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PubMed2005

Effects of daunorubicin, mitomycin C, azathioprine and cyclosporin A on human retinal pigmented epithelial, corneal endothelial and conjunctival cell lines.

BACKGROUND: We wished to investigate the toxicity of four immunosuppressant and antimetabolic drugs, which are known to influence postoperative wound healing, on three different human ocular cell lines. METHODS: Acute toxicity to cyclosporin A, azathioprine, mitomicyn C and daunorubicin was assessed in Chang cells by monitoring their uptake of propidium iodide during a 3-h period. Chronic toxicity was assessed by monitoring the proliferation and viability of subconfluent cultures of Chang cells, human corneal endothelial cells (HCECs) and retinal pigmented epithelial (RPE) cells after continuous exposure to the drugs for 7 days. RESULTS: Acute toxicity testing revealed no obvious effects. However, the chronic toxicity tests disclosed a narrow concentration range over which cell proliferation decreased dramatically but calcein metabolism was sustained. Although the three lines reacted similarly to each agent, HCECs were the most vulnerable to daunorubicin and mitomycin. At a daunorubicin concentration of 0.05 microg/ml, a 75% decrease in calcein metabolism (P < 0.001) and a > or = 95% cell loss (P < 0.001) were observed. At a mitomycin concentration of 0.01 mug/ml, cell density decreased by 61% (P < 0.001) without a change in calcein metabolism, but at 0.1 microg/ml, the latter parameter decreased to 12% (P = 0.00014). At this concentration the proliferation of Chang and RPE cells decreased by more than 50%, whilst calcein metabolism was largely sustained. Cyclosporin inhibited cell proliferation moderately at lower concentrations (< 5 microg/ml; P=0.05) and substantially at higher ones, with a corresponding decline in calcein metabolism. Azathioprine induced a profound decrease in both parameters at concentrations above 5 microg/ml. CONCLUSION: Daunorubicin, cyclosporin and azathioprine could be used to inhibit excessive intraocular scarring after glaucoma and vitreoretinal surgery without overly reducing cell viability. The attributes of immunosuppressants lie in their combined antiproliferative and immunomodulatory effects.

Antimetabolites↗
PubMed2005

Intranuclear microtubules are hallmarks of an unusual form of cell death in cisplatin-treated C6 glioma cells.

We describe an unusual form of non-accidental cell death marked by ectopic microtubules in the nucleus of a subpopulation of cisplatin-treated C6 glioma astrocytes in culture. At electron microscopy, the perinuclear condensed chromatin did not completely adhere to the nuclear envelope of these cells being separated by single or loosely bundled 20-nm-thick microtubules located in an electron-lucid slit-like zone; the presence of alpha-tubulin lining the inner membrane of the nuclear envelope was confirmed by immunolabeling at confocal microscopy. Since tufts of microfilaments-like fibers also occurred in their central nuclear areas, these cells are referred to as CIMMs (Cells with Intranuclear Microtubules and Microfilaments). The nuclear reorganization of CIMMs also involved nucleolar segregation and formation of heterogeneous ectopic ribonucleoprotein (RNP)-derived structures, indicating disruption of the RNP-based transcription machinery. The cytoplasmic organelles of CIMMs were structurally intact, and propidium iodide did not accumulate intracellularly under vital conditions while the plasma membrane was often Annexin V-positive. All these findings suggest that CIMMs were lethally damaged and committed to an atypical programmed cell death resembling early apoptosis (this is also supported by the presence of a limited number of TUNEL-positive CIMMs). CIMMs appeared well before the main cisplatin-induced cycling arrest of the cell population (G2/M block at 72 h) and had mostly G1 DNA content: this suggests that they may represent the cohort of cells which passed cisplatin-altered mitoses with intranuclear retention of microtubules from an incompletely disassembled mitotic spindle.

Actin Cytoskeleton↗
PubMed2007

Cytotoxic oxysterols induce caspase-independent myelin figure formation and caspase-dependent polar lipid accumulation.

Oxysterols, mainly those oxidized at the C7 position, induce a complex mode of cell death exhibiting some characteristics of apoptosis associated with a rapid induction of lipid rich multilamellar cytoplasmic structures (myelin figures) observed in various pathologies including atherosclerosis. The aim of this study was to determine the relationships between myelin figure formation, cell death, and lipid accumulation in various cell lines (U937, THP-1, MCF-7 [caspase-3 deficient], A7R5) treated either with oxysterols (7-ketocholesterol [7KC], 7beta-hydroxycholesterol, cholesterol-5alpha,6alpha-epoxide, cholesterol-5beta,6beta-epoxide, 25-hydroxycholesterol) or cytotoxic drugs (etoposide, daunorubicin, tunicamycin, rapamycin). Cell death was assessed by the measurement of cellular permeability with propidium iodide, characterization of the morphological aspect of the nuclei with Hoechst 33342, and identification of myelin figures by transmission electron microscopy. Nile Red staining (distinguishing neutral and polar lipids) was used to identify lipid content by flow cytometry and spectral imaging microscopy. Whatever the cells considered, myelin figures were only observed with cytotoxic oxysterols (7KC, 7beta-hydroxycholesterol, cholesterol-5beta, 6beta-epoxide), and their formation was not inhibited by the broad spectrum caspase inhibitor z-VAD-fmk. When U937 cells were treated with oxysterols or cytotoxic drugs, polar lipid accumulation was mainly observed with 7KC and 7beta-hydroxycholesterol. The highest polar lipid accumulation, which was triggered by 7KC, was counteracted by z-VAD-fmk. These findings demonstrate that myelin figure formation is a caspase-independent event closely linked with the cytotoxicity of oxysterols, and they highlight a relationship between caspase activity and polar lipid accumulation.

Apoptosis↗
PubMed2001

Fluorescent dyes for cell viability: an application on prefixed conditions.

In recent years increasing attention has been given to apoptosis for its role in pathologic, organogenetic and homeostatic phenomena. Acridine orange (AO), Hoechst 33342 (HO) and propidium iodide (PI) are among the most used fluorescent dyes used to analyse cell culture viability. In fact, they respectively show specificity for living, apoptotic and late apoptosis/necrosis states. We explored whether HO, AO and PI can be used on prefixed monolayers of three commonly used cell lines. Here we mainly describe the metachromatic effects obtained by fluorescence microscopy with double and triple dye combinations. Furthermore, we propose an easy staining method in which a balanced sequential treatment with HO, AO and PI allows identification of different viability states onto fixed cells by using a long-pass FITC filter. This method extends the spectrum of suitable applications for these dyes in fluorescence viability detection onto previously fixed (prefixed) samples.

Acridine Orange↗
PubMed1998

Cellular taurine release triggered by stimulation of the Fas(CD95) receptor in Jurkat lymphocytes.

One of the hallmarks of apoptosis is cell shrinkage which appears to be important for cell death. The mechanisms mediating cell volume decrease have, however, not been addressed. Mechanisms employed by swollen cells to decrease their cell volume include activation of ion transport pathways, such as ion channels and KCl cotransport, and release of cellular osmolytes, such as taurine, sorbitol, betaine and inositol. The present study has been performed to test for release of taurine. To this end Jurkat human T-lymphocytes were loaded with [3H]taurine and apoptotic cell death induced by triggering the Fas(CD95) receptor with monoclonal crosslinking antibody. Triggering the Fas(CD95) receptor led to a release of 60+/-5% of cellular taurine within 90 min. The release did not occur prior to 45 min. The release coincided with cell shrinkage as evidenced from forward scatter in FACS analysis and preceeded DNA fragmentation according to propidium iodide staining. The delay of taurine release was not influenced by exchange of medium and thus was not due to extracellular accumulation of a stimulator. The Fas(CD95)-induced taurine release, cell shrinkage and DNA fragmentation were blunted by lowering of ambient temperature to 23 degreesC. Following pretreatment of cells with Fas(CD95) antibody at 23 degreesC rewarming led to rapid taurine release, cell shrinkage and DNA fragmentation, indicating that the temperature-sensitive step is distal to the mechanisms accounting for the delay. Osmotic cell swelling led to an immediate release of taurine. In conclusion, Fas(CD95) triggering leads to delayed taurine release through a temperature-sensitive mechanism.

Antibodies, Monoclonal↗
PubMed2003

Chlamydia trachomatis-infected macrophages induce apoptosis of activated T cells by secretion of tumor necrosis factor-alpha in vitro.

Chlamydia trachomatis-infected macrophages induce T cell apoptosis. This ability might promote intracellular survival of Chlamydia and perpetuate chronic chlamydial infection. The purpose of this study was to examine the molecular mechanisms by which C. trachomatis-infected macrophages induce T cell apoptosis. Monocytes and T cells were isolated from the peripheral blood of healthy donors. Macrophages were infected with C. trachomatis, and autologous T cells were stimulated by mitogen. After 6 days, both populations were cultured together using a two-chamber transwell membrane system to differentiate between mechanisms involving either cell-to-cell contact or secretion of apoptotic factors. Apoptotic T cells were identified by propidium iodide through-flow cytometry, and tumor necrosis factor-alpha (TNF-alpha) concentrations were measured by enzyme-linked immunosorbent assay. Antagonists of TNF-alpha, the Fas (CD95) molecule, TNF-related apoptosis-inducing ligand (TRAIL), and catalase were added to differentiate between the pathways of apoptosis. C. trachomatis-infected macrophages significantly induced T cell apoptosis by cell-to-cell contact (mean +/- standard deviation, 30+/-4%; P<0.001) and by humoral mechanisms (mean +/- standard deviation, 22+/-3%, P<0.001). Humoral apoptosis was mediated by secretion of TNF-alpha from infected macrophages. Inhibition of secretory TNF-alpha by the monoclonal anti-TNF-alpha antibody adalimumab (D2E7) blocked T cell death in vitro. In contrast, T cell apoptosis mediated by cell-to-cell contact was not inhibited by the different anti-apoptotic reagents. In summary, TNF-alpha derived from infected macrophages is an important apoptosis factor for T cell apoptosis induced by C. trachomatis-infected cells.

Adalimumab↗
PubMed2007

Effect of lycopene on insulin-like growth factor-I, IGF binding protein-3 and IGF type-I receptor in prostate cancer cells.

PURPOSE: Prostate cancer is the second most common cancer that leads to death in elderly men. The risk of prostate cancer prevalence is often associated with the elevated level of insulin-like growth factor-I (IGF-I) and decreased level of IGF-binding protein 3 (IGFBP-3). Lycopene, a carotenoid, reduces the proliferation of cancer cells and induces apoptosis. Hence, higher intake of lycopene can be associated with the lower risk of prostate cancer. However, the mechanism of action of lycopene in the prevention of prostate cancer is still unclear. The present study was carried out to study the effects of lycopene on the components of IGF system and apoptosis in androgen-independent prostate cancer cells (PC-3 cells). METHODS: PC-3 cells were treated with various concentrations of lycopene, (20, 40 and 60 microM) for 24, 48, 72 and 96 h. IGF-I, IGFBP-3 and IGF-I receptor (IGF-IR) levels in lycopene-treated cells were evaluated. Annexin V and propidium iodide (PI) binding studies were done to assess apoptosis. RESULTS: PC-3 cells treated with lycopene showed a significant decrease in cell proliferation. Lycopene, at a dose of 40 microM, significantly increased the level of IGFBP-3. Lycopene-induced apoptosis was confirmed by annexin V and PI binding. Lycopene-induced DNA fragmentation was absent after 24 h treatment whereas the same was observed after 48 h treatment. There was a significant decrease in the IGF-IR expression after the cells were treated with lycopene and IGF-I. CONCLUSION: The data obtained suggest that the components of the IGF system may act as a positive regulator of lycopene-induced apoptosis in PC-3 cells. Thus, the observed lycopene-induced biological effects and their associated mechanisms are encouraging and may lead to the development of a highly successful drug for the treatment of prostate cancer.

Annexin A5↗
PubMed2003

Purification of Sarcocystis neurona sporocysts from opossum (Didelphis virginiana) using potassium bromide discontinuous density gradient centrifugation.

This report describes a new, inexpensive procedure for the rapid and efficient purification of Sarcocystis neurona sporocysts from opossum small intestine. S. neurona sporocysts were purified using a discontinuous potassium bromide density gradient. The procedure provides a source of sporocyst wall and sporozoites required for reliable biochemical characterization and for immunological studies directed at characterizing antigens responsible for immunological responses by the host. The examined isolates were identified as S. neurona using random amplified polymorphic DNA primers and restriction endonuclease digestion assays. This method allows the collection of large numbers of highly purified S. neurona sporocysts without loss of sporocyst viability as indicated by propidium iodide permeability and cell culture infectivity assays. In addition, this technique might also be used for sporocyst purification of other Sarcocystis spp.

Animals↗
PubMed2005

Hemocyte differentiation in the hematopoietic organs of the silkworm, Bombyx mori: prohemocytes have the function of phagocytosis.

Hemocytes isolated from the larval hematopoietic organs of the silkworm were classified following staining with acridine orange and propidium iodide. Among the hemocytes isolated from the hematopoietic organs of whole fifth larval and wandering stages, most were prohemocytes (60%-70%) and oenocytoids (30%-40%). Granulocytes comprised only about 0.5%-1% at the wandering stage and were even rarer at other stages; no spherulocytes or plasmatocytes were found. Therefore, hemocyte differentiation inside larval hematopoietic organs is not as extensive as previously thought. Following 10-30 min in vitro culture of hemocytes isolated from larval hematopoietic organs, many young granulocytes and plasmatocytes appeared. Furthermore, during phagocytosis assays, prohemocytes were seen to adopt the morphology of plasmatocytes, containing fragments of phagocytosed cells. Our results underline the similarities between Drosophila and Bombyx hematopoiesis.

Acridine Orange↗
PubMed2005

Selective visualisation of neuroepithelial bodies in vibratome slices of living lung by 4-Di-2-ASP in various animal species.

Pulmonary neuroepithelial bodies (NEBs) are extensively innervated organoid groups of neuroendocrine cells that lie in the epithelium of intrapulmonary airways. Our present understanding of the morphology of NEBs is comprehensive, but direct physiological studies have so far been challenging because the extremely diffuse distribution of NEBs makes them inaccessible in vivo and because a reliable in vitro model is lacking. Our aim has been to optimise an in vitro method based on vibratome slices of living lungs, a model that includes NEBs, the surrounding tissues and at least part of their complex innervation. This in vitro model offers satisfactory access to pulmonary NEBs, provided that they can be differentiated from other tissue elements. The model was first optimised for living rat lung slices. Neutral red staining, reported to stain rabbit NEBs, proved unsuccessful in rat slices. On the other hand, the styryl pyridinium dye, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4-Di-2-ASP), showed brightly fluorescent cell groups, reminiscent of NEBs, in the airway epithelium of living lung slices from rat. In addition, nerve fibres innervating the NEBs were labelled. The reliable and specific labelling of pulmonary NEBs by 4-Di-2-ASP was corroborated by immunostaining for protein gene-product 9.5. Live cell imaging and propidium iodide staining further established the acceptable viability of 4-Di-2-ASP-labelled NEB cells in lung slices, even over long periods. Importantly, the in vitro model and 4-Di-2-ASP staining procedure for pulmonary NEBs appeared to be equally reproducible in mouse, hamster and rabbit lungs. Diverse immunocytochemical procedures could be applied to the lung slices providing an opportunity to combine physiological and functional morphological studies. Such an integrated approach offers additional possibilities for elucidating the function(s) of pulmonary NEBs in health and disease.

Animals↗
PubMed2006

Mechanisms of programmed cell death during oogenesis in Drosophila virilis.

We describe the features of programmed cell death occurring in the egg chambers of Drosophila virilis during mid-oogenesis and late oogenesis. During mid-oogenesis, the spontaneously degenerating egg chambers exhibit typical characteristics of apoptotic cell death. As revealed by propidium iodide, rhodamine-conjugated phalloidin staining, and the TUNEL assay, respectively, the nurse cells contain condensed chromatin, altered actin cytoskeleton, and fragmented DNA. In vitro caspase activity assays and immunostaining procedures demonstrate that the atretic egg chambers possess high levels of caspase activity. Features of autophagic cell death are also observed during D. virilis mid-oogenesis, as shown by monodansylcadaverine staining, together with an ultrastructural examination by transmission electron microscopy. During the late stages of oogenesis in D. virilis, once again, the two mechanisms, viz., nurse cell cluster apoptosis and autophagy, operate together, manifesting features of cell death similar to those detailed above. Moreover, an altered form of cytochrome c seems to be released from the mitochondria in the nurse cells proximal to the oocyte. We propose that apoptosis and autophagy function synergistically during oogenesis in D. virilis in order to achieve a more efficient elimination of the degenerated nurse cells and abnormal egg chambers.

Actins↗
PubMed2002

Domoic acid neurotoxicity in hippocampal slice cultures.

The neurotoxicity of domoic acid was studied in 2-3 week old rat hippocampal slice cultures, derived from 7 day old rat pups. Domoic acid 0.1-100 microM was added to the culture medium for 48 hrs, alone or together with the glutamate receptor antagonists NS-102 (5-Nitro-6,7,8,9-tetrahydrobenzo[G]indole-2,3-dione-3-oxime), NBQX (2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline) or MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate), followed by transfer of the cultures to normal medium for additional 48 hrs. Neuronal degeneration in the fascia dentata (FD), CA3 and CA1 hippocampal subfields was monitored and EC(50) values estimated by densitometric measurements of the cellular uptake of propidium iodide (PI). The CA1 region was most sensitive to domoic acid, with an EC(50) value of 6 microM domoic acid, estimated from the PI-uptake at 72 hrs. Protective effects of 10 microM NBQX against 3 and 10 microM domoic acid were observed for both dentate granule cells and CA1 and CA3c pyramidal cells. NS102 and MK 801 only displayed protective effects when combined with NBQX. MK801 significantly increased the combined neuroprotective effect of NBQX and NS102 against 10 microM domoic acid in both CA1 and FD, but not in CA3. We conclude, that domoic acid neurotoxicity in CA3 and in hippocampal slice cultures in general primarily involves AMPA/kainate receptors. At high concentrations (10 microM domic acid) NMDA receptors are, however, also involved in the toxicity in CA1 and FD.

Animals↗
PubMed2000

Excitatory amino acid neurotoxicity and modulation of glutamate receptor expression in organotypic brain slice cultures.

Using organotypic slice cultures of hippocampus and cortex-striatum from newborn to 7 day old rats, we are currently studying the excitotoxic effects of kainic acid (KA), AMPA and NMDA and the neuroprotective effects of glutamate receptor blockers, like NBQX. For detection and quantitation of the induced neurodegeneration, we have developed standardized protocols, including--a) densitometric measurements of the cellular uptake of propidium iodide (PI), --b) histological staining by Flouro-Jade, --c) lactate dehydrogenase (LDH) release to the culture medium, --d) immunostaining for microtubulin-associated protein 2, and --e) general and specific neuronal and glial cell stains. The results show good correlation between the different markers, and are in accordance with results obtained in vivo. Examples presented in this review will focus on the use of PI uptake to monitor the excitotoxic effects of --a) KA and AMPA (and NMDA) in hippocampal slice cultures, and --b) KA and AMPA in corticostriatal slice cocultures, with demonstration of differentiated neuroprotective effects of NBQX in relation to cortex and striatum and KA and AMPA. A second set of studies include modulation of hippocampal KA-induced excitotoxicity and KA-glutamate receptor subunit mRNA expression after long-term exposure to low, non-toxic doses of KA and NBQX. We conclude that organotypic brain slice cultures, combined with standardized procedures for quantitation of cell damage and receptor subunit changes is of great potential use for studies of excitotoxic, glutamate receptor-induced neuronal cell death, receptor modulation and related neuroprotection.

Animals↗
PubMed2001

Artificial Pseudomonas aeruginosa biofilms and confocal laser scanning microscopic analysis.

Bacterial biofilms may be formed at various sites, including mucous membranes, teeth, and infectious lesions. To elucidate the structure and the function of biofilms, artificial biofilms of mucoid-type Pseudomonas aeruginosa organisms (strain PT1252) were made by centrifuging the organisms onto the surface of a coverglass and culturing further in broth media supplied continuously (45 ml/h). The biofilm structure at 4, 8, 12, and 24 h was visualized with fluorescent staining (SYTO9, propidium iodide [PI], and/or fluorescein isothiocyanate-concanavalin A [FITC-ConA]) by confocal laser scanning microscopy (CLSM). It was clearly demonstrated that the number of bacteria (10(4)--10(6)/ml) could be estimated by their fluorescence intensity. Sectional analysis of each biofilm layer (1-microm thickness) made it possible to demonstrate the three-dimensional development of biofilms, and revealed that the biofilms were 9 microm in height after 12 h. The live and dead organisms were differentiated by SYTO9 and PI, respectively, in situ in biofilms, and about 13% of the organisms were dead in 12-h-old biofilms. When 12-h-old biofilms were exposed to ciprofloxacin at minimum bactericidal concentration (6.26 microg/ml) for 90 min, all the organisms were killed, but some organisms (11 +/- 1.3%; n = 3) in 24-h-old biofilms with thicker and denser structure were still alive after exposure for 120 min. These results indicate that the CLSM analysis of artificial biofilms was useful for elucidating bacterial functions in biofilms, and may lead to a new quantitative system for estimating the bactericidal efficacy of antibacterial drugs in biofilms.

Anti-Infective Agents↗
PubMed2005

The use of new probes and stains for improved assessment of cell viability and extracellular polymeric substances in Candida albicans biofilms.

Phenotypic and genotypic cell differentiation is considered an important feature that confers enhanced antifungal resistance in candidal biofilms. Particular emphasis has been placed in this context on the viability of biofilm subpopulations, and their heterogeneity with regard to the production of extracellular polymeric substances (EPS). We therefore assessed the utility of two different labeled lectins Erythrina cristagalli (ECA) and Canavalia ensiformis (ConA), for EPS visualization. To evaluate the viability of candidal biofilms, we further studied combination stains, SYTO9 and propidium iodide (PI). The latter combination has been successfully used to assess bacterial, but not fungal, viability although PI alone has been previously used to stain nuclei in fungal cells. Candida albicans biofilms were developed in a rotating disc biofilm reactor and observed in situ using confocal scanning laser microscopy (CSLM). Our data indicate that SYTO9 and PI are reliable vital stains that may be used to investigate C. albicans biofilms. When used together with ConA, the lectin ECA optimized EPS visualization and revealed differential production of this material in mature candidal biofilms. The foregoing probes and stains and the methodology described should help better characterize C. albicans biofilms in terms of cell their viability, and EPS production.

Biofilms↗
PubMed2006

Application of fluorescent probes to study structural changes in Aspergillus fumigatus exposed to amphotericin B, itraconazole, and voriconazole.

The broad objective of this study was to document patterns of structural changes following antifungal treatment, and to determine any relationship with minimum inhibitory concentration (MIC) of an antifungal. Three clinical isolates of Aspergillus fumigatus, with high, intermediate, and low amphotericin B (AB), itraconazole (IZ), and voriconazole (VZ) MICs were studied in 24-well plates with cover slips. The fluorescent probes used were Calcofluor White (cell wall), propidium iodide (nucleus), and MitoTracker Green FM (mitochondria). Fluorescent microscopy as early as 3-h after exposure revealed that AB treated hyphae had intact cell wall with deformed mitochondria and nuclei while IZ and VZ treated hyphae revealed no intact cell wall, and deformation of mitochondria and nuclei. At 48 h, AB treated cells revealed rupture of hyphae and disintegration of mitochondria, and nuclei, IZ treated hyphae were swollen with disintegration of mitochondria, and nuclei while VZ treated hyphae showed rupture and disintegration of mitochondria and nuclei. The structural changes for the three strains studied were similar in fluorescent microscopy as long as the incubation time and their respective MICs were used. Thus, AB, IZ, and VZ induced gross organelle defects in A. fumigatus nuclei, mitochondria, and cell wall, which were consistent with respective MICs of antifungals used.

Amphotericin B↗
PubMed2005

Estradiol protects against oxygen and glucose deprivation in rat hippocampal organotypic cultures and activates Akt and inactivates GSK-3beta.

Here we investigated the neuroprotective effect of 17beta-estradiol in an in vitro model of ischemia. We used organotypic hippocampal slice cultures, acute or chronically treated with 17beta-estradiol (10 nM), and exposed to oxygen and glucose deprivation (OGD). Cellular death was quantified by measuring uptake of propidium iodide (PI), a marker of dead cells. In OGD exposed cultures, treated only with vehicle, about 70% of the CA1 area of hippocampus was labeled with PI, indicating a great percentage of cellular death. When cultures were treated with 17beta-estradiol (acute or chronically), this cellular death was reduced to 15%. This effect was prevented by LY294002 but was not by PD98059. Immunoblotting revealed that both, chronic and acute, treatments with 17beta-estradiol induced the phosphorylation/activation of Akt and the phosphorylation/inactivation of GSK-3beta. Our results show a clear neuroprotective effect of 17beta-estradiol and suggest that this effect could involve PI3-K pathway.

Animals↗
PubMed2005

Intracellular delivery of Bak BH3 peptide by microbubble-enhanced ultrasound.

PURPOSE: To investigate the possibility of intracellular delivery of Bak BH3 peptide using sonoporation effect by microbubble-enhanced ultrasound. METHODS: HeLa and BJAB cells were exposed to 1.696-Mhz focused ultrasound with 2% microbubble contrast agents (OPTISON). Cell-impermeable calcein was used as an indicator for successful sonoporation, and propidium iodide staining was used for cell viability assessment. Peptides were also exposed to ultrasound with OPTISON and analyzed with mass spectrometry for evaluation of stability under ultrasound exposure. The effect of transduced Bak BH3 peptide was evaluated by the cell viability of successfully sonoporated cells. RESULTS: Bak BH3 peptides did not undergo mechanical degradation with microbubble-enhanced ultrasound exposure. With the increase of acoustic energy exposure, the sonoporation efficiency saturated both in BJAB and HeLa cells, while direct cell death rate by ultrasound exposure tended to increase. When BJAB cells were treated with 100 microM Bak BH3 peptides, and ultrasound exposure with ultrasound contrast agents (OPTISON), an increased 35% cell death was confirmed. On the other hand, although HeLa cells had a similar trend, they failed to exhibit statistical significance. CONCLUSIONS: Our results suggest that microbubble-enhanced focused ultrasound peptide transduction is possible. Further optimization of ultrasound exposure conditions may be necessary.

Antineoplastic Agents↗