Alpha-lipoic acid ameliorates tauopathy-induced oxidative stress, apoptosis, and behavioral deficits through the balance of DIAP1/DrICE ratio and redox homeostasis: Age is a determinant factor.

Tauopathies belong to a heterogeneous class of neuronal diseases resulting in the metabolic disturbance. A disulfide natural compound of Alpha-Lipoic acid (ALA) has shown numerous pharmacologic, antioxidant, and neuroprotective activities under neuropathological conditions. The aim of this study was to investigate the neuroprotective effects of ALA on the tauopathy-induced oxidative disturbance and behavioral deficits. The transgenic Drosophila model of tauopathy induced by human tauR406W using GAL4/UAS system and effects of ALA (0.001, 0.005, and 0.025 % w/w of diet) on the neuropathology of tau in younger (20 days) and older (30 days) adults were investigated via biochemical, molecular, behavioral and in-situ tissue analyses. Expression of apoptosis-related proteins involving Drosophila Cyt-c-d (trigger of intrinsic apoptosis) and DrICE (effector caspase) were upregulated in both ages (20 and 30 days) and DIAP1 (caspase inhibitor) has reduced only in older model flies compared to the controls. Remarkably, all doses of ALA increased DIAP1 and glutathione (GSH) as well as reducing Cyt-c-d and lipid peroxidation (LPO) in the younger flies compared to the model flies. Moreover, the higher doses of ALA were able to decrease thiol concentrations, to increase total antioxidant capacity, and to improve the behavioral deficits (locomotor function, olfactory memory, and ethanol sensitivity) in the younger flies. On the other hand, only a higher dose of ALA was able to decrease DrICE, Cyt-c-d, LPO, and thiol as well as increasing antioxidant capacity and decreasing ethanol sensitivity (ST50, RT50) in the older flies. TUNEL assay showed that all doses of ALA could potentially increase the DIAP1/DrICE ratio and exert anti-apoptotic effects on younger, but not on the older adults. Furthermore, data obtained from the in-situ ROS assay confirmed that only a higher dose of ALA significantly decreased the ROS level at both ages. Our data showed that an effective neuroprotective dose of ALA and its mechanism of action on this model of tauopathy could potentially be influenced by longevity. Moreover, it was shown that ALA prevents apoptosis and decreases the redox homeostasis, and this partially explains the mechanism by which ALA diminishes behavioral deficits.

Non-apoptotic enteroblast-specific role of the initiator caspase Dronc for development and homeostasis of the Drosophila intestine.

The initiator caspase Dronc is the only CARD-domain containing caspase in Drosophila and is essential for apoptosis. Here, we report that homozygous dronc mutant adult animals are short-lived due to the presence of a poorly developed, defective and leaky intestine. Interestingly, this mutant phenotype can be significantly rescued by enteroblast-specific expression of dronc+ in dronc mutant animals, suggesting that proper Dronc function specifically in enteroblasts, one of four cell types in the intestine, is critical for normal development of the intestine. Furthermore, enteroblast-specific knockdown of dronc in adult intestines triggers hyperplasia and differentiation defects. These enteroblast-specific functions of Dronc do not require the apoptotic pathway and thus occur in a non-apoptotic manner. In summary, we demonstrate that an apoptotic initiator caspase has a very critical non-apoptotic function for normal development and for the control of the cell lineage in the adult midgut and therefore for proper physiology and homeostasis.

Combing metabolomics with bioanalysis methods to study the antitumor mechanism of the new acridone derivative 8q on CCRF-CEM cells: 8q induced mitochondrial-mediated apoptosis and targeted the PI3K/AKT/FOXO1 pathway.

A novel acridone derivative, N-(2-(dimethylamino)ethyl)-1-((3-methoxybenzyl)amino)-5- nitro-9-oxo-9,10-dihydro-acridine-4-carboxamide (8q), which was synthesized in our lab, showed potent anti-leukaemia activity against CCRF-CEM cells. Moreover, in silico predictions showed that 8q conformed to the rule of five and displayed low toxicity. However, the mechanism of anti-leukaemia action remains unclear. The aim of this research was to reveal the probable anti-leukaemia mechanism of 8q on CCRF-CEM cells. Flow cytometry assay demonstrated that 8q induced apoptosis. The expression of caspase family proteins results showed that 8q could only promote cleaved caspase-3, 7 and 9 expressions without affecting cleaved caspase-8 protein, hinting that 8q induced mitochondrial-mediated apoptosis. Further, we detected 3 indicators of mitochondrial lesions, including increased of Cyt-C release, with a decrease in MMP and ATP levels. Next, metabolomics were introduced to assist in the research of the anti-leukaemia mechanism of 8q. The metabolomics results showed that 100 nM 8q could increase the level of GSH, and decrease its oxidation products. These indicated 8q could influence the ROS, which derived by mitochondria. Then we examined the effect of 8q on intracellular ROS levels. What is particularly interesting is that 8q inhibited cell ROS stress at low concentration and stimulated ROS stress at high concentration. The pro-apoptosis mechanisms of 8q were then explored. 8q significantly decreased anti-apoptotic proteins Bcl-2 and Bcl-xL expression, whereas it up-regulated the pro-apoptotic proteins Bax, Bak, Bad, Bik and Puma expression. In addition, 8q dramatically inhibited the expression of FASN, which is related to fatty acid metabolism. Furthermore, PI3K, AKT and FOXO1 were inactivated, and the expression of total AKT was also inhibited by 8q treatment, which promoted intrinsic apoptosis. In conclusion, these findings demonstrate that 8q can induce mitochondrial lesions and promote mitochondrial-mediated pathway apoptosis by regulating the expression of Bcl-2 family proteins and inhibiting the activity of the PI3K/AKT/FOXO1 signaling pathway.

Endoribonuclease ENDU-2 regulates multiple traits including cold tolerance via cell autonomous and nonautonomous controls in Caenorhabditis elegans.

Environmental temperature acclimation is essential to animal survival, yet thermoregulation mechanisms remain poorly understood. We demonstrate cold tolerance in Caenorhabditis elegans as regulated by paired ADL chemosensory neurons via Ca2+-dependent endoribonuclease (EndoU) ENDU-2. Loss of ENDU-2 function results in life span, brood size, and synaptic remodeling abnormalities in addition to enhanced cold tolerance. Enzymatic ENDU-2 defects localized in the ADL and certain muscle cells led to increased cold tolerance in endu-2 mutants. Ca2+ imaging revealed ADL neurons were responsive to temperature stimuli through transient receptor potential (TRP) channels, concluding that ADL function requires ENDU-2 action in both cell-autonomous and cell-nonautonomous mechanisms. ENDU-2 is involved in caspase expression, which is central to cold tolerance and synaptic remodeling in dorsal nerve cord. We therefore conclude that ENDU-2 regulates cell type-dependent, cell-autonomous, and cell-nonautonomous cold tolerance.

Analysis the effect of hyperbaric oxygen preconditioning on neuronal apoptosis, Ca2+ concentration and caspases expression after spinal cord injury in rats.

OBJECTIVE: To investigate the effects of hyperbaric oxygen preconditioning (HBO-PC) on neuronal apoptosis, Ca2+ concentration, and Caspases expression after spinal cord injury (SCI) in rats. MATERIALS AND METHODS: A total of 36 rats were randomly divided into control group (CON group), hyperbaric oxygen preconditioning group (HBO-PC group) and spinal cord injury group (SCI group), with 12 rats in each group. Rats in group HBO-PC were given HBO-PC intervention before modeling. SCI model was established by modified Allen method in group HBO-PC and group SCI. Basso-Beattie-Bresnahan (BBB) locomotor rating scale and motor evoked potential (MEP) examination were used to assess the neurological function. The expression of apoptosis gene caspase (3, 7, 8, 12) mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). The concentration of Ca2+ in spinal cord tissue of each group was detected. RESULTS: CON group, HBO-PC group, and SCI group were gradually diminishing in BBB score and potential value and amplitude of MEP, respectively. The differences between groups were statistically significant (p<0.05). The expressions of Caspase-3 and 7, 8 and 12 mRNA in SCI group were significantly higher than those in CON group and HBO-PC group, respectively (p<0.05). There was no significant difference between CON group and HBO-PC group (p>0.05). The concentrations of Ca2+ in the CON group, HBO-PC group and SCI group were gradually increased; differences between groups were statistically significant (p<0.05). CONCLUSIONS: HBO-PC can reduce the loss of motor function of SCI rats, which may inhibit the activation of endoplasmic reticulum pathway of neural apoptosis, and reduce the calcium overload through inhibiting the expressions of pro-apoptotic proteins (Caspase-3/7/8/12), thus reducing the cell apoptosis and protecting neurons.

Apoptosis induction in human breast cancer cell lines by synergic effect of raloxifene and resveratrol through increasing proapoptotic genes.

AIMS: Breast cancer is the most common cancer of women. The aim of this study was to investigate the synergic effect of raloxifene (Ral) and resveratrol (Res) on apoptosis of breast cancer cell lines (MCF7 and MDA-MB-231). MAIN METHODS: Cells were treated with Ral and Res alone and in combination. Cell viability (MTT assay), apoptosis (TUNEL assay) and nitric oxide (NO) production (Griess method) were investigated. Expression of proapoptotic gene (Bax and p53), anti-apoptotic gene (Bcl2) and caspases-3, caspase -8 were evaluated. One-way ANOVA test was used for data analysis. KEY FINDINGS: The viabilities of MCF7 and MDA-MB-231 cells treated by Ral (1 µM) and Res (20 µM) decreased significantly (p = 0.000) and their synergic use showed more reduction. Nitric oxide production by MCF7 and MDA-MB-231 cells exposed upon each drug alone and in combination showing a significant reduction (p = 0.000). There was also an increase in apoptosis in the cells treated with combination use of Ral and Res in both cell lines. Moreover, reduced expression of Bcl2 and increased expression of Bax and p53 genes were observed. SIGNIFICANCE: The synergic effects of Ral and Res through increased ratio of Bcl2/Bax and expressions of p53, caspase-3 and caspase-8 genes indicating a better therapeutic effect on breast cancer cells relative to each drug alone. Combination of Res and Ral via increased expression of apoptotic genes including Bax, p53 and caspase-3 and caspase-8 is able to promote apoptosis as a mitochondrial dependent pathway in MCF7 and MDA-MB-231. The synergic effect was more potent in MCF7 estrogen receptor positive cell line.

5-aminolevulinic acid mediated photodynamic therapy inhibits survival activity and promotes apoptosis of A375 and A431 cells.

OBJECTIVES: The purpose of this study was to investigate the effects of 5-aminolaevulinic acid mediated photodynamic therapy (ALA-PDT) on the survival activity and apoptosis of human melanoma cell line A375 and non-melanoma skin carcinoma cell line A431 cells. The mechanism for cellular apoptosis was explored. METHODS: The cell survival activity was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and the proportion of apoptotic cells was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The expression levels of Bcl-2, Bax, caspase-3, caspase-8 and caspase-9 protein were assessed by western blot. The subcellular localization of cytochrome c was comparatively investigated by immunohistochemistry between pre-ALA-PDT and post- ALA-PDT. RESULTS: ALA-PDT significantly inhibited the survival activity of A375 cells and A431 cells in a dose- and time-dependent manner. The optimum inhibition efficiencies for A375 cells and A431 cells were obtained at 0.6 mM ALA at 4 h and 8 h after ALA-PDT, respectively. The phenomena of apoptosis were observed in ALA-PDT treated cells by TUNEL assay. The apoptotic rates of A375 cells and A431 cells were 90.0% and 61.5% at 6 h after ALA-PDT, respectively. Apoptosis induced by ALA-PDT involved in down-regulation of Bcl-2 protein, up-regulation of Bax protein and cleaved-PARP protein. It was observed that the expression of cleaved- caspase-3, caspase-8 and caspase-9 proteins in A375 cells and A431 cells gradually increased in 2 h and 4 h but decreased at 4-6 h and 6-8 h after ALA-PDT, respectively. In apoptosis cells immunohistochemical localization show that cytochrome C diffused from the mitochondria into the cytosol. CONCLUSION: ALA-PDT could significantly inhibit the survival activity of A375 and A431 cells. The apoptosis induced by ALA-PDT in A375 and A431 cells was related to the caspase-dependent death-receptor pathway and Cytochrome c-dependent mitochondrial pathway.

LQFM030 reduced Ehrlich ascites tumor cell proliferation and VEGF levels.

AIMS: This study reports the biological properties of LQFM030 in vivo, a molecular simplification of the compound nutlin-1. MAIN METHODS: Ehrlich ascites tumor (EAT)-bearing mice were treated intraperitoneally with LQFM030 (50, 75 or 150mg/kg) for 10days to determine changes in ascites tumor volume, body weight, cytotoxicity and angiogenesis. Moreover, flow cytometric expression of p53 and p21 proteins and caspase-3/7, -8 and -9 activation were investigated in EAT cells from mice treated. Acute oral systemic toxicity potential of LQFM030 in mice was also investigated using an alternative method. KEY FINDINGS: Treatment of EAT-bearing mice with LQFM030 resulted in a marked decline in tumor cell proliferation and the vascular endothelial growth factor (VEGF) levels along with enhanced survival of the mice. Apoptotic tumor cell death was detected through p53 and p21 modulation and increase of caspase-3/7, -8 and -9 activity. LQFM030 also showed orally well tolerated, being classified in the UN GHS category 5 (LD50>2000-5000mg/Kg). SIGNIFICANCE: LQFM030 seems to be a promising antitumor candidate for combinatory therapy with typical cytotoxic compounds, reducing the toxicity burden while allowing a superior anticancer activity. Moreover, these data also open new perspectives for LQFM030 as an antiangiogenic agent for treatment of diseases involving VEGF overexpression.

Caspase14 expression is associated with triple negative phenotypes and cancer stem cell marker expression in breast cancer patients.

BACKGROUND AND OBJECTIVES: The Caspase14 (CASP14) was reported that the low expression of CASP14 in ovarian cancer and colon cancer was associated with cancer progression, on the other hand, that the CASP14 expression in breast cancer was higher than that of non-cancerous tissues. The purpose of this study is to determine the clinical significance of CASP14 in breast cancer. METHODS: We performed immunohistochemistry for CASP14, ER, PgR, HER2, Ki67, EGFR, CK5/6, CD44, CD24, ALDH1, claudins, and androgen receptor in 222 breast cancer patients including 55 TNBC cases, and evaluated the relationship of CASP14, above-mentioned markers, and prognosis. Using public microarray database of breast cancer, the prognostic value of CASP14 was calculated. RESULTS: High CASP14 expression was significantly associated with TNBC subtype (P = 0.015), nuclear grade (P = 0.006), Ki67, EGFR (P < 0.001, P = 0.016), ALDH1, CD44 and CD24 (P < 0.001, P < 0.001, P = 0.001) in 222 breast cancer cases, and the high expression of claudin1 (P = 0.017), and androgen receptor (P = 0.002) in TNBC cases was related to the high CASP14. According to the public database, survival in the high CASP14 breast cancer patients was shorter than low CASP14 patients. CONCLUSIONS: High CASP14 expression is a marker of breast cancer aggressiveness in association with proliferation, TNBC phenotype, and cancer stemness.

Neutral sphingomyelinase inhibition alleviates apoptosis, but not ER stress, in liver ischemia-reperfusion injury.

Previous studies have revealed the activation of neutral sphingomyelinase (N-SMase)/ceramide pathway in hepatic tissue following warm liver ischemia reperfusion (IR) injury. Excessive ceramide accumulation is known to potentiate apoptotic stimuli and a link between apoptosis and endoplasmic reticulum (ER) stress has been established in hepatic IR injury. Thus, this study determined the role of selective N-SMase inhibition on ER stress and apoptotic markers in a rat model of liver IR injury. Selective N-SMase inhibitor was administered via intraperitoneal injections. Liver IR injury was created by clamping blood vessels supplying the median and left lateral hepatic lobes for 60 min, followed by 60 min reperfusion. Levels of sphingmyelin and ceramide in liver tissue were determined by an optimized multiple reactions monitoring (MRM) method using ultrafast-liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Spingomyelin levels were significantly increased in all IR groups compared with controls. Treatment with a specific N-SMase inhibitor significantly decreased all measured ceramides in IR injury. A significant increase was observed in ER stress markers C/EBP-homologous protein (CHOP) and 78 kDa glucose-regulated protein (GRP78) in IR injury, which was not significantly altered by N-SMase inhibition. Inhibition of N-SMase caused a significant reduction in phospho-NF-kB levels, hepatic TUNEL staining, cytosolic cytochrome c, and caspase-3, -8, and -9 activities which were significantly increased in IR injury. Data herein confirm the role of ceramide in increased apoptotic cell death and highlight the protective effect of N-SMase inhibition in down-regulation of apoptotic stimuli responses occurring in hepatic IR injury.

Effects of intrauterine growth restriction during late pregnancy on the cell apoptosis and related gene expression in ovine fetal liver.

This study investigated the effect of intrauterine growth restriction (IUGR) during late pregnancy on the cell apoptosis and related gene expression in ovine fetal liver. Eighteen time-mated Mongolian ewes with singleton fetuses were allocated to three groups at d 90 of pregnancy: Restricted Group 1 (RG1, 0.18 MJ ME kg BW-0.75 d-1, n = 6), Restricted Group 2 (RG2, 0.33 MJ ME kg BW-0.75 d-1, n = 6) and a Control Group (CG, ad libitum, 0.67 MJ ME kg BW -0.75 d -1, n = 6). Fetuses were recovered at slaughter on d 140. Fetal liver weight, DNA content and protein/DNA ratio, proliferation index, cytochrome c, activities of Caspase-3, 8, and 9 were examined, along with relative expression of genes related to apoptosis. Fetuses in both restricted groups exhibited decreased BW, hepatic weight, DNA content, and protein/DNA ratio when compared to CG (P < 0.05), as well as reduced proliferation index (P < 0.05). However, the increased numbers of apoptotic cells in fetal liver were observed in both restricted groups (P < 0.05). Fetuses with severe IUGR (RG1) exhibited increased (P < 0.05) activities of Caspase-3, 8, 9, as higher levels of mitochondrial cytochrome c in fetal liver; intermediate changes were found in RG2 fetuses, but the difference were not significant (P > 0.05). Hepatic expression of gene related to apoptosis showed reduced protein 21 (P21), B-cell lymphoma 2 (Bcl-2) and apoptosis antigen 1 ligand (FasL) expression in RG1 and RG2 (P < 0.05). In contrast, the increased hepatic expression of protein 53 (P53), Bcl-2 associated X protein (Bax) and apoptosis antigen 1 (Fas) in both IUGR fetuses were found (P < 0.05). These results indicate that the fetal hepatocyte proliferation were arrested in G1 cell cycle, and the fetal hepatocyte apoptosis was sensitive to the IUGR resulted from maternal undernutrition. The cell apoptosis in IUGR fetal liver were the potential mechanisms for its retarded proliferation and impaired development.

Arsenic sulfide induces apoptosis and autophagy through the activation of ROS/JNK and suppression of Akt/mTOR signaling pathways in osteosarcoma.

Osteosarcoma is a common primary malignant bone tumor, the cure rate of which has stagnated over the past 25-30 years. Arsenic sulfide (As2S2), the main active ingredient of the traditional Chinese medicine realgar, has been proved to have antitumor efficacy in several tumor types including acute promyelocytic leukemia, gastric cancer and colon cancer. Here, we investigated the efficacy and mechanism of As2S2 in osteosarcoma both in vitro and in vivo. In this study, we demonstrated that As2S2 potently suppressed cell proliferation by inducing G2/M phase arrest in various osteosarcoma cell lines. Also, treatment with As2S2 induced apoptosis and autophagy in osteosarcoma cells. The apoptosis induction was related to PARP cleavage and activation of caspase-3, -8, -9. As2S2 was demonstrated to induce autophagy as evidenced by formation of autophagosome and accumulation of LC3II. Further studies showed that As2S2-induced apoptosis and autophagy could be significantly attenuated by ROS scavenger and JNK inhibitor. Moreover, we found that As2S2 inhibited Akt/mTOR signaling pathway, and suppressing Akt and mTOR kinases activity can increase As2S2-induced apoptosis and autophagy. Finally, As2S2in vivo suppressed tumor growth with few side effects. In summary, our results revealed that As2S2 induced G2/M phase arrest, apoptosis, and autophagy via activing ROS/JNK and blocking Akt/mTOR signaling pathway in human osteosarcoma cells. Arsenic sulfide may be a potential clinical antitumor drugs targeting osteosarcoma.

Oxidative stress and cytotoxic effects of silver ion in mouse lung macrophages J774.1 cells.

Silver is commonly used as a disinfectant, and chronic exposure to silver may cause argyria, resulting in a gray-blue discoloration of human skin. However, the mechanism for cellular toxicity of silver has not been well explained. We studied the mode of cell death, the ratio of glutathione disulfide/glutathione, induction of metallothionein and activation of mitogen-activated protein kinases in J774.1 cells together with activation of antioxidant responsive element and nuclear factor-κB in CHO cells following exposure to silver ion (Ag+ ) to investigate the mechanism by which Ag+ causes lethal effects. Ag+ increased phosphorylation levels of extracellular signal-regulated, c-Jun N-terminal and p38 mitogen-activated protein kinases and remarkably increased the ratio of glutathione disulfide/glutathione in both a time- and concentration-dependent manner. Luciferase reporter gene assays revealed that antioxidant responsive element and nuclear factor-κB were activated following exposure to Ag+ . In addition, exposure to Ag+ increased the mRNA and protein levels of metallothionein. We investigated whether or not Ag+ killed J774.1 cells by inducing apoptosis. Ag+ increased the activity of caspase-3/7 which was abrogated by caspase 3 and pan-caspase inhibitors. However, these inhibitors did not ameliorate the cytotoxic effects of Ag+ , suggesting that Ag+ causes oxidative stress, which leads to necrotic rather than apoptotic cell death in J774.1 cells by decreasing functional sulfhydryl groups including glutathione in the cells. Copyright © 2016 John Wiley & Sons, Ltd.

Mitochondria and Caspases Tune Nmnat-Mediated Stabilization to Promote Axon Regeneration.

Axon injury can lead to several cell survival responses including increased stability and axon regeneration. Using an accessible Drosophila model system, we investigated the regulation of injury responses and their relationship. Axon injury stabilizes the rest of the cell, including the entire dendrite arbor. After axon injury we found mitochondrial fission in dendrites was upregulated, and that reducing fission increased stabilization or neuroprotection (NP). Thus axon injury seems to both turn on NP, but also dampen it by activating mitochondrial fission. We also identified caspases as negative regulators of axon injury-mediated NP, so mitochondrial fission could control NP through caspase activation. In addition to negative regulators of NP, we found that nicotinamide mononucleotide adenylyltransferase (Nmnat) is absolutely required for this type of NP. Increased microtubule dynamics, which has previously been associated with NP, required Nmnat. Indeed Nmnat overexpression was sufficient to induce NP and increase microtubule dynamics in the absence of axon injury. DLK, JNK and fos were also required for NP. Because NP occurs before axon regeneration, and NP seems to be actively downregulated, we tested whether excessive NP might inhibit regeneration. Indeed both Nmnat overexpression and caspase reduction reduced regeneration. In addition, overexpression of fos or JNK extended the timecourse of NP and dampened regeneration in a Nmnat-dependent manner. These data suggest that NP and regeneration are conflicting responses to axon injury, and that therapeutic strategies that boost NP may reduce regeneration.

Tricholoma matsutake Aqueous Extract Induces Hepatocellular Carcinoma Cell Apoptosis via Caspase-Dependent Mitochondrial Pathway.

Tricholoma matsutake, one of widely accepted functional mushrooms, possesses various pharmacological activities, and its antitumor effect has become an important research point. Our study aims to evaluate the cytotoxicity activities of T. matsutake aqueous extract (TM) in HepG2 and SMMC-7721 cells. In in vitro experiments, TM strikingly reduced cell viability, promoted cell apoptosis, inhibited cell migration ability, induced excessive generation of ROS, and caused caspases cascade and mitochondrial membrane potential dissipation in hepatocellular carcinoma cells. In in vivo experiments, 14-day TM treatment strongly suppressed tumor growth in HepG2 and SMMC-7721-xenografted nude mice without influence on their body weights and liver function. Furthermore, TM increased the levels of cleaved poly-ADP-ribose polymerase (PARP), Bad, and Bax and reduced the expressions of B-cell lymphoma 2 (Bcl-2) in treated cells and tumor tissues. All aforementioned results suggest that caspase-dependent mitochondrial apoptotic pathways are involved in TM-mediated antihepatocellular carcinoma.

Mutational analysis of metacaspase CaMca1 and decapping activator Edc3 in the pathogenicity of Candida albicans.

Candida albicans, an opportunistic fungal pathogen, displays apoptotic cell death in response to various stresses and a wide range of antifungal treatments. CaMca1, which is the only metacaspase in C. albicans, has been described as a key player in apoptotic cell death. Edc3 is an mRNA decapping activator and a scaffold protein of processing bodies. Edc3 was previously shown to regulate CaMCA1 expression and oxidative stress-induced apoptosis. In this study, we analyzed the contribution of the catalytic residues of the CaMca1 to the oxidative stress-induced apoptosis and pathogenicity of C. albicans. The CaMCA1C292A mutation decreased caspase activity to a level similar to that observed in the Camca1/Camca1 deletion strain and over-expression of CaMCA1C292A failed to suppress the oxidative-stress phenotypes of the edc3/edc3 mutant strain. The edc3/edc3, Camca1/Camca1, and CaMCA1C292A mutant strains were not virulent in a murine candidiasis model. Filamentation defects were observed in the Camca1/Camca1 mutant cells, whereas this defect was only partial in CaMCA1C292A mutant cells. These results suggest that CaMca1 and Edc3 play essential roles in the oxidative stress-induced apoptosis and virulence of C. albicans, and also support the notion that Edc3 is a key regulator of CaMca1 expression.

Effects of flavopiridol on critical regulation pathways of CD133high/CD44high lung cancer stem cells.

BACKGROUND: Flavopiridol a semisynthetic flavone that inhibits cyclin-dependent kinases (CDKs) and has growth-inhibitory activity and induces a blockade of cell-cycle progression at G1-phase and apoptosis in numerous human tumor cell lines and is currently under investigation in phase II clinical trials. Cancer stem cells (CSCs) are comprised of subpopulation of cells in tumors that have been proposed to be responsible for recurrence and resistance to chemotherapy. The aim of the present study was to investigate the effects of flavopiridol in cancer stem cell cytoskeleton, cell adhesion, and epithelial to mesenchymal transition in CSCs. METHODS: The cells were treated with flavopiridol to determine the inhibitory effect. Cell viability and proliferation were determined by using the WST-1 assay. Caspase activity and immunofluorescence analyses were performed for the evaluation of apoptosis, cell cytoskeleton, and epithelial-mesenchymal transition (EMT) markers. The effects of flavopiridol on the cell cycle were also evaluated. Flow cytometric analysis was used to detect the percentages of CSCs subpopulation. We analyzed the gene expression patterns to predict cell cycle and cell cytoskeleton in CSCs by RT-PCR. RESULTS: Flavopiridol-induced cytotoxicity and apoptosis at the IC50 dose, resulting in a significant increase expression of caspases activity. Cell cycle analyses revealed that flavopiridol induces G1 phase cell cycle arrest. Flavopiridol significantly decreased the mRNA expressions of the genes that regulate the cell cytoskeleton and cell cycle components and cell motility in CSCs. CONCLUSION: Our results suggest that Flavopiridol has activity against lung CSCs and may be effective chemotherapeutic molecule for lung cancer treatment.

Programmed Cell Death During Caenorhabditis elegans Development.

Programmed cell death is an integral component of Caenorhabditis elegans development. Genetic and reverse genetic studies in C. elegans have led to the identification of many genes and conserved cell death pathways that are important for the specification of which cells should live or die, the activation of the suicide program, and the dismantling and removal of dying cells. Molecular, cell biological, and biochemical studies have revealed the underlying mechanisms that control these three phases of programmed cell death. In particular, the interplay of transcriptional regulatory cascades and networks involving multiple transcriptional regulators is crucial in activating the expression of the key death-inducing gene egl-1 and, in some cases, the ced-3 gene in cells destined to die. A protein interaction cascade involving EGL-1, CED-9, CED-4, and CED-3 results in the activation of the key cell death protease CED-3, which is tightly controlled by multiple positive and negative regulators. The activation of the CED-3 caspase then initiates the cell disassembly process by cleaving and activating or inactivating crucial CED-3 substrates; leading to activation of multiple cell death execution events, including nuclear DNA fragmentation, mitochondrial elimination, phosphatidylserine externalization, inactivation of survival signals, and clearance of apoptotic cells. Further studies of programmed cell death in C. elegans will continue to advance our understanding of how programmed cell death is regulated, activated, and executed in general.

A mouse retinal explant model for use in studying neuroprotection in glaucoma.

The study aims to validate mouse retinal explants for use as an efficient tool to investigate neuroprotective therapies for treatment in diseases such as glaucoma. Eyes from C57BL/6 mice were enucleated immediately post-mortem to make two retinal explants per eye. Explants were cultured at an air/medium interface on membrane inserts for 7 days ex-vivo. Explants were treated either with Z-VAD-FMK (a pan-caspase inhibitor; 100 µM) or vehicle. Retinal Ganglion cell (RGC) density was analysed by ßIII tubulin and RNA-binding protein with multiple splicing (RBPMS) immunohistochemistry. Caspase activity was measured using Caspase 3/7 glo assay and western blot. Caspase-3 expression was quantified using RT-PCR and western blotting. Retinal explants treated with Z-VAD-FMK demonstrated a 1.5-fold (p = 0.027) increase in number of surviving RGCs on day 4 compared to the control treatment using ßIII tubulin staining. RGC viability was 2-fold (p = 0.002) higher in RGC stained with RBPMS on day 1 compared to control. There was no RBPMS staining of RGCs beyond day 1 in either treatment. The caspase activity was 4.75 and 5.5-fold (p = 0.002 and 0.004 respectively) higher in control as compared to treatment with Z-VAD-FMK on day 1, 2 respectively. Increase in caspase activity in control group was also confirmed by western blot for day 1 protein lysates. Caspase-3 mRNA expression was 4.75-fold higher in Z-VAD-FMK treated explants compared to control on day 1 (p < 0.001). Culture conditions appropriate to retinal explant culture for investigation of RGC apoptosis was identified. Retinal cultures at day 4 were ideal for detecting neuroprotection using ßIII tubulin staining. RBPMS acts as a viability marker as well as to define best time point for investigation of apoptosis related signalling pathways which is day 1. These findings suggest that mouse retinal explants are good model for studying ganglion cell specific apoptosis and are applicable to diseases such as glaucoma.

Apoptosis-associated protein expression in human salivary gland morphogenesis.

OBJECTIVE: Salivary gland (SG) development is based on branching morphogenesis, in which programmed cell death has been proposed to play a role in cell signalling and organ shaping. In the mouse salivary gland apoptosis has been suggested to play a key role in lumen formation, removing the central cells of the epithelial stalks. Here we analyse the expression of several anti- and pro-regulators of apoptosis during human SG development in a range of developmental stages. DESIGN: Foetal SGs obtained from the University of São Paulo were analysed by immunohistochemistry to assess the expression of apoptosis-associated proteins: caspases (caspase-6, -7, -9 and cleaved caspase-3), Bcl-2 family members (Bax, Bak, Bad, Bid, Bcl-2, Bcl-x and Bcl-xL), Survivin (BIRC5), Cytochrome C and Apaf-1. RESULTS: Nuclear expression of Bax and Bak was identified in presumptive luminal areas at initial stages, while Bcl-xL showed the most relevant anti-apoptotic activity. Caspase-6, -7 and -9 were expressed during all stages, while interestingly cleaved caspase-3 showed no prominent expression, indicating that caspase-7 is the main effector. Apoptosome complex components Apaf-1 and Cytochrome C, as well as survivin were all positive in developing glands. CONCLUSIONS: The particular expression pattern of several apoptotic regulators in human SG development suggests the existence of a fundamental role for apoptosis during duct formation. The absence of Bad and Bid expressions indicates that the instrinsic pathway is more active then the extrinsic during human gland formation. The subcellular localisation of intrinsic-apoptosis proteins correlated with apoptotic activity, but also suggested additional non-apoptotic functions.