Double Staining Assay Research Articles

Simvastatin Induces Apoptosis in Castrate Resistant Prostate Cancer Cells by Deregulating Nuclear Factor-κB Pathway

In castrate resistant prostate cancer cells we investigated the cytotoxic effect of simvastatin and the mechanism involved. After treating PC3 and DU-145 cells with simvastatin, cell viability and apoptosis were determined using tetrazolium salt based colorimetric assay and annexin-V-fluorescein isothiocyanate/propidium iodide double staining assay, respectively. To determine whether simvastatin affects the nuclear factor-κB pathway, we assessed IκBα and phosphorylated IκBα expression, and p65 and phosphorylated p65 subcellular localization by Western blot analysis. Also, changes in nuclear factor-κB transcriptional activity were assessed using a luciferase reporter assay. After treating PC3 and DU-145 cells with 0, 20 or 40 μM simvastatin for 24, 48 or 72 hours, the proportion of viable cells decreased and the proportion of apoptotic cells increased in a dose and time dependent manner. Western blot analysis showed that simvastatin inhibited IκBα phosphorylation and degradation. It also demonstrated that simvastatin increased p65 protein levels in cytoplasmic fractions and decreased phosphorylated p65 protein levels in nuclear fractions but did not change p65 protein levels in cytoplasm. Luciferase reporter assay showed that simvastatin dose dependently reduced nuclear factor-κB activity. Reverse transcriptase-polymerase chain reaction and Western blot revealed that simvastatin inhibited nuclear factor-κB regulated cIAP-1 and 2, cFLIP-S and XIAP expression in dose and time dependent fashion. Simvastatin inhibited castrate resistant prostate cancer cell growth by inducing apoptosis. These effects were probably mediated by the inhibition of IκBα phosphorylation and nuclear translocation of p50/p65 dimer in the nuclear factor-κB pathway.

HYPOCHLOROUS ACID, A MACROPHAGES PRODUCT, INDUCES ENDOTHELIAL APOPTOSIS: THE ROLE OF ENDOPLASMIC RETICULUM STRESS

ObjectivesSuperficial erosion of coronary plaques due to endothelial loss causes acute coronary syndromes (ACS). Hypochlorous acid (HOCl), a macrophages product can induce endothelialapoptosis. Disturbing (ER) function results in ER stress...

THE ROLES OF PRESENILIN-2 GENE IN THE APOPTOSIS OF MYOCARDIAL CELL

ObjectivesPresenilin (PS) gene is a novel gene family of causative gene related with familial Alzheimer's disease. They have been detected not only in the brain, but also in some peripheral...

Synthesis and bioevaluation of novel arylnaphthalene lignans as anticancer agents

Novel arylnaphthalene lignans were synthesized and their structures were established by 1H NMR, 13C NMR, and HRMS. These compounds were evaluated for their in vitro cytotoxicity against cancer cell lines by MTT assay. Compound 5d possessed the highest cytotoxicity against KB cells. Apoptosis of KB cells treated with 5d was observed by acridine orange and ethidium bromide double staining assay. Western blot analysis disclosed that 5d induced apoptosis via mitochondrial pathway accompanied by an increased expression of Bax and a decreased expression of Bcl-2.

Inhibitory effect of selaginellin on high glucose-induced apoptosis in differentiated PC12 cells: Role of NADPH oxidase and LOX-1

Hyperglycemia clearly plays a key role in the development and progression of diabetic neuropathy. Hyperglycemia induces oxidative stress to generate reactive oxygen species in diabetic neurons resulting in neuronal damage and dysfunction. Apoptosis has been proposed as a possible mechanism for high glucose-induced neural dysfunction and neuronal cell injury. High glucose per se enhances lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) expression via activation of NADPH oxidase/reactive oxygen species pathway in endothelial cells. Selaginellin, a component extracted from Saussurea pulvinata (Hook. et Grev.) Maxim, was assessed for its ability to protect rat pheochromocytoma (PC12) cells against oxidative toxicity induced by high glucose. The differentiated PC12 cells were pretreated with various concentrations (10−7, 3×10−7 or 10−6M) of selaginellin for 1h and then co-treated with selaginellin and d-glucose (75mM) for 72h. Selaginellin was shown to protect differentiated PC12 cells against high glucose toxicity, as determined by characteristic morphological features, cell viability, and apoptosis as evaluated by Hoechst 33,258 staining assay, annexin V-propidium iodide double staining assay and caspase-3 activity. In addition, the increase in NADPH oxidase activity, mRNA expression of NADPH oxidase subunits (NOX-1 and NOX-2) and LOX-1, and reactive oxygen species production induced by high glucose were significantly inhibited by selaginellin or by anti-LOX-1 antibody. The present study demonstrated that inhibitory effect of selaginellin on high glucose-induced cell injury and apoptosis in differentiated PC12 cells is related to inhibition of LOX-1/NADPH oxidase–reactive oxygen species/caspase-3 signaling pathway.

Experimental study of IL-17 in mice infected with Mycoplasma suis

At present, only a few studies are available for the diagnosis of the T help cell 17(Th-17) about Mycoplasma suis. The objective of this study was to investigate the variation of interleukin-17(IL-17) in peripheral blood and spleen of M. suis-infected BALB/c mice. The variation of IL-17 were detected by using the method of ELISA, RT-PCR, Immunofluorescence double staining assays after infecting 1, 3, 5, 7 and 9 days. The results showed that after one day, three days, five days, seven days, nine and days the expression of IL-17 of mice infected with M. suis was respectively and significantly higher than the control group (P < 0.05). Five days later, IL-17 concentration raised to the highest point (P < 0.05). In conclusion, IL-17 concentration of BALB/c mice increased, and raised to the highest point at the fifth day. The results assist scientific data for function of Th-17 cells in the occurrence, development and turnover of M. suis and provided a theoretical basis for clinical immunotherapy.

Corynebacterium diphtheriae 67-72p hemagglutinin, characterized as the protein DIP0733, contributes to invasion and induction of apoptosis in HEp-2 cells

Although Corynebacterium diphtheriae has been classically described as an exclusively extracellular pathogen, there is growing evidence that it may be internalized by epithelial cells. The aim of the present report was to investigate the nature and involvement of the surface-exposed non-fimbrial 67–72 kDa proteins (67-72p), previously characterized as adhesin/hemagglutinin, in C. diphtheriae internalization by HEp-2 cells. Transmission electron microscopy and bacterial internalization inhibition assays indicated the role of 67-72p as invasin for strains of varied sources. Cytoskeletal changes with accumulation of polymerized actin in HEp-2 cells beneath adherent 67-72p-adsorbed microspheres were observed by the Fluorescent actin staining test. Trypan blue staining method and Methylthiazole tetrazolium reduction assay showed a significant decrease in viability of HEp-2 cells treated with 67-72p. Morphological changes in HEp-2 cells observed after treatment with 67-72p included vacuolization, nuclear fragmentation and the formation of apoptotic bodies. Flow cytometry revealed an apoptotic volume decrease in HEp-2 cells treated with 67-72p. Moreover, a double-staining assay using Propidium Iodide/Annexin V gave information about the numbers of vital vs. early apoptotic cells and late apoptotic or secondary necrotic cells. The comparative analysis of MALDI-TOF MS experiments with the probes provided for 67-72p CDC-E8392 with an in silico proteome deduced from the complete genome sequence of C. diphtheriae identified with significant scores 67-72p as the protein DIP0733. In conclusion, DIP0733 (67-72p) may be directly implicated in bacterial invasion and apoptosis of epithelial cells in the early stages of diphtheria and C. diphtheriae invasive infection.

Over-expression of uPA increases risk of liver injury in pAAV-HBV transfected mice

To investigate the relationship between over-expression of urokinase plasminogen activator (uPA) and hepatitis B virus (HBV) related liver diseases in a transgenic mouse model. Albumin-tetracycline reverse transcriptional activator and tetO-uPA transgenic mice were generated respectively through pronuclear injection and crossed to produce the double transgenic in-alb-uPA mice, for which doxycycline (Dox)-inducible and liver-specific over-expression of uPA can be achieved. Hydrodynamic transfection of plasmid adeno-associated virus (AAV)-1.3 HBV was performed through the tail veins of the Dox-induced in-alb-uPA mice. Expression of uPA and HBV antigens were analyzed through double-staining immunohistochemical assay. Cytokine production was detected by enzyme linked immunosorbent assay and α-fetoprotein (AFP) mRNA level was evaluated through real-time quantitative polymerase chain reaction. Plasmid AAV-1.3 HBV hydrodynamic transfection in Dox-induced transgenic mice not only resulted in severe liver injury with hepatocarcinoma-like histological changes and hepatic AFP production, but also showed an increased serum level of HBV antigens and cytokines like interleukin-6 and tumor necrosis factor-α, compared with the control group. Over-expression of uPA plays a synergistic role in the development of liver injury, inflammation and regeneration during acute HBV infection.

Effects of Chicken Selenoprotein W on H2O2-Induced Apoptosis in CHO-K1 Cells

Selenoprotein W (SelW) is expressed in various tissues of many animals and acts as an oxidoreductase in mammals. However, little is known about the role of the SelW in birds. To investigate the role of the chicken SelW on H(2)O(2)-induced apoptosis in CHO-K1 cells, overexpression of a chicken SelW cell lines (CHO-K1/SelW) were constructed. Using acridine orange/ethidium bromide (AO/EB) double staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays, as well as WST-1 cell viability assay, we monitored the extent of the H(2)O(2)-induced apoptosis and detected the abundance of the caspase-3, caspase-8, and fas mRNA by real-time quantitative reverse transcription PCR (qPCR). We here found that overexpression of SelW cells, compared with the wild-type cells, resulted in a markedly decrease in sensitivity to H(2)O(2)-induced oxidative stress and had a lower apoptotic cell death in AO/EB and TUNEL assays. Cell viability revealed that overexpression of SelW cells had higher cell viability than wild-type cells. qPCR results found that overexpression of SelW cells had a lower levels of caspase-3, caspase-8, and fas mRNA than wild-type cells. Taken together, our findings suggested that SelW could reduce the oxidative damage induced by H(2)O(2) and had an important protective function in against oxidative damage.

Anticancer effect and apoptosis induction by quercetin in the human lung cancer cell line A-549

The aim of the present study was to investigate the anticancer effect of quercetin (QC) in the human lung cancer cell line A-549 and further study the mechanism of apoptosis induction by QC. Low differentiation potential A-549 human lung cancer cells were treated with QC at different doses and for different times, and the growth inhibitory rates were detected by MTT assay. Apoptosis induced by QC in A-549 cells was observed by Annexin V/PI double staining and flow cytometric assay. The relative tumor growth ratio of the treated/control tumors (T/C) (%) was chosen to represent the tumor growth inhibition of A-549 cell nude mouse xenografts by QC. Apoptosis of the nude mouse xenografts was observed by Annexin V/PI double staining and flow cytometric assay and DNA fragmentation assay. To further determine the molecular mechanism of apoptosis induced by QC, changes in the expression of bcl-2 and bax genes were detected by RT-PCR. Following incubation with QC, the cell growth of the low differentiation potential A-549 human lung cancer cells was dramatically inhibited in a dose-dependent manner. After the cells were exposed to QC for 24, 48 and 72 h, the IC50 value was 1.02 ± 0.05, 1.41 ± 0.20 and 1.14 ± 0.19 µmol/l, respectively. Apoptosis in the A-549 cells induced by QC was noted. The apoptotic subpopulation of A-549 cells was approximately 12.96 and 24.58%, respectively, when cells were incubated with 1.2 µmol/l QC for 48 and 72 h. T/C (%) of A-549 nude mouse xenografts was 44.3, when the nude mice were treated with QC (8 mg/kg). Meanwhile, apoptosis induced by QC was observed in the A-549 nude mouse xenografts. Increased expression of the bax gene and decreased expression of the bc1-2 gene were noted using RT-PCR. Our results provide further evidence of the growth inhibition of the A-549 human lung adenocarcinoma cancer cell line by QC. This effect is associated with the induction of apoptosis in A-549 cells and the molecular mechanism may be related to the reduction in expression of the apoptosis-regulating gene bcl-2, and increase in expression of the apoptosis-regulating gene bax. These results were also confirmed in vivo.

Effects of nicotine on proliferation and osteoblast differentiation in human alveolar bone marrow-derived mesenchymal stem cells

Aims Nicotine is a risk factor for various diseases, including osteoporosis, oral cancer, and periodontal disease. Numerous studies have elucidated the effects of nicotine on cell proliferation and differentiation. The purpose of this study was to determine the effects of nicotine on the proliferation and osteoblast differentiation of human alveolar bone marrow-derived mesenchymal stem cells (hABMMSCs). Main methods In this study, we treated hABMMSCs with different doses (1 μM to 5 mM) of nicotine. The survival and proliferation of hABMMSCs were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay and crystal violet assay. TUNEL and propidium iodide (PI) double staining assay were also performed. The effect of nicotine on osteoblast differentiation of hABMMSCs was determined by measuring calcium accumulation using alizarin red-sulfate (AR-S) staining, measurement of alkaline phosphatase (ALP) activity, and semi-quantitative PCR of osteoblast markers. Key findings The survival and proliferation of hABMMSCs did not differ when they were exposed to nicotine at concentrations ranging from 1 μM to 100 μM; however, cell proliferation increased when the cells were exposed to nicotine at concentrations of 1–2 mM and decreased significantly when exposed to 5 mM of nicotine. A number of cells were stained by PI but not by TUNEL, and membrane vacuolization was observed in hABMMSCs treated with 5 mM nicotine. Calcium accumulation; ALP activity; and mRNA levels of ALP, bone sialoprotein (BSP), collagen type I α 1 (Col1αI), and runt-related transcription factor 2 (Runx2) were significantly decreased by treatment with 2 mM of nicotine, while osteocalcin transcripts decreased by treatment with 1 to 2 mM of nicotine. Significance These results suggest that nicotine has a bimodal effect on the proliferation and osteoblast differentiation in hABMMSCs.

Reactive oxygen species–mitochondria pathway involved in FV-429-induced apoptosis in human hepatocellular carcinoma HepG2 cells

FV-429 is a newly synthesized flavonoid with a bis(2-hydroxyethyl) amino propoxy substitution. In this study, we investigate the anticancer effect of FV-429 both in vivo and in vitro. These data have shown that FV-429 could significantly inhibit tumor growth in mice inoculated with Heps hepatoma cells without evident toxicity. After the treatment of FV-429 (40 mg/kg), the inhibitory rate of tumor weight was 52.12%. Then, we performed diamidinophenylindole staining and annexin V/propidium iodide double-staining assay to investigate the apoptosis induced by FV-429 in HepG2 cells. Further research revealed that FV-429 induced apoptosis through the mitochondrial apoptotic pathway, as indicated by a change in Bax/Bcl-2 ratios, collapse of mitochondrial membrane potential, the transposition of apoptotic-inducing factor and cytochrome c, caspase-3 and caspase-9 activation, and degradation of poly (ADP-ribose) polymerase. The accumulation of reactive oxygen species induced by FV-429 in HepG2 cells was also observed. Moreover, the mitogen-activated protein kinases, the downstream effect of reactive oxygen species accumulation including c-Jun N-terminal kinase and p38 mitogen-activated protein kinases, could be activated by FV-429. Taken together, our results provided a mechanistic framework for further exploration of FV-429 as a novel chemotherapy for human tumors.

Differential cytotoxic responses to low- and high-dose photodynamic therapy in human gastric and bladder cancer cells

Here, we present differential cytotoxic responses to two different doses of photodynamic therapies (PDTs; low-dose PDT [LDP] and high-dose PDT [HDP]) using a chlorin-based photosensitizer, DH-II-24, in human gastric and bladder cancer cells. Fluorescence-activated cell sorting analysis using Annexin V and propidium iodide (PI) showed that LDP induced apoptotic cell death, whereas HDP predominantly caused necrotic cell death. The differential cytotoxic responses to the two PDTs were further confirmed by a DiOC(6) and PI double-staining assay via confocal microscopy. LDP, but not HDP, activated caspase-3, which was inhibited by Z-VAD, Trolox, and BAPTA-AM. LDP and HDP demonstrated opposite effects on intracellular reactive oxygen species (ROS)/Ca(2+) signals; LDP stimulated intracellular ROS production, contributing to a transient increase of intracellular Ca(2+) , whereas HDP induced a massive and prolonged elevation of intracellular Ca(2+) responsible for the transient production of intracellular ROS. In addition, the two PDTs also increased in situ transglutaminase 2 (TG2) activity, with a higher stimulation by HDP, and this increase in activity was prevented by Trolox, BAPTA-AM, and TG2-siRNA. LDP-induced apoptotic cell death was strongly inhibited by Trolox and TG2-siRNA and moderately suppressed by BAPTA-AM. However, HDP-mediated necrotic cell death was partially inhibited by BAPTA-AM but not by TG2-siRNA. Thus, these results demonstrate that LDP and HDP induced apoptotic and necrotic cell death by differential signaling mechanisms involving intracellular Ca(2+) , ROS, and TG2.

Triptolide-induced apoptosis by inactivating nuclear factor-kappa B apoptotic pathway in multiple myeloma in vitro

The effect of triptolide on proliferation and apoptosis of human multiple myeloma RPMI-8226 cells in vitro, as well as the roles of nuclear factor-kappa B (NF-κB) and IκBα was investigated. The effect of tritptolide on the growth of RPMI-8226 cells was studied by MTT assay. Apoptosis was detected by Hoechest 33258 staining and Annexin V/PI double staining assay. The expression of NF-κB and IκBα was observed by Western blot and confocal microscopy. The results showed that triptolide inactivated NF-κB apoptotic pathway in human multiple myeloma RPMI-8226 cells. Triptolide at nM range induced proliferation inhibition in a dose- and time-dependent manner and apoptosis in a dose-dependent fashion in RPMI-8226 cells. Besides, we observed the inhibition of NF-κB /p65 in the nuclear fraction was correlated with the increase in the protein expression of IκBα in the cytosol. These results suggested that triptolide might exhibit its strong anti-tumor effects via inactivation of NF-κB/p65 and IκBα.

Effect of miR-221 on the viability and apoptosis of hepatocellular carcinoma HepG2 cells

To investigate the effect of microRNA-221 (miR-221) on cell viability and apoptosis of hepatocellular carcinoma HepG2 cells. MiR-221 inhibitors and mimics were transfected into HepG2 cells. The expression of miR-221 was detected by real time quantitative RT-PCR. CellTiter-blue cell viability kit, Hoechst 33342/propidium iodide (PI) double staining assay, flow cytometry and Apo-ONE homogeneous caspase-3/7 kit were used to measure cell viability and apoptosis. MiR-221 inhibitors significantly inhibited the cell growth and miR-221 mimics increased cell viability 48 hours post-transfection measured by both CellTiter-blue cell viability kit and Hoechst 33342/PI assay (P is less than to 0.05). There was a positive correlation between these two assays (r = 0.993, P is less than to 0.01). With miR-221 mimics, the number of G1 stage cells (47.67%+/-1.53%) was significantly reduced as compared to the blank control (59.00%+/-1.00%) and the negative control (58.00%+/-1.00%, F = 81.77, P is less than to 0.01), and it was significantly raised in S stage (20.33%+/-1.15%) than in blank control (11.00%+/-1.00%) and negative control (12.00%+/-1.00%, F = 70.9, P is less than to 0.01) with flow cytometry analysis. More cell apoptosis and necrosis were significantly induced by miR-221 inhibitors 48 hours post-transfection detected by both Hoechst 33342/PI assay and flow cytometry PE Annexin V kit (P is less than to 0.05). The result from Apo-ONE homogeneous caspase-3/7 kit was consistent with the above two apoptotic assays, which showed that with miR-221 inhibitors, the activity of caspase-3/7 was significantly enhanced (P is less than to 0.05). MiR-221 contributes to the growth of hepatocellular carcinoma cells and miR-221 inhibition can induce cell apoptosis. miR-221 has the potential to become one of the new molecular targets for liver cancer therapy.

The extract of Paris polyphylla exerts apoptotic induction and synergic antiproliferative effect with anticancer drugs in SMMC-7721 human liver cancer cells

Paris polyphylla is a famous traditional Chinese medicine with anti-inflammatory and hematischesis properties and has been shown recently to possess anticancer activities. However, the molecular mechanism underlying the anticancer effect of the herb is poorly understood. In this study, its ability to induce apoptosis and exert a synergistic antiproliferative effect by a combination with 5-fluorouracil (5-Fu) and Oxaliplatin (Oxa) were investigated in human SMMC-7721 liver cancer cells. Our findings have shown that the 95% ethanol extract of Paris polyphylla (PPE) inhibited the proliferation of SMMC-7721 cells dose- and time-dependently, as shown by loss of cell viability. The apoptotic induction was determined by Annexin-V/PI double staining assay, apoptotic body and swollen mitochondria observed under electron microscope, and breakdown of mitochondrial membrane potentials detected by JC-1 staining. Moreover, apoptosis in the SMMC-7721 cells was accompanied by S phase cell cycle arrest, the activation of pro-caspase 3, and a marked increase in Bax/Bcl-2 ratios due to upregulation of Bax and downregulation of Bcl-2 proteins expression by western blotting. In addition, we have observed that PPE increased the inhibitory effect of 5-Fu and Oxa on the proliferation of SMMC-7721 cells in vitro using Media-effect analysis. This combination regimen was more potent than either agent alone. Furthermore, the mRNA levels of thymidylate synthase (TS) and human excision repair cross-complementing (ERCC1) were suppressed when incubation with PPE, indicating that the effect of PPE on TS and ERCC1 mRNA levels at least partly caused the synergic effect. Overall, these results offer a potential mechanism for PPE-induced apoptosis and its synergic effect with anticarcinogenic drugs in SMMC-7721 cancer cells and indicate Paris polyphylla as a promising herb for chemoprevention and chemotherapy of hepotoma.

Expansion of Intronic GGCCTG Hexanucleotide Repeat in NOP56 Causes SCA36, a Type of Spinocerebellar Ataxia Accompanied by Motor Neuron Involvement

Autosomal-dominant spinocerebellar ataxias (SCAs) are a heterogeneous group of neurodegenerative disorders. In this study, we performed genetic analysis of a unique form of SCA (SCA36) that is accompanied by motor neuron involvement. Genome-wide linkage analysis and subsequent fine mapping for three unrelated Japanese families in a cohort of SCA cases, in whom molecular diagnosis had never been performed, mapped the disease locus to the region of a 1.8 Mb stretch (LOD score of 4.60) on 20p13 (D20S906-D20S193) harboring 37 genes with definitive open reading frames. We sequenced 33 of these and observed a large expansion of an intronic GGCCTG hexanucleotide repeat in NOP56 and an unregistered missense variant (Phe265Leu) in C20orf194, but we found no mutations in PDYN and TGM6. The expansion showed complete segregation with the SCA phenotype in family studies, whereas Phe265Leu in C20orf194 did not. Screening of the expansions in the SCA cohort cases revealed four additional occurrences, but none were revealed in the cohort of 27 Alzheimer disease cases, 154 amyotrophic lateral sclerosis cases, or 300 controls. In total, nine unrelated cases were found in 251 cohort SCA patients (3.6%). A founder haplotype was confirmed in these cases. RNA foci formation was detected in lymphoblastoid cells from affected subjects by fluorescence in situ hybridization. Double staining and gel-shift assay showed that (GGCCUG)n binds the RNA-binding protein SRSF2 but that (CUG)(6) does not. In addition, transcription of MIR1292, a neighboring miRNA, was significantly decreased in lymphoblastoid cells of SCA patients. Our finding suggests that SCA36 is caused by hexanucleotide repeat expansions through RNA gain of function.

Protective effects of early hypoxic post-conditioning in cultured cortical neurons

Primary objective: Recent evidence suggests that delayed hypoxic post-conditioning is neuroprotective. The aim of the present study was to test whether early post-conditioning applied immediately after hypoxia could protect cultured neurons from hypoxia/reoxygenation (H/R)-induced injuries.Methods: Primary cortical neuronal culture depleted of microglia was exposed to H/R. Post-conditioning started immediately after hypoxia and consisted of three cycles of 15-minutes of reoxygenation and 15-minutes of hypoxia. Cell viability assay was performed using Cell Counting Kit-8 (CCK-8). Apoptosis was evaluated by Hoechst 33258 staining, FITC-Annexin V/PI double staining and Western blot assay (testing the cleaved caspase-3 expression). Reactive oxygen species (ROS), intracellular Ca2+ and mitochondrial membrane potential (MMP) were examined using confocal laser-scanning microscopy.Main results: H/R significantly reduced cell viability and increased neuronal apoptosis and necrosis. Furthermore, the expression of cleaved caspase-3, ROS production and intracellular Ca2+ were increased. MMP was attenuated. Injuries induced by H/R were substantially attenuated by early hypoxic post-conditioning. Changes in cleaved caspase-3 expression, ROS production, intracellular Ca2+ level and MMP in response to H/R were significantly decreased by the post-conditioning.Conclusions: The findings demonstrated that early hypoxic post-conditioning could protect neurons against H/R-induced injuries independent of microglial cells, possibly by inhibiting ROS over-production and intracellular Ca2+ accumulation and maintaining MMP.

Abstract 1623: The spliceosome as a new therapeutic target for anticancer treatment

Abstract Background: Bioinformatics analyses of pathways that are differentially expressed between malignant and benign lesions could allow discovering new therapeutic targets. In the present study, we identified spliceosome assembly components as candidate target for cancer treatment. The spliceosome, an intracellular ribonucleoprotein complex, is involved in eukaryotic pre-mRNA processing by splicing out intronic nucleic acids. Experimental procedures: Pathway analyses were done on exon and gene expression arrays datasets. Differential gene expression between malignant and benign lesions was applied to four public datasets. Small nuclear ribonucleoprotein E (SNRPE) downregulation was achieved by short interfering RNA technology in SKBr-3 and MDA-MB-231 human breast cancer cells. Functional effects of siRNA-induced knockdown of SNRPE expression on cell viability, cell cycle distribution and apoptosis were respectively determined using WST-1 assay, FACS analysis and annexin-V/7-AAD double-staining assay. Autophagy was investigated by electron microscopy (EM). We also evaluated alternative RNA splicing at the exon level using SpliceArray microarray. Results: Pathway analyses with BioCarta database identified spliceosome assembly component (SAC) as the most differentially expressed pathway between breast malignant and benign lesions (permutation p=0.002). The differential expression of SAC was confirmed in four different public dataset on breast, ovarian, lung and skin cancers. Based on these data we investigated whether SNRPE, a component of spliceosome, could be a candidate target. Knockdown of SNRPE protein expression resulted in a high inhibition of SKBr-3 (70%) and MDA-MB-231 (48%) cell proliferation as compared to non-targeting siRNA-transfected cells. Splice array analyses suggested a deregulation of alternative splicing, together with a dramatic reduction of mTOR transcripts. mTOR downregulation, together with a decrease in phosphorylation of downstream protein in the mTOR pathway (i.e 4E-BP1) was confirmed by western blot analyses. We then investigated the mechanisms of cell death. siRNA-mediatd SNRPE knockdown was not found to induce typical apoptosis as annexin-V-positive cells were not detected. However, the presence of numerous vesicular organelle of autophagy revealed by EM is strongly amplified in SNRPE-transfected cells. This observed deregulation of the mTOR pathway in SNRPE-depleted cells might in part explain the induction of autophagy following SNRPE silencing. Conclusion : Our data report that specific inactivation of SNRPE protein expression induces mTOR downregulation and autophagy in cancer cell lines, suggesting that the spliceosome could represent a new potential target that may open new perspectives in cancer therapy. Molecular mechanisms that lead to mTOR dowregulation by SNRPE extinction are being investigated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1623. doi:10.1158/1538-7445.AM2011-1623

Group B Streptococcus serotypes III and V induce apoptosis and necrosis of human epithelial A549 cells

Although group B Streptococcus (GBS) has been classically described as an exclusively extracellular pathogen, growing evidence suggests that it may be internalized by epithelial cells. However, the fates of intracellular GBS and of infected respiratory epithelial cells remain unclear. Little is known about the bacterial components involved in these processes. The present study investigated the bacterial internalization by A549 cells and the apoptosis/necrosis of the infected human epithelial cells. The morphological changes in A549 cells observed from 2 h post-infection with GBS included vacuolization and the formation of apoptotic bodies. Flow cytometry revealed that 81.2% of apoptotic A549 cells were infected with GBS serotype III 90356-liquor. Moreover, a double-staining assay using propidium iodide (PI)/Annexin V (AV) gave information about the numbers of viable (PI-/AV-) (18.27%) vs. early apoptotic (PI-/AV+) (73.83%) and late apoptotic cells (PI+/AV+) (7.37%) during infection of A549 cells with GBS III 90356-liquor. In addition, 37% necrotic cells were observed in A549 cells infected with GBS serotype V 90186-blood. In conclusion, GBS serotypes III and V induce apoptosis of epithelial cells in the early stages of GBS infection, resulting in tissue destruction, bacterial spreading and, in consequence, invasive disease or systemic infection.