Fluorescein Isothiocyanate-annexin Research Articles

Short-term effects of calcium ions on the apoptosis and onset of mineralization of human dental pulp cells in vitro and in vivo.

Calcium ions (Ca2+) are a major constituent of most pulp-capping materials and have an important role in the mineralization of human dental pulp cells (hDPCs). A previous study by our group has shown that increased levels of Ca2+ can promote hDPC-mediated mineralization in long-term cultures (21 days). However, the initiation of mineralization occurs in the early stage of osteogenic inductive culture, and the effects of Ca2+ on the mineralization of hDPCs in short-term cultures (five days) have not been studied in detail. Furthermore, the underlying mechanism by which Ca2+ stimulates the mineralization of hDPCs has remained controversial. A strong correlation between mineralization and cell apoptosis and/or death has been identified. Thus, the present study hypothesized that Ca2+ may promote the onset of hDPC-mediated mineralization through inducing their apoptosis and/or death. To verify this hypothesis, Ca2+ was added to the growth culture medium and osteogenic culture medium at various concentrations. Alizarin Red S staining and reverse transcription-polymerase chain reaction analysis were used to evaluate the onset of mineralization. Furthermore, the cell counting kit-8 and fluorescein isothiocyanate-Annexin V/propidium iodide double-staining method were adopted to detect the proliferation and apoptosis of hDPCs in the growth culture medium. An animal experiment and scanning electron microscopic observation of ceramic graft implants were applied to measure the mineralization in vivo. The results showed that 5.4 and 9.0 mM Ca2+ accelerated the onset of mineralized matrix nodule formation, promoted osteopontin mRNA expression and induced marked cell apoptosis and necrosis, but had no obvious effect on cell proliferation. These findings indicated a positive association between cell apoptosis and/or death and the timing of formation as well as the quantity of extracellular mineralization induced by Ca2+ in short-term cultured hDPCs.

Identification of indole-3-carboxylic acids as non-ATP-competitive Polo-like kinase 1 (Plk1) inhibitors

A series of indole-3-carboxylic acids were designed as novel small molecular non-ATP-competitive Plk1 inhibitors. The designed compounds were synthesized and evaluated. Most of the targeted compounds showed potent Plk1 inhibitory activities and anti-proliferative characters. Particularly, 4f and 4g showed Plk1 inhibitory activity with IC50 values of 0.41 and 0.13μM, which were about 5 and 17 times more potent compared to thymoquinone, respectively. Compound 4g also showed inhibitory activity to HeLa and MCF-7 cell lines with IC50 values of 0.72 and 1.15μM, which was almost 3 and 4 times more potent than thymoquinone. Study of mechanism of action suggested that 4g was an ATP-independent and substrate-dependent Plk1 inhibitor. Moreover, 4g showed excellent Plk1 inhibitory selectivity against Plk2 and Plk3. Fluorescein isothiocyanate Annexin V/propidium iodide (PI) double-staining assay and western-blot results indicate that induction of apoptosis by 4g is involved in its anti-tumor activity. This study may provide a support for further optimization of non-ATP-competitive Plk1 inhibitors.

Neuroprotective Effects of Neuregulin-1 ß on Oligodendrocyte Type 2 Astrocyte Progenitors Following Oxygen and Glucose Deprivation

BackgroundHypoxic-ischemic brain injury in neonates, especially in premature infants, is one of the main contributors to the mortality of newborns and can cause nervous system dysfunction in children. The major pathogenesis seems to be cerebral ischemia/reperfusion in the immature white matter that preferentially targets vulnerable premyelinating oligodendrocytes. ObjectivesThe goal of this study was to culture oligodendrocyte type 2 astrocyte cells in an oxygen and glucose deprivation environment to simulate ischemia injury and examine the cellular and molecular mechanisms involved in the neuroprotective effects of neuregulin-1ß on ischemia-induced immature oligodendrocytes. MethodsOligodendrocyte type 2 astrocyte cells were cultured from neonatal Sprague-Dawley rat cerebra. The cells were divided into two groups: one was subjected to oxygen and glucose deprivation for 9 hours and the other was treated with 50 ng/mL or 100 ng/mL neuregulin-1β during oxygen and glucose deprivation. Cell survival was determined by Trypan Blue staining and cell apoptosis were observed by fluorescein isothiocyanate-Annexin V and propidium iodide double staining. To study if the PI3K-Akt signaling pathway was involved in the mechanism of protective effect of neuregulin-1ß, Western blot analysis was used to quantitative the changes of protein. ResultsTreatment with neuregulin-1ß within the period of oxygen and glucose deprivation significantly increased cell survival and also resulted in a significant decrease in cell apoptosis. The neuroprotective effects of neuregulin-1ß were prevented by treatment with Ly294002, an inhibitor of the phosphatidylinositol-3-kinase/Akt pathway. ConclusionsThese results suggest that neuregulin-1ß could protect the oligodendrocyte type 2 astrocyte progenitors against hypoxic injury, and the mechanism may be associated with the PI3K-Akt signaling pathway.

Response to 'TNF/TNFR signal transduction pathway-mediated anti-apoptosis and anti-inflammatory effects of sodium ferulate on IL-1β-induced rat osteoarthritis chondrocytes in vitro'

Sodium ferulate (SF) is a natural component of traditional Chinese herbs. Our previous study shows that SF has a protective effect on osteoarthritis (OA). The objective of this study was to investigate the effect of SF on the TNF/TNF receptor (TNFR) signal transduction pathway of rat OA chondrocytes.Primary rat articular chondrocytes were co-treated with IL-1β and SF. Chondrocyte apoptosis was assessed by fluorescein isothiocyanate-annexin V/propidium iodide assay. The PCR array was used to screen the expression of 84 key genes involved in apoptosis. The release of TNFα and prostaglandin E2 were analyzed by ELISA. Expressions of proteins were assessed by western blotting. The activity of NF-κB was determined by electrophoretic mobility shift assay (EMSA). Gene expression of inducible nitric oxide synthase (iNOS) was evaluated by real-time quantitative PCR. The nitric oxide content was measured with the Griess method.After treatment with SF, the apoptosis rate of chondrocytes significantly attenuated (P < 0.01). Results of the apoptosis PCR array suggested that mRNA expression of some core proteins in the TNF/TNFR pathway showed valuable regulation. The protein expressions of TNFα, TNFR-1, TNF receptor-associated death domain, caspase-8 and caspase-3 were prevented by SF in a concentration-dependent manner. SF also inhibited activities of caspase-8 and caspase-3 compared with the OA model control (P < 0.01). TNF receptor-associated factor-2 expression, phosphorylations of inhibitor of NF-κB kinase (IKK) subunits alpha and beta, and NF-κB inhibitor, alpha (IκBα) were all concentration-dependently suppressed by SF treatment. The results of EMSA showed that SF inhibited the activity of NF-κB. In addition, the expressions of cycloxygenase-2 and iNOS and the contents of prostaglandin E2 and NO were attenuated with the treatment of SF (P < 0.01).SF has anti-apoptosis and anti-inflammatory effects on an OA model induced by IL-1β in vitro, which were due to inhibitory actions on the caspase-dependent apoptosis pathway and the IKK/NF-κB signal transduction pathway of the TNF/TNFR pathway.

Vitamin C induces apoptosis in AGS cells by down-regulation of 14–3-3σ via a mitochondrial dependent pathway

Ascorbic acid (vitamin C) is an essential component of most living cells. Apart from antioxidant activity, it has been reported to inhibit cancer cell growth in vitro in human cancer cells. However, the cellular mechanism underlying anticancer activity has not been fully elucidated. In this study, vitamin C showed a cytotoxic effect on human gastric cancer cell line AGS (LD50 300μg/ml). Further, flow cytometry analysis showed that vitamin C increased the sub-G1 (apoptosis) population and apoptosis confirmed by fluorescein isothiocyanate–Annexin V double staining in AGS cells. Moreover, specific immuno-blotting revealed the expression of the phosphorylated form of Bad (S136), 14–3-3σ, pro-caspases-3, -6, -8, and-9 protein levels were significantly decreased and Bax/Bcl-xL ratio was increased in a dose-dependent manner. Also, wound healing assay results showed that vitamin C inhibited AGS cell proliferation. These findings suggest that vitamin C induces apoptosis and might be a potential therapeutic agent for gastric cancer.

Role of zebrafish thrombocyte and non-thrombocyte microparticles in hemostasis

Hemostasis is a defense mechanism that protects an organism from bleeding in the event of injury. We have previously demonstrated the utility of the zebrafish as a model to study human hemostasis. However, there are no studies on the role of microparticles in hemostasis in early vertebrates. Studying microparticles in zebrafish may provide insight into the evolution of microparticle function in hemostasis and may lead to direct observation of these microparticles in zebrafish larvae due to transparency of the vessels. In this investigation we demonstrate the presence of cellular microparticles in fish blood by both immunostaining as well as by using zebrafish whose thrombocytes are labeled with green fluorescent protein. Further investigation showed that microparticles were also labeled by fluorescein isothiocyanate annexin V, suggesting that these particles are derived via apoptosis. A portion of the fluorescein isothiocyanate annexin V labeled microparticles was also labeled by DiI-C18. Labeling by DiI-C18 suggests that some microparticles are derived from young thrombocytes. Additionally, GpIIb antibody labels almost all thrombocyte-derived microparticles and a greater percentage of microparticles are labeled by GpIIb antibody than by DiI-C18. This suggests that thrombocyte microparticles are derived from both young and mature thrombocytes. Furthermore, the increase of microparticles by adding excessive microparticles into blood in vitro and through intravenous injections led to an increased hemostatic response. In addition, treatment with tumor necrosis factor alpha resulted in an increased number of thrombocyte microparticles and enhanced hemostasis; in contrast, treatment with zVAD-FMK, a caspase inhibitor, resulted in a decrease in thrombocyte microparticles and decreased hemostasis. We also found that thrombocyte microparticles agglutinate, along with other cells and cellular microparticles, in the presence of an excess of either ristocetin or ultra-large von Willebrand factor. Also, stimulation of von Willebrand factor release in vivo resulted in clusters of thrombocyte microparticles in the veins. Moreover, thrombocyte microparticles were the first to appear at the site of arterial injury. We found that thrombocyte microparticles are functionally equivalent to platelet microparticles. The microparticles initiate arterial thrombus formation in a von Willebrand factor-dependent manner and further enhance thrombus formation by forming clusters of microparticles in venous thrombosis. This finding may have applications for understanding the role of platelet microparticles in humans and may have diagnostic applications.

TNF/TNFR signal transduction pathway-mediated anti-apoptosis and anti-inflammatory effects of sodium ferulate on IL-1β-induced rat osteoarthritis chondrocytes in vitro

IntroductionSodium ferulate (SF) is a natural component of traditional Chinese herbs. Our previous study shows that SF has a protective effect on osteoarthritis (OA). The objective of this study was to investigate the effect of SF on the TNF/TNF receptor (TNFR) signal transduction pathway of rat OA chondrocytes.MethodsPrimary rat articular chondrocytes were co-treated with IL-1β and SF. Chondrocyte apoptosis was assessed by fluorescein isothiocyanate-annexin V/propidium iodide assay. The PCR array was used to screen the expression of 84 key genes involved in apoptosis. The release of TNFα and prostaglandin E2 were analyzed by ELISA. Expressions of proteins were assessed by western blotting. The activity of NF-κB was determined by electrophoretic mobility shift assay (EMSA). Gene expression of inducible nitric oxide synthase (iNOS) was evaluated by real-time quantitative PCR. The nitric oxide content was measured with the Griess method.ResultsAfter treatment with SF, the apoptosis rate of chondrocytes significantly attenuated (P < 0.01). Results of the apoptosis PCR array suggested that mRNA expression of some core proteins in the TNF/TNFR pathway showed valuable regulation. The protein expressions of TNFα, TNFR-1, TNF receptor-associated death domain, caspase-8 and caspase-3 were prevented by SF in a concentration-dependent manner. SF also inhibited activities of caspase-8 and caspase-3 compared with the OA model control (P < 0.01). TNF receptor-associated factor-2 expression, phosphorylations of inhibitor of NF-κB kinase (IKK) subunits alpha and beta, and NF-κB inhibitor, alpha (IκBα) were all concentration-dependently suppressed by SF treatment. The results of EMSA showed that SF inhibited the activity of NF-κB. In addition, the expressions of cycloxygenase-2 and iNOS and the contents of prostaglandin E2 and NO were attenuated with the treatment of SF (P < 0.01).ConclusionSF has anti-apoptosis and anti-inflammatory effects on an OA model induced by IL-1β in vitro, which were due to inhibitory actions on the caspase-dependent apoptosis pathway and the IKK/NF-κB signal transduction pathway of the TNF/TNFR pathway.

Effects of varied ionic calcium and phosphate on the proliferation, osteogenic differentiation and mineralization of human periodontal ligament cells in vitro

A number of bone-filling materials containing calcium (Ca(2+) ) and phosphate (P) ions have been used in the repair of periodontal bone defects; however, the effects that local release of Ca(2+) and P ions has on biological reactions are not fully understood. In this study, we investigated the effects of various levels of Ca(2+) and P ions on the proliferation, osteogenic differentiation and mineralization of human periodontal ligament cells (hPDLCs). The hPDLCs were obtained using an explant culture method. Defined concentrations and ratios of ionic Ca(2+) to inorganic P were added to standard culture and osteogenic induction media. The ability of hPDLCs to proliferate in these growth media was assayed using the Cell Counting Kit-8. Cell apoptosis was evaluated by the fluorescein isothiocyanate-annexin V/propidium iodide double-staining method. Osteogenic differentiation and mineralization were investigated by morphological observations, alkaline phosphatase activity and Alizarin Red S/von Kossa staining. The mRNA expression of osteogenic related markers was analysed using RT-PCR. Within the ranges of Ca(2+) and P ion concentrations tested, we observed that increased concentrations of Ca(2+) and P ions enhanced cell proliferation and formation of mineralized matrix nodules, whereas alkaline phosphatase activity was reduced. The RT-PCR results showed that elevated concentrations of Ca(2+) and P ions led to a general increase of Runx2 mRNA expression and decreased alkaline phosphatase mRNA expression, but gave no clear trend on osteocalcin mRNA levels. The concentrations and ratios of Ca(2+) and P ions could significantly influence proliferation, differentiation and mineralization of hPDLCs. Within the range of concentrations tested, we found that the combination of 9.0 mm Ca(2+) ions and 4.5 mm P ions were the optimal concentrations for proliferation, differentiation and mineralization in hPDLCs.

Dihydroartemisinin Induces Endoplasmic Reticulum Stress-Mediated Apoptosis in HepG2 Human Hepatoma Cells

Previous studies showed that dihydroartemisinin (DHA) possessed antitumor activity in many human tumor cells through the induction of apoptosis. The aim of this study was to investigate the effects of DHA on apoptosis in the human hepatocellular carcinoma cell line HepG2 and the possible molecular mechanisms involved. The inhibitory effect of DHA on HepG2 cells was measured by MTT assay. The percentage of apoptotic cells was detected by flow cytometry with double staining of fluorescein isothiocyanate-annexin V/propidium iodide. The intracellular production of reactive oxygen species (ROS) and intracellular Ca2+ concentration ([Ca2+]i) were detected by fluorescence spectrophotometry. Protein expression of GADD153, Bcl-2 and Bax in HepG2 cells was examined by Western blot and immunocytochemistry. DHA significantly inhibited proliferation of HepG2 cells in a dose- and time-dependent manner. The apoptosis rates in HepG2 cells treated with 0, 50, 100 and 200 μmol/L DHA for 24 hours were 2.53 ± 0.88%, 24.85 ± 3.63%, 35.27 ± 5.92% and 48.53 ± 7.76%, respectively. Compared with the control group, DHA significantly increased ROS generation and [Ca2+]i level (P <0.05), with the generation of ROS preceding the increase in [Ca2+]i. An increase in GADD153 and Bax expression and a decrease in Bcl-2 were observed in DHA-treated cells. Pretreatment with the antioxidant N-acetylcysteine could attenuate the effects of DHA in the experiments. DHA could inhibit proliferation and induce apoptosis in HepG2 cell lines through increasing the intracellular production of ROS and [Ca2+]i. Endoplasmic reticulum stress-induced apoptosis may contribute to this effect by regulating the expression of GADD153, proapoptotic Bax, and antiapoptotic Bcl-2.

Induction of Nicotinamide–Adenine Dinucleotide Phosphate Oxidase and Apoptosis by Biodegradable Polymers in Macrophages: Implications for Stents

The drug-eluting stent platform has a limited surface area, and a polymer carrier matrix is coated to enable sufficient loading of drugs. The development of a suitable polymer has been challenging because it must exhibit biocompatibility with the intravascular milieu. The use of biodegradable polymers seems to be attractive because it enables drug release as it degrades and is eventually eliminated from the body leaving the permanent metallic stent polymer-free. The aim of this study was to investigate the biocompatibility of biodegradable polymers using the human monocyte cell line. Cultured monocytes differentiated into functional macrophages (THP-1) were incubated with various polymers including poly-L-lactide (PLA), polycaprolactone (PCL), or poly-D, L-lactide-co-glycolide (PLGA) for up to 5 days. Exposure of cells to the polymers resulted in macrophage-polymer adhesion and induced marked pro-oxidant species as measured by calcein AM uptake assay and flow cytometric analysis of 2',7'-dichlorofluorescin fluorescence, respectively. Real-time reverse-transcription polymerase chain reaction and Western blot analysis of expression of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases revealed enhanced expression of NADPH oxidase subunits in response to PLA and PLGA compared with that of PCL. Flow cytometric analysis of fluorescein isothiocyanate-Annexin V and propium iodide-stained PLA and PGLA polymer-exposed THP-1 cells showed early and late apoptotic changes. Similarly, exposure to the PLA and PGLA polymers, but not to the PCL polymer, resulted in enhanced staining for cleaved poly(ADP-ribose) polymerase-1, a protein fragment produced by caspase cleavage. These results indicate that biodegradable polymers are associated with cell adhesion, NADPH oxidase-induced generation of reactive oxygen species and excess apoptosis.

Peroxisome proliferator-activated receptor gamma and early folliculogenesis during an acute hyperandrogenism condition

Acute hyperandrogenism decreases serum P levels and induces early apoptosis of antral follicles by a mechanism mediated by the peroxisome proliferator-activated receptor gamma system and independent of the steroidogenic acute regulator protein.

DYRK1A and DYRK3 Promote Cell Survival through Phosphorylation and Activation of SIRT1

DYRK1A (the dual specificity tyrosine phosphorylation-regulated kinase 1A) plays an important role in body growth and brain physiology. Overexpression of this kinase has been associated with the development of Down syndrome in both human and animal models, whereas single copy loss-of-function of DYRK1A leads to increased apoptosis and decreased brain size. Although more than a dozen of DYRK1A targets have been identified, the molecular basis of its involvement in neuronal development remains unclear. Here we show that DYRK1A and another pro-survival member of the DYRK family, DYRK3, promote cell survival through phosphorylation and activation of SIRT1, an NAD(+)-dependent protein deacetylase that is essential in a variety of physiological processes including stress response and energy metabolism. DYRK1A and DYRK3 directly phosphorylate SIRT1 at Thr(522), promoting deacetylation of p53. A SIRT1 phosphorylation mimetic (SIRT1 T522D) displays elevated deacetylase activity, thus inhibiting cell apoptosis. Conversely, a SIRT1 dephosphorylation mimetic (SIRT1 T522V) fails to mediate DYRK-induced deacetylation of p53 and cell survival. We show that knockdown of endogenous DYRK1A and DYRK3 leads to hypophosphorylation of SIRT1, sensitizing cells to DNA damage-induced cell death. We also provide evidence that phosphorylation of Thr(522) activates SIRT1 by promoting product release, thereby increasing its enzymatic turnover. Taken together, our findings provide a novel mechanism by which two anti-apoptotic DYRK members promote cell survival through direct modification of SIRT1. These findings may have important implications in understanding the molecular mechanisms of tumorigenesis, Down syndrome, and aging.

Magnetic Fe3O4 nanoparticles and chemotherapy agents interact synergistically to induce apoptosis in lymphoma cells

The purpose of this study was to investigate the potential effects of combination therapy using magnetic nanoparticles of Fe3O4 (MNP-Fe3O4) and chemotherapeutic drugs on lymphoma cells. Proliferation, inhibition, and viability of Raji cells were detected by MTT and trypan blue exclusion. The percentage of cells undergoing apoptosis was detected by flow cytometry using fluorescein isothiocyanate-annexin V and propidium iodide staining. p53 and nuclear factor-κB (NF-κB) protein levels were measured by Western blot. The results showed that proliferation of Raji cells was inhibited by adriamycin or daunorubicin in a dose-and time-dependent manner. Cell sensitivity was improved and the 50% inhibitory concentrations of adriamycin and daunorubicin decreased when combined with a MNP-Fe3O4 carrier. Interestingly, increased apoptosis in Raji lymphoma cells was accompanied by upregulation of p53 protein and downregulation of NF-κB protein. Furthermore, the combination of MNP-Fe3O4 with adriamycin or daunorubicin increased p53 protein levels and decreased NF-κB protein levels more than adriamycin or daunorubicin alone, indicating that MNP-Fe3O4 could enhance the effect of chemotherapeutic drugs on p53 and NF-κB. Similar results for cell apoptosis and protein expression were not observed for the groups treated with dexamethasone ± MNP-Fe 3O4 (P > 0.05). These findings suggest a potential clinical application for MNP-Fe3O4 in combination with daunorubicin or adriamycin in the treatment of lymphoma.

Effects of Ethanol on Mouse Embryonic Stem Cells

Fetal alcohol syndrome (FAS) reflects a constellation of congenital abnormalities caused by excess maternal consumption of alcohol. It is likely that interference with embryonic development plays a role in the pathogenesis of the disorder. Ethanol-induced apoptosis has been suggested as a causal factor in the genesis of FAS. Mouse embryonic stem (mES) cells are pluripotent cells that differentiate in vitro to cell aggregates termed embryoid bodies (EBs), wherein differentiation capacity and gene expression profile are similar to those of the early embryo. To investigate the effects of ethanol during differentiation, mES cells were cultured on a gelatin surface in the presence of leukemia inhibitory factor which maintains adherent undifferentiated cells or in suspension to promote formation of EBs. All cells were treated (1-6 days) with 80 mM ethanol. The pluripotency and differentiation of mES cells were evaluated by western blotting of stage-specific embryonic antigen (SSEA-1), transcription factors Oct-3/4, Sox-2, and Nanog, using alkaline phosphatase staining. Apoptosis (early to late stages) was assessed by fluorescence-activated cell sorting using TdT-mediated biotin-dUTP nick-end labelling assay and fluorescein isothiocyanate-Annexin V/propidium iodide staining. Ethanol increased apoptosis during in vitro differentiation of mES cells to EBs, whereas undifferentiated cells were not affected. Ethanol exposure also interfered with pluripotency marker patterns causing an upregulation of SSEA-1 under self-renewal conditions. In EBs, ethanol delayed the downregulation of SSEA-1 and affected the regulation of transcription factors during differentiation. Our findings suggest that ethanol may contribute to the pathogenesis of FAS by triggering apoptotic pathways during differentiation of embryonic stem cells and deregulating early stages of embryogenesis.

Low-intensity ultrasound adjuvant therapy: enhancement of doxorubicin-induced cytotoxicity and the acoustic mechanisms involved.

In this study, the effects of low-intensity pulsed ultrasound (LIU) as an adjuvant to doxorubicin (DOX) treatment was further investigated in comparison to hyperthermia as another widely used adjuvant. The effects were compared with respect to cell killing and apoptosis induction in U937 cells. Human primary liver cancer (PLC) cells were also used to evaluate the effects of the combinations. The use of an echo contrast agent was investigated for further enhancement of cytotoxicity. Finally, the acoustic mechanisms involved were investigated. The effects of different treatment regimens on cell viability were determined using the Trypan blue dye-exclusion test. Apoptosis induction was detected by flow cytometry using fluorescein isothiocyanate-annexin V and propidium iodide staining. The mechanistic study involved electron paramagnetic spin trapping for detecting free radical formation as an indicator of the occurrence of inertial cavitation and spectrophotometry for sucrose hydrolysis as an indicator for noncavitational effects. The combination treatments exerted synergistic effects on cytotoxicity depending on the acoustic conditions used. The use of LIU as an adjuvant to DOX treatment was shown to be superior to the use of hyperthermia as an adjuvant. Moreover, the combination seems to be promising for other cancer types provided that the acoustic conditions are properly selected with respect to drug concentration. The key ultrasound mechanism responsible for the synergism observed was shown to be the production of free radicals by inertial cavitation. Non-cavitational forces were also shown to contribute to the effect. This study is motivating to engage in in vivo research with various cancer types as a step toward clinical applicability and is emphasizing on the importance of developing therapeutic protocols for setting LIU parameters with respect to other therapeutic conditions.

Involvement of Protective Autophagy in TRAIL Resistance of Apoptosis-defective Tumor Cells

Targeting TRAIL receptors with either recombinant TRAIL or agonistic DR4- or DR5-specific antibodies has been considered a promising treatment for cancer, particularly due to the preferential apoptotic susceptibility of tumor cells over normal cells to TRAIL. However, the realization that many tumors are unresponsive to TRAIL treatment has stimulated interest in identifying apoptotic agents that when used in combination with TRAIL can sensitize tumor cells to TRAIL-mediated apoptosis. Our studies suggest that various apoptosis defects that block TRAIL-mediated cell death at different points along the apoptotic signaling pathway shift the signaling cascade from default apoptosis toward cytoprotective autophagy. We also obtained evidence that inhibition of such a TRAIL-mediated autophagic response by specific knockdown of autophagic genes initiates an effective mitochondrial apoptotic response that is caspase-8-dependent. Currently, the molecular mechanisms linking disabled autophagy to mitochondrial apoptosis are not known. Our analysis of the molecular mechanisms involved in the shift from protective autophagy to apoptosis in response to TRAIL sheds new light on the negative regulation of apoptosis by the autophagic process and by some of its individual components.

Direct Effect of Human Immunodeficiency Virus Protease Inhibitors on Neutrophil Function and Apoptosis via Calpain Inhibition

Impairment of neutrophil functions and high levels of apoptotic neutrophils have been reported in human immunodeficiency virus (HIV) patients. The aim of the present study was to investigate the direct in vitro effects of the different HIV protease inhibitors (PIs) on neutrophil functions and apoptosis and to explore their mechanisms of action. The effects of nelfinavir (NFV), saquinavir (SQV), lopinavir (LPV), ritonavir (RTV), and amprenavir (APV) in the range of 5 to 100 microg/ml on neutrophil function, apoptosis, and mu-calpain activity were studied. The neutrophil functions studied included superoxide production stimulated by 5 ng/ml phorbol myristate acetate, 5 x 10(-7) M N-formyl-methionyl-leucyl-phenylalanine, and 1 mg/ml opsonized zymosan; specific chemotaxis; random migration; and phagocytosis. Apoptosis was determined by DNA fragmentation, fluorescein isothiocyanate-annexin V binding, and nuclear morphology. All three neutrophil functions, as well as apoptosis, were similarly affected by the PIs. SQV and NFV caused marked inhibition and LPV and RTV caused moderate inhibition, while APV had a minor effect. mu-Calpain activity was not affected by the PIs in neutrophil lysate but was inhibited after its translocation to the membranes after cell stimulation. SQV, which was the most potent inhibitor of neutrophil functions and apoptosis, caused significant inhibition of calpain activity, while APV had no effect. The similar patterns of inhibition of neutrophil functions and apoptosis by the PIs, which coincided with inhibition of calpain activity, suggest the involvement of calpain activity in the regulation of these processes.

Functional Linkage between NOXA and Bim in Mitochondrial Apoptotic Events

NOXA is a BH3-only protein whose expression is induced by certain p53-depenent or independent apoptotic stimuli. Both NOXA and Bim are avid binders of Mcl-1, but a functional linkage between these BH3-only proteins has not yet been reported. In this study, we demonstrate that Mcl-1 binding of endogenously induced NOXA interferes with the ability of Mcl-1 to efficiently sequester endogenous Bim, as Bim is displaced from its complex with Mcl-1. Induced NOXA significantly enhances the UV sensitivity of cells, and the ensuing mitochondrial depolarization is entirely abrogated by Bim knockdown. These results demonstrate a Mcl-1-mediated cross-talk between endogenous NOXA and Bim that occurs upstream of the Bak/Bax-dependent execution of UV-induced mitochondrial depolarization. The current findings demonstrate that the mitochondrial response to an induced expression of NOXA is executed by endogenous Bim and suggest a plausible mechanism for the observed NOXA-Bim linkage.

Macrophage-mediated Bystander Effect Triggered by Tumor Cell Apoptosis

Restoration of apoptosis is an important therapeutic strategy for cancer, but bystander effects may be crucial to treatment success. We examined the involvement of bystander effects in the outcome of pro-apoptotic treatments and investigated the role of macrophages. Using a murine N202 breast cancer chamber model and intravital microscopy, we observed bystander apoptosis in vivo in mixed spheroids consisting of bystander N202 cells plus modified N202 cells overexpressing the p14ARF N-terminal region, which promotes p53-mediated apoptosis. The effect was not observed in cocultures in vitro, and could not be transferred through conditioned medium from modified N202 cells. However, if macrophages were also included in the N202 co-cultures, bystander apoptosis was restored, and correlated with elevated surface expression of phosphatidyl serine, a macrophage recognition molecule, on the modified N202 cells. Bystander killing was not observed in cocultures of N202 cells plus macrophages plus cisplatin-treated or 5-fluorouracil-treated N202 cells, where apoptosis induction in the target cell population was inefficient, suggesting that specific activation of macrophages by apoptotic tumor cells was required. The results suggest that pro-apoptotic therapies benefit both from the intrinsic vulnerability of cancer cells to apoptosis and from an innate immune response that amplifies the therapeutic effect.

The ESAT6 protein of Mycobacterium tuberculosis induces apoptosis of macrophages by activating caspase expression

The secreted Mycobacterium tuberculosis protein, ESAT6, has been studied extensively in pathogenicity and vaccine experiments. Despite these studies little is known about the function of this protein. In this report, we demonstrate that ESAT6 induces apoptosis in THP-1 human macrophages using fluorescein isothiocyanate-Annexin V and intracellular caspase staining. We show that the induction of apoptosis by ESAT6 is dependent on the dose of the protein and the expression of caspase genes. Using real-time RT-PCR, we found that expression of caspase-1, -3, -5, -7 and -8 genes was upregulated in cells treated with ESAT6 relative to untreated cells. Furthermore, we show that while infection of THP-1 cells with wild-type M. tuberculosis strain H37Rv resulted in significant apoptosis 48 h post infection, a deletion mutant that does not express ESAT6 failed to induce significant apoptosis. Finally, experimental results using a cell impermeable fluorescent stain suggests that the formation of membrane pores may be a primary mechanism by which ESAT6 evokes an apoptotic response.