC binding and cell morphology. This study confirmed the mitochondria as a primary target of photodynamic action. The multifaceted action of hypericin involves the alteration of mitochondria-bound hexokinase, initiating a cascade of events that converge to alter the energy metabolism of glioma cells and their survival. In view of the complex mechanism of action of hypericin, further exploration is warranted in a perspective of its clinical application as a potential phototoxic agent in the treatment of glioma tumors.

Potentiation of lonidamine and diazepam, two agents acting on mitochondria, in human glioblastoma treatment.

BACKGROUND: Cellular metabolism in glioblastoma multiforme, the most common primary brain tumor in humans, is characterized by a high rate of aerobic glycolysis that is dependent on mitochondria-bound hexokinase. Moreover, high levels of glucose utilization and tumor aggressiveness in glioblastoma are associated with a high density of mitochondrial benzodiazepine receptors. We sought to inhibit glioblastoma metabolism by simultaneously inhibiting hexokinase with lonidamine and binding benzodiazepine receptors with diazepam. METHODS: Cellular glioblastoma metabolism in five glioblastoma cell lines was assessed in vitro by measuring cell proliferation (by use of a tetrazolium-based colorimetric assay, measurement of DNA synthesis, and assessment of cell cycle distribution), by measuring membrane fluidity (by fluorescence polarization measurement of cells stained with a fluorescent probe), and by measuring changes in intracellular pH. Immunodeficient nude mice bearing subcutaneous xenografts of human glioblastoma cells were used to assess the antitumor activities of lonidamine and diazepam; the mice were treated twice daily with lonidamine (total daily dose of 160 mg/kg body weight) and/or diazepam (total daily dose of 1 mg/kg body weight) for 10 consecutive days. RESULTS: When used in combination, the two drugs had a stronger effect on glioblastoma cell proliferation and metabolism in vitro than did either agent used alone. In vivo, the combination of lonidamine and diazepam was significantly more effective in reducing glioblastoma tumor growth than either drug alone (two-sided P<.01, Mann-Whitney U test, comparing growth of treated tumors with that of untreated tumors); this tumor growth retardation was maintained as long as treatment was given. CONCLUSION: The combination of lonidamine and diazepam--drugs that target two distinct mitochondrial sites involved in cellular energy metabolism--potentiates the effects of the individual drugs and may prove useful in the treatment of human glioblastomas.

Hypocrellin A photosensitization involves an intracellular pH decrease in 3T3 cells.

The fluorescent pH probe carboxy-seminaphtorhodafluor-1 (C-Snarf-1) has been used for laser microspectro-fluorometric assays of intracellular pH in 3T3 mouse fibroblasts treated with hypocrellin A. These results are compared to those previously obtained with the structurally related hydroxylated polycyclic quinone, hypericin (Sureau et al., J. Am. Chem. Soc. 118, 9484-9487, 1996). A mean local intracellular pH drop of 0.6 units has been observed in the presence of 1 microM hypocrellin A after 90 s of exposure to 0.1 microW of laser irradiation at 514.5 nm. The time evolution of the cytoplasm acidification for hypocrellin A-treated cells is faster than that for cells treated by hypericin. Thus, release of protons from an excited state of hypocrellin A appears to be more efficient than that from hypericin. In addition, the pH dependence of the quenching of C-Snarf-1 fluorescence in 3T3 cells under continuous irradiation has been observed. It is shown here that under continuous illumination, a pH decrease is able to induce a modification of the intracellular binding equilibrium of C-Snarf-1 that results in an increase of C-Snarf-1 fluorescence intensity. This latter observation suggests that the protons generated upon the photoexcitation of hypericin or its analogs may be involved in the production of other photoreactive species. Finally, we suggest that, just as for hypericin, this pH drop may be involved in the antiviral and antitumor activity of hypocrellin A.

Intracellular pH governs the subcellular distribution of hexokinase in a glioma cell line.

Hexokinase plays a key role in regulating cell energy metabolism. Hexokinase is mainly particulate, bound to the mitochondrial outer membrane in brain and tumour cells. We hypothesized that the intracellular pH (pH1) controls the intracellular distribution of hexokinase. Using the SNB-19 glioma cell line, pH1 variations were imposed by incubating cells in a high-K+ medium at different pH values containing specific ionophores (nigericin and valinomycin), without affecting cell viability. Subcellular fractions of cell homogenates were analysed for hexokinase activity. Imposed pH1 changes were verified microspectrofluorimetrically by using the pH1-sensitive probe SNARF-1-AM (seminaphtho-rhodafluor-1-acetoxymethyl ester). Imposition of an acidic pH1 for 30 min strongly decreased the particulate/total hexokinase ratio, from 63% in the control sample to 31%. Conversely, when a basic pH1, was imposed, the particulate/total hexokinase ratio increased to 80%. The glycolytic parameters, namely lactate/pyruvate ratio, glucose 6-phosphate and ATP levels, were measured concomitantly. Lactate/pyruvate ratio and ATP level were both markedly decreased by acidic pH1 and increased by basic pH1. Conversely, the glucose 6-phosphate level was increased by acidic pH1 and decreased by basic pH1. To demonstrate that the change of hexokinase distribution was not due to altered metabolite levels of glycolysis, a pH1 was imposed for a 5 min incubation time. Modification of the hexokinase distribution was similar to that noted after a 30 min incubation, whereas metabolite levels of glycolysis were not affected. These results provide evidence that the intracellular distribution of hexokinase is highly sensitive to variations of the pH1, and regulates hexokinase activity.

Subcellular distribution of hypericin in human cancer cells.

Confocal laser microspectrofluorometric measurements on human T47D mammary tumor cells have been performed to assess the intracellular distribution of hypericin within the various cell compartments: cytoplasmic membrane, cytoplasm and nucleus. Confocal fluorescence measurements obtained from microvolumes (approximately 1 micron3) located within the three sites of interest show that, while being primarily located in the cell membrane and cytoplasm after a short-term incubation in a 10(-6) M hypericin-containing culture medium, hypericin actually reaches the inside of the cell nucleus after a long-term incubation (210 min). Moreover, owing to the relative fluorescence quantum yields of hypericin determined in vitro when the molecule interacts with DNA, membrane and protein model systems, it is assumed that there is a significant accumulation of the drug into the cell nucleus. Consequently, the nucleus has to be considered as a possible target for the toxic action of hypericin.

Microspectrofluorometry of the protonation state of ellipticine, an antitumor alkaloid, in single cells.

The protonation state and intracellular distribution of ellipticine were investigated in single human mammary T47D cells by confocal laser microspectrofluorimetry. In the cell nucleus, only the protonated form of ellipticine was detected as a direct consequence of its apparent pK increase upon DNA binding. Both protonated and neutral forms were present in the aqueous cytoplasm, where the pH is close to the drug pK. When cells were incubated in high concentrations of K+, a condition that depolarizes the plasma membrane potential, ellipticine cellular accumulation was reduced. In the cytoplasm, ellipticine was mainly bound to mitochondria, and its protonation equilibrium was shifted toward the neutral form. The fluorescence spectrum of ellipticine bound to mitochondria was insensitive to valinomycin, whereas it was markedly shifted toward the protonated form after carbonyl cyanide p-trifluoromethoxy-phenylhydrazone or nigericin addition. Similar studies with ellipticine bound to isolated mitochondria suggest that it behaves as a fluorescent probe of mitochondrial pH in both isolated mitochondria and single living cells.

Characterization of a c-met proto-oncogene activated in human xeroderma pigmentosum cells after treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).

Human xeroderma pigmentosum (XP) fibroblasts were transformed with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The transformed cells, called ASKMN, were immortalized, grew in agar and were tumorigenic in nude mice. A trp-met oncogene was identified in ASKMN cells, after transfection of high molecular weight DNA on 3T3 mouse cells. The ASKMN cells and the 3T3 transformants expressed the 5-kb mRNA transcribed by the tpr-met oncogene and its p65tpr-met phosphorylated protein. Using the polymerase chain reaction (PCR) technique followed by hybridization with synthetic probes or direct sequencing, we showed that the sequence encompassing the 'rearranged breakpoint' was the same as that previously described in the tpr-met oncogene present in the MNNG-HOS cells. However, G to A transitions found in the tpr or met sequences of the ASKMN oncogene, probably the result of the specific mutagenic activity of MNNG, were absent in the MNNG-HOS gene. Apparently normal chromosomes 1 and 7 were identified in the ASKMN cell metaphases using several cytogenetic techniques.

Interactions of iron-anthracycline complexes with living cells: a microspectrofluorometric study.

The interaction of iron-anthracycline complexes with tumor cells has been studied using microspectrofluorometry. The anthracyclines used were adriamycin, 4'-O-tetrahydropyranyladriamycin and daunorubicin. In every case, a 1:3 Fe(III)-anthracycline complex is formed. The three daunorubicin molecules that bind to one Fe(III) are not chemically modified through complexation with iron. In the case of the Fe(III)-adriamycin and Fe(III)-4'-O-tetrahydropyranyladriamycin complexes, about one of the three anthracycline molecules is chemically modified, yielding a highly lipophilic derivative, the 7,8-dehydro-9,10-desacetyladriamycin. The others molecules remain unchanged, i.e., highly hydrophilic in the case of adriamycin. These two species have a different fluorescent spectrum and can be identified inside the cell, using microspectrofluorometry. In the case of the Fe(III)-adriamycin complex, the lipophilic derivative is more rapidly internalized in the cell than the hydrophilic one. Diffusion into the plasmic membrane is the limiting step for the uptake of anthracycline by cells; this means that the plasmic membrane speeds up the dissociation of the Fe(III)-anthracycline complex.

Assessment of membrane potential using confocal microspectrofluorimetry.

Some slow potentiometric dyes, e.g., 3,3'-dipropylthiacarbocyanine and tetramethylrhodamine methyl ester, exhibit fluorescence spectral changes on redistributing from aqueous medium to cells. This effect has been used for spectroscopic discrimination of the emissions from free and bound dyes in cells. Such a discrimination can, in principle, allow for the assessment of cell membrane potential in individual cells, using the Nernst equation applied to the ratio of free dye fluorescence intensities inside and outside of the cell.

SNARF-1 as an intracellular pH indicator in laser microspectrofluorometry: a critical assessment.

The use of SNARF-1-AM (seminaphtorhodafluor-1-acetoxymethylester) to measure the internal pH of a single living cell by laser microspectrofluorometry has been analyzed with a lymphocyte murine B cell line A20. After incubation of the cells at 37 degrees C in the presence of 10 microM SNARF-1-AM, the internal concentration of SNARF-1 was approximately 200 microM. The enhancement of fluorescent intensity of the probe is concomitant with its leakage out of the cells. During the measurement period, this induces a continuous increase of the contribution of the external probe to the total fluorescence intensity. This prevented classical spectrofluorometry measurements, but did not preclude microspectrofluorometry measurements of internal pH. The ratio R was calculated from fluorescence intensities at 635 and 590 nm and used as an indicator of the intracellular pH. Calibration curves of the intracellular pH were obtained in the presence of nigericin and valinomycin. It appeared that both the fluorescence intensity and the ratio R were lower inside the cell than those values obtained in aqueous solutions. Possible interactions with the main biological macromolecules (i.e., DNA, proteins, membranes) were investigated as well as a possible compartmentation of the probe in cellular organelles. The modifications of probe characteristics inside the cells were attributed to the binding of the probe to cellular proteins. The intracellular pH of A20 cells, measured by SNARF-1 on 84 cells, was found to be 7.18 +/- 0.10 (with an external pH of 7.40 +/- 0.05), which corresponded with values obtained by conventional fluorometric methods.

Microspectrofluorimetric study of the kinetics of cellular uptake and metabolization of benzo(a)pyrene in human T 47D mammary tumor cells: evidence for cytochrome P1450 induction.

The kinetics of penetration, activation and detoxification of benzo(a)pyrene were determined by near U.V. microspectrofluorimetric measurements on single living cells. This technique allows one to monitor the different intracellular fluorescent species present in a subcellular microvolume by using spectral decomposition of the fluorescence data. The T47-D cell line was chosen for its high capability of metabolization. The penetration involves a simple diffusion transfer through the cytoplasmic membrane of the cell, with a half-time of approximately 2 min. The metabolization process gives rise, with more than a one hour delay after intracellular incorporation of the hydrocarbon, to a rapid conversion of B(a)P into unconjugated metabolites, leading to a transient accumulation of the 3OH-B(a)P metabolite in the cell. This feature may be related to the enhancement of cytochrome P1450 activity, induced by the B(a)P itself. The ability of the cell to increase its Cyt-P1450 level, after exposure to B(a)P, gives indirect evidence for the presence of the Ah gene complex in the T47-D cell line.