Inhibitor Of Caspase-activated DNase Research Articles

Inhibition of Caspase 3 and Caspase 9 Mediated Apoptosis: A Multimodal Therapeutic Target in Traumatic Brain Injury.

Traumatic brain injury (TBI) is one of the significant causes of death and morbidity, and it is hence a focus of translational research. Apoptosis plays an essential part in the pathophysiology of TBI, and its inhibition may help overcome TBI's negative consequences and improve functional recovery. Although physiological neuronal death is necessary for appropriate embryologic development and adult cell turnover, it can also drive neurodegeneration. Caspases are principal mediators of cell death due to apoptosis and are critical for the required cleavage of intracellular proteins of cells committed to die. Caspase-3 is the major executioner Caspase of apoptosis and is regulated by a range of cellular components during physiological and pathological conditions. Activation of Caspase-3 causes proteolyzation of DNA repair proteins, cytoskeletal proteins, and the inhibitor of Caspase-activated DNase (ICAD) during programmed cell death, resulting in morphological alterations and DNA damage that define apoptosis. Caspase-9 is an additional crucial part of the intrinsic pathway, activated in response to several stimuli. Caspases can be altered post-translationally or by modulatory elements interacting with the zymogenic or active form of a Caspase, preventing their activation. The necessity of Caspase-9 and -3 in diverse apoptotic situations suggests that mammalian cells have at least four distinct apoptotic pathways. Continued investigation of these processes is anticipated to disclose new Caspase regulatory mechanisms with consequences far beyond apoptotic cell death control. The present review discusses various Caspase-dependent apoptotic pathways and the treatment strategies to inhibit the Caspases potentially.

Incomplete Caspase 3 Activation and Mitigation of Apoptosis in Hibernating Ground Squirrels, Spermophilus lateralis.

AbstractHibernating golden-mantled ground squirrels, Spermophilus [Callospermophilus] lateralis, tolerate proapoptotic conditions, such as low body temperature, anorexia, acidosis, and ischemia/reperfusion. Avoiding widespread apoptosis is critical for hibernator survival. Caspase 3, the key executioner of apoptosis, cleaves a majority of apoptotic targets. Under proapoptotic conditions, inactive procaspase 3 (32 kDa) is activated when cleaved into 17- and 12-kDa fragments (p32, p17, and p12, respectively). Caspase 3 activation results in extreme enzymatic activation. Activity increases >10,000-fold followed by apoptotic execution. Is widespread apoptosis occurring during the proapoptotic hibernation season? Western blots showed p17 increased ∼2-fold during hibernation, indicating caspase 3 activation. However, in vitro caspase 3 activity assays found no extreme increases in activity. Downstream caspase 3 targets ICAD (inhibitor of caspase-activated deoxyribonuclease) and PARP (poly (ADP-ribose) polymerase) did not experience elevated cleavage during hibernation, which is inconsistent with caspase 3 activation. TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) assays from multiple tissues found only 0.001%-0.009% of cells were TUNEL positive during winter, indicating negligible apoptosis during hibernation. Typically, caspase 3 activation generates a strong commitment toward apoptosis. We found that despite a ∼2-fold increase in active caspase 3, hibernators experience no downstream caspase 3 activity or widespread apoptosis. A systems-level approach suggests an incomplete signaling cascade wherein some caspase 3 activation during hibernation does not necessarily lead to bona fide apoptosis.

Abstract 10926: Importance of Mitochondrial DNA Damage in Cigarette Smoke Extract-Induced Activation of Innate Immune System

Objective: Smoking is a well-known risk factor for atherosclerosis, yet the mechanism by which smoking causes atherosclerosis remains unclear. It has been reported that inflammation and DNA damage are involved in the formation and development of atherosclerosis. We have previously reported that nuclear DNA damage was increased in peripheral mononuclear cells of smokers compared to those of non-smokers, and that DNA damage was accumulated in atherosclerotic lesions. In this study, to clarify the mechanism by which smoking induces inflammation in vascular endothelial cells, we investigated the effect of cigarette smoke extract (CSE) on both nuclear and mitochondrial DNA damage and the subsequent cellular response in human umbilical vein endothelial cells (HUVEC). Methods and Results: CSE increased double-strand breaks in HUVEC. Notably, this increase was detected relatively late (after 3 days of CSE exposure) compared to other genotoxic agents. CSE also increased oxidative DNA damage both in the nucleus and mitochondria and induced mitochondrial dysfunction. Mitochondrial dysfunction decreased cytosolic protein levels of ICAD, an inhibitor of caspase-activated DNase (CAD), which causes nuclear DNA fragmentation, and increased CAD in the nucleus, suggesting that this is one of the mechanisms of nuclear DNA double-strand breaks. In addition, continuous stimulation by CSE induced the accumulation of cytosolic DNA. Interestingly, real-time PCR analysis revealed that the accumulated DNA in the cytosol was derived not only from the nucleus but also from mitochondria. We further examined whether DNA accumulated in the cytosol activated cytosolic DNA-sensing pathways. The production of cGAMP, a second messenger in cGAS (a DNA-sensing receptor) signaling, was increased by CSE. We also found that the mRNA expression of IL-6 was increased by CSE, and that the increase was suppressed by si-cGAS. Conclusions: This study showed that continuous exposure to CSE induces not only nuclear but also mitochondrial DNA damage, which leads to cytosolic DNA accumulation, and evokes chronic inflammation via the cGAS-STING pathway.

SpICAD contributes to WSSV infection by suppressing apoptosis in mud crab (Scylla paramamosain)

ICAD is the inhibitor of caspase-activated deoxyribonuclease and has been widely investigated for its apoptotic regulatory function in higher animals, whereas apoptosis is tightly relevant to immune defense. However, the involvement of ICAD during virus infection has not been addressed in invertebrates. In this study, SpICAD identified in mud crab (Scylla paramamosain) was characterized, we found that the expression of SpICAD was upregulated during WSSV (White Spot Syndrome Virus) infection in mud crab, then, the role of SpICAD in immunoregulation was further explored via RNAi approach, the data revealed that SpICAD could inhibit caspase signaling and mitochondrial membrane potential (⊿ψm) and thus suppress apoptosis process. Moreover, our results showed that virus infection could activate SpICAD to reduce the apoptosis rate of hemocyte, which eventually contribute to WSSV replication in mud crab. The current study therefore provides novel insights into the anti-viral immune response of mud crabs.

Apoptosis in cerebrospinal fluid as outcome predictors in severe traumatic brain injury: An observational study.

Traumatic brain injury (TBI), due to its high mortality and morbidity, is an important research topic. Apoptosis plays a pathogenic role in a series of neurological disorders, from neurodegenerative diseases to acute neurological lesions.In this study, we analyzed the association between apoptosis and the Glasgow Outcome Scale (GOS), to examine the potential of apoptosis as a biomarker for a TBI outcome. Patients with severe TBI were recruited at the Department of Neurosurgery, Wujin Hospital Affiliated with Jiangsu University, between January 2018 and December 2019. As a control group, healthy subjects were recruited. The concentrations of caspase-3, cytochrome c, sFas, and caspase-9 in the cerebrospinal fluid (CSF) were analyzed by enzyme-linked immunosorbent assay (ELISA). The association between the GOS and the clinical variables age, sex, initial Glasgow Coma Scale (GCS) score, intracranial pressure (ICP), cerebral perfusion pressure (CPP), initial computed tomography (CT) findings, and apoptotic factors was determined using logistic regression. The area under the receiver operator characteristic (ROC) curve (AUC), and thus the sensitivity and specificity of each risk factor, were obtained.The levels of caspase-3, cytochrome c, sFas, and caspase-9 in the TBI group were significantly higher than those in the control group (P < .05). The logistic regression results showed that ICP and caspase-3 were significant predictors of outcome at 6 months post-TBI (P < .05). The AUC was 0.925 and 0.888 for ICP and caspase-3, respectively. However, the AUC for their combined prediction was 0.978, with a specificity and sensitivity of 96.0% and 95.2%, respectively, showing that the combined prediction was more reliable than that of the 2 separate factors.We demonstrated that caspase-3, cytochrome C, sFas, and caspase-9 were significantly increased in the CSF of patients following severe TBI. Furthermore, we found that ICP and caspase-3 were more reliable for outcome prediction in combination, rather than separately.

Induction of Melanoma Cell-Selective Apoptosis Using Anti-HER2 Antibody-Conjugated Gold Nanoparticles.

PurposeThis study was conducted to verify the induction and mechanism of selective apoptosis in G361 melanoma cells using anti-HER2 antibody-conjugated gold nanoparticles (GNP-HER2).Materials and MethodsFollowing GNP-HER2 treatment of G361 cells, cell cycle arrest and apoptosis were measured by WST-1 assay, Hemacolor staining, Hoechst staining, immunofluorescence staining, fluorescence-activated cell sorting analysis, and Western blotting.ResultsG361 cells treated with GNP-HER2 showed condensation of nuclei, which is an apoptotic phenomenon, and translocation of apoptosis-inducing factor and cytochrome c from mitochondria into the nucleus and cytoplasm, respectively. Increases in BAX in cells undergoing apoptosis, activation of caspase-3 and -9, and fragmentation of poly (ADP-ribose) polymerase and DNA fragmentation factor 45 (inhibitor of caspase-activated DNase) were observed upon GNP-HER2 treatment. Following GNP-HER2 treatment, an increase of cells in sub-G1 phase, which is a signal of cell apoptosis, was observed. This resulted in the down-regulation of cyclin A, cyclin D1, cyclin E, cdk2, cdk4, and cdc2 and the up-regulation of p21. Thus, GNP-HER2 treatment was confirmed to induce the cessation of cell cycle progression. Also, decreases in phospho-focal adhesion kinase and phospho-human epidermal growth factor receptor, which activate cellular focal adhesion, and decreases in phospho-paxillin, which stimulates the disassembly of filamentous actin, were observed. Reduced cell adhesion and disassembly of the intracellular structure indicated cell deactivation.ConclusionGNP-HER2 can selectively kill G361 melanoma cells without affecting normal cells. The mechanism of G361 cell death upon treatment with GNP-HER2 was apoptosis accompanied by activation of caspases.

Antiproliferative activity of chrysin (5, 7-dihydroxyflavone) from Indigofera tinctoria on human epidermoid carcinoma (A431) cells

IntroductionIndigofera tinctoria widely used in Indian and Chinese medicinal system to cure various diseases. Based on the ethnomedicinal value of this plant, the present investigation was aimed to isolate the bioactive compound from Indigofera tinctoria and evaluate the antiproliferative activity against human epidermoid carcinoma (A431) cells. MethodsChrysin (CH) was purified from the chloroform extract of I. tinctoria leaves by silica gel column chromatography and its antiproliferative effect against A431 cells was evaluated by MTT assay. Apoptotic effects were analysed by DNA fragmentation, nucleic acid content and acridine orange/ethidium bromide staining. Cell cycle arrest was analysed by flow cytometry. The apoptotic markers such as cytochrome c, caspase 9, caspase 3 and inhibitor of caspase activated DNase (ICAD) expression were analysed by western blot. ResultsMTT assay results revealed that the CH has inhibited the proliferation of A431 cells with IC50 value of 23.52 μg/mL and exhibited potent cytotoxicity; where as there is no promising toxic effect against normal HaCaT cells. DNA fragmentation analysis indicated that the CH treated A431 cells displayed the fragmented DNA. Further, CH induced remarkable cell death through apoptosis (87.65 ± 1.80%) and arrested the cell cycle at Sub G1 phase. Moreover, the CH could enhance the expression of cytochrome c, caspase 9, caspase 3 and inhibit the ICAD in a dose dependent manner. ConclusionThe present investigation concluded that the CH purified from I. tinctoria has potentially inhibited the proliferation of human epidermoid carcinoma (A431) cells and induce the apoptosis through caspase dependent manner.

Effect of electroacupuncture on learning and memory in rats with cerebral ischemia-reperfusion and its mechanism

Objective To explore the effect and possible mechanism of electroacupuncture on learning and memory impairment in rats with cerebral ischemia reperfusion. Methods 90 SD rats used for step-down test were divided into normal control group, simple cerebral ischemia reperfusion of 7 d, 14 d, 30 d, 60 d group (I/R) and cerebral ischemia reperfusion of 7 d, 14 d, 30 d, 60 d and electroacupuncture intervention group (I/R+ EA), through simple random sampling method (10 rats in each group); 70 SD rats used for molecular biology experiments were randomly divided into normal control group, simple cerebral ischemia and reperfusion of 3 h, 6 h, 12 h group (I/R) and cerebral ischemia reperfusion of 3 h, 6 h, 12 h and electroacupuncture intervention group (I/R+ EA), n=10.Use suture method to make model of cerebral ischemia reperfusion in rats.Take the rats' baihui acupoint and shuigou acupoint to acupuncture.The final strength was 5 mA and the duration was 30 minutes.Step down test was used to observe the restoration of learning and memory in rats.Western blot was used to detect the expression of procaspase-3, procaspase-12, PARP and ICAD in rat cerebral cortex and striatum.Immunofluorescence histochemical method was used to detect the number of GRP94 positive cells. Results The results of the platform showed that the learning and memory abilities of the rats were obviously improved after electroacupuncture treatment.Electric shock incubation period of rats in electroacupuncture of 7 d and 14 d group was (90.60±17.01) and (128.80±8.85) respectively.It was significantly higher than that of cerebral ischemia reperfusion of 7 d group(51.20±5.09) and 14 d group(105.00±10.39). The number of mistakes in single foot jump of rats in electroacupuncture of 7 d and 14 d group was (3.90±0.99) and (2.70±0.67) respectively.It was significantly lower than that of 7 d group(9.20±1.61) and 14 d group(6.10±0.99). Through SPSS13.0 statistical software one-way ANOVA, the difference was statistically significant (P<0.05). Western blot results showed that the expression of procaspase-3, procaspase-12, PARP protein precursor and ICAD protein, upregulation increased after electroacupuncture(P<0.05). The results of immunofluorescence histochemistry showed that the number of positive cells of GRP94 protein was significantly decreased after 3 h, 6 h and 12 h after electroacupuncture intervention compared with the corresponding I/R group. Conclusion Electroacupuncture can effectively improve the learning and memory ability of rats with ischemia-reperfusion, which may be related to the apoptosis of cerebral cells after cerebral ischemia reperfusion injury by blocking the apoptosis pathway mediated by endoplasmic reticulum and inflammatory factors. Key words: Electroacupuncture; Cerebral ischemia reperfusion; Apoptosis; Learning and memory; Rat

Caspase-mediated cleavage of X-ray repair cross-complementing group 4 promotes apoptosis by enhancing nuclear translocation of caspase-activated DNase

X-ray repair cross-complementing group 4 (XRCC4), a repair protein for DNA double-strand breaks, is cleaved by caspases during apoptosis. In this study, we examined the role of XRCC4 in apoptosis. Cell lines, derived from XRCC4-deficient M10 mouse lymphoma cells and stably expressing wild-type XRCC4 or caspase-resistant XRCC4, were established and treated with staurosporine (STS) to induce apoptosis. In STS-induced apoptosis, expression of wild-type, but not caspase-resistant, XRCC4 in XRCC4-deficient cells enhanced oligonucleosomal DNA fragmentation and the appearance of TUNEL-positive cells by promoting nuclear translocation of caspase-activated DNase (CAD), a major nuclease for oligonucleosomal DNA fragmentation. CAD activity is reportedly regulated by the ratio of two inhibitor of CAD (ICAD) splice variants, ICAD-L and ICAD-S mRNA, which, respectively, produce proteins with and without the ability to transport CAD into the nucleus. The XRCC4-dependent promotion of nuclear import of CAD in STS-treated cells was associated with reduction of ICAD-S mRNA and protein, and enhancement of phosphorylation and nuclear import of serine/arginine-rich splicing factor (SRSF) 1. These XRCC4-dependent, apoptosis-enhancing effects were canceled by depletion of SRSF1 or SR protein kinase (SRPK) 1. In addition, overexpression of SRSF1 in XRCC4-deficient cells restored the normal level of apoptosis, suggesting that SRSF1 functions downstream of XRCC4 in activating CAD. This XRCC4-dependent, SRPK1/SRSF1-mediated regulatory mechanism was conserved in apoptosis in Jurkat human leukemia cells triggered by STS, and by two widely used anti-cancer agents, Paclitaxel and Vincristine. These data imply that the level of XRCC4 expression could be used to predict the effects of apoptosis-inducing drugs in cancer treatment.

Signaling pathways of docosahexenoic acid-induced apoptosis of prostate cancer cells

Objective To explore how docosahexenoic acid (DHA) changes apoptosis signaling pathways to further lead to apoptosis of prostate cancer cells. Methods RT2 Profiler PCR Arrays were used to screen the targets of DHA-induced prostate cancer cells apoptosis (10 apoptosis genes were up-regulated and 5 apoptosis genes were down-regulated). Real-time quantitative polymerase chain reaction (Real-time PCR) was used as reliable tools to validate the results. Results As compared with bovine serum albumin (BSA), after incubation with DHA for 24 h, the transcriptional level of Caspase family: Caspase-1, Caspase-3 and Caspase-9 in DU145 cells were increased 2.06, 4.88 and 12.10 times respectively; The transcriptional level of pro-apoptotic genes B cell lymphoma/leukemia-2 associated X protein (bax) was increased 2.93 times and B cell lymphoma/leukemia-2 (bcl-2)/bax ratio increased. Cell death inducing related genes CIDEA and DFFA were increased 2.34, 3.21 times respectively; Tumor necrosis factor related genes LTA and tumor necrosis factor (TNF) were increased 2.04 and 2.24 times respectively; The expression of tumor protein P53 (TP53) gene mRNA increased 2.97 times. In addition, as compared with the BSA control group, after incubation with DHA for 24 h, the transcriptional levels of apoptosis inducing factor 1 (AIFM1), Akt1, BH3 locus death inducing gene (BID), BIRC6 and X-linked inhibitor of apoptosis protein (XIAP) were decreased. After DHA treatment for 24 h, the mRNA expression of Caspase-1, Caspase-3, Caspase-9 and Caspase-12 was increased respectively. Moreover, the expression of Caspase-3, Caspase-9 and Caspase-12 had a dose-dependent relationship with DHA concentration. The mRNA expression of bax, CIDEA, DFFA, TP53 and TNF normalized with TATA box binding protein (TBP) in 24 h DHA-treated DU145 cells was increased obviously, and the expression of bax, CIDEA and TNF had a dose-dependent relationship with DHA concentration. The mRNA expression of AIFM1, protein kinase B (Akt1), BID, BIRC6 and XIAP was decreased obviously after incubation with different concentrations of DHA for 24 h, and the expression of bax, CIDEA and TNF had a dose-dependent relationship with DHA concentration. Conclusion DHA can change the related genes expression of apoptosis signaling pathways in Caspase family, by this way it can induce the apoptosis of prostate cancer cells. Key words: Prostatic carcinoma; Androgen resistance prostate cancer; Polyunsaturated fatty acids; Docosahexenoic acid

The caspase‐activated DNase: apoptosis and beyond

Organismal development and function requires multiple and accurate signal transduction pathways to ensure that proper balance between cell proliferation, differentiation, inactivation, and death is achieved. Cell death via apoptotic caspase signal transduction is extensively characterized and integral to this balance. Importantly, the view of apoptotic signal transduction has expanded over the previous decades. Subapoptotic caspase signaling has surfaced as mechanism that can promote the adoption of a range of cellular fates. An emerging mechanism of subapoptotic caspase signaling is the activation of the caspase-activated DNase (CAD) through controlled cleavage of the inhibitor of CAD (ICAD). CAD-induced DNA breaks incite a DNA damage response, frequently invoking p53 signaling, that transduces a change in cell fate. Cell differentiation and senescence are fates demonstrated to arise from CAD-induced DNA breaks. Furthermore, an apparent consequence of CAD activity is also emerging, as a potential source of oncogenic mutations. This review will discuss the mechanisms underlying CAD-induced DNA breaks and highlight how CAD activity promotes diverse cell fates.

Marein protects against methylglyoxal-induced apoptosis by activating the AMPK pathway in PC12 cells

Diabetic encephalopathy, which is characterized by cognitive decline and dementia, commonly occurs in patients with long-standing diabetes. Previous studies have suggested that methylglyoxal (MG), an endogenous toxic compound, plays an important role in diabetic complications such as cognitive impairment. MG induces neuronal apoptosis. To clarify whether marein, a major compound from the hypoglycemic plant Coreopsis tinctoria, prevents PC12 cell damage induced by MG, we cultured PC12 cells in the presence of MG and marein. Marein attenuated MG-induced changes in the mitochondrial membrane potential (ΔΨm), mitochondrial permeability transition pores (mPTPs), intracellular Ca2+ levels, the production of reactive oxygen species (ROS), glutathione (GSH)/glutathione disulfide (GSSG) and adenosine triphosphate (ATP), and the increase in the percentage of apoptotic cells. Marein also increased glyoxalase I (Glo1) activity, phospho-AMPKα (Thr172) and Bcl-2 expression and diminished the activation of Bax, caspase-3 and inhibitor of caspase-activated deoxyribonuclease (ICAD). Importantly, pretreatment of cells with marein diminished the compound C-induced inactivation of p-AMPK. Molecular docking simulation showed that marein interacted with the γ subunit of AMPK. In conclusion, we found for the first time that the neuroprotective effect of marein is due to a reduction of damage to mitochondria function and activation of the AMPK signal pathway. These results indicate that marein may be a potent compound for preventing/counteracting diabetic encephalopathy.

Anesthetic Isoflurane Induces DNA Damage Through Oxidative Stress and p53 Pathway.

DNA damage is associated with aging and neurological disorders, including Alzheimer's disease. Isoflurane is a commonly used anesthetic. It remains largely unknown whether isoflurane induces DNA damage. Phosphorylation of the histone protein H2A variant X at Ser139 (γH2A.X) is a marker of DNA damage. We therefore set out to assess the effects of isoflurane on γH2A.X level in H4 human neuroglioma cells and in brain tissues of mice. Oxidative stress, caspase-activated DNase (CAD), and the p53 signaling pathway are involved in DNA damage. Thus, we determined the interaction of isoflurane with reactive oxygen species (ROS), CAD, and p53 to illustrate the underlying mechanisms. The cells were treated with 2% isoflurane for 3 or 6h. The mice were anesthetized with 1.4% isoflurane for 2h. Western blot, immunostaining and live cell fluorescence staining were used in the experiments. We showed that isoflurane increased levels of γH2A.X, cleaved caspase-3, and nucleus translocation of CAD and decreased levels of inhibitor of CAD (ICAD) and p53. Isoflurane enhanced the nucleus level of γH2A.X. Moreover, caspase inhibitor Z-VAD and ROS generation inhibitor N-acetyl-L-cysteine (NAC) attenuated the isoflurane-induced increase in γH2A.X level. However, NAC did not significantly alter the isoflurane-induced reduction in p53 level. Finally, p53 activator (actinomycin D) and inhibitor (pifithrin-α) attenuated and potentiated the isoflurane-induced increase in γH2A.X level, respectively. These findings suggest that isoflurane might induce DNA damage, as represented by increased γH2A.X level, via induction of oxidative stress and inhibition of the repair of DNA damage through the p53 signaling pathway.

Transcriptome Analysis of Peripheral Blood in Chronic Inflammatory Demyelinating Polyradiculoneuropathy Patients Identifies TNFR1 and TLR Pathways in the IVIg Response.

We have studied the response to intravenous immunoglobulins (IVIg) by a transcriptomic approach in 11 chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) patients (CIDP duration = 6 [0.83–6.5] years). RNA was extracted from cells in whole blood collected before and 3 weeks after IVIg treatment, and hybridized on Illumina chips. After RNA quality controls, gene expression was analyzed using statistical tests fitted for microarrays (R software, limma package), and a pathway analysis was performed using DAVID software. We identified 52 genes with expression that varied significantly after IVIg (fold change [FC] > 1.2, P < 0.001, false discovery rate [FDR] <0.05). Among these 52 genes, 7 were related to immunity, 3 were related to the tumor necrosis factor (TNF)-α receptor 1 (TNFR1) pathway (inhibitor of caspase-activated DNase (ICAD): FC = 1.8, P = 1.7E-7, FDR = 0.004; p21 protein-activated kinase 2 [PAK2]: FC = 1.66, P = 2.6E-5, FDR = 0.03; TNF-α-induced protein 8-like protein 1 [TNFAIP8L1]: P = 1.00E-05, FDR = 0.026), and 2 were related to Toll-like receptors (TLRs), especially TLRs 7 and 9, and were implicated in autoimmunity. These genes were UNC93B1 (FC = 1.6, P = 2E-5, FDR = 0.03), which transports TLRs 7 and 9 to the endolysosomes, and RNF216 (FC = 1.5, P = 1E-05, FDR = 0.03), which promotes TLR 9 degradation. Pathway analysis showed that the TNFR1 pathway was significantly lessened by IVIg (enrichment score = 24, Fischer exact test = 0.003). TNF-α gene expression was higher in responder patients than in nonresponders; however, it decreased after IVIg in responders (P = 0.04), but remained stable in nonresponders. Our data suggest the actions of IVIg on the TNFR1 pathway and an original mechanism involving innate immunity through TLRs in CIDP pathophysiology and the response to IVIg. We conclude that responder patients have stronger inflammatory activity that is lessened by IVIg.

Decreased Expression of Inhibitor of Caspase-Activated DNase (ICAD) in Renal Cell Carcinoma - Tissue Microarray of Human Samples.

Although primary localised tumours of renal cell carcinoma (RCC) can be treated relatively successfully with surgery, metastatic RCC has poor prognosis because of late diagnosis and resistance to therapies. In the present study, we were interested in profiling the protein expression of “inhibitor of caspase-activated DNase” (ICAD), an apoptosis inhibitor, in kidney cancer and its paired normal kidney. Immunohistochemistry with automated batch staining and morphometry using digital pathology were used to compare ICAD in 121 RCC specimens with their paired normal kidney tissue. Tissue microarray of formalin-fixed, paraffin-embedded archival tissue was used. Intensity and localisation of ICAD were compared between normal and cancer samples, and against grading within the cancers. The results demonstrated that, in this cohort, ICAD was highly expressed in the proximal tubular epithelium of normal kidney, and significantly decreased in clear cell RCC tissue (p < 0.05) as well as other subtypes of RCC (p < 0.01) compared with normal kidney. There was a tendency towards nuclear localisation of ICAD in clear cell RCC, but not in other subtypes of RCC. No significant association was found between ICAD intensity and grade of RCC. In summary, down-regulation of ICAD occurs in RCC. ICAD normally inhibits DNA fragmentation and apoptosis; thus, its down-regulation was unexpected in a cancer known for its resistance to apoptosis. However, these RCC samples were from primary, not metastatic, RCC sites, and down-regulated ICAD may be part of a progressive pathway that promotes RCC metastasis.

Ulmus Macrocarpa 열수 추출물에 의한 비장세포 수명 연장

Ulmus macrocarpa has been used in Korean medicinal food material to physical disorder or tonic material. The purpose of the present study was to evaluate splenocyte life span expansion effects of Ulmus macrocarpa water extract (UMWE) in general cell culture condition. Splenocytes were handled in the presence of 100 μg/ml UMWE for several different time conditions. Live cells were detected with Hoechst 33,342 dye and cell survival molecules were identified through Western blot. Changes in level of cytokine synthesis were evaluated by ELISA. UMWE showed an effect on increased splenocyte survival. UMWE elevated slightly PI3K phosphorylation and ERK1/2 phosphorylation used at 48 hr and 96 hr. Moreover, Bcl-2 was elevated at 48 hr and 96 hr in UMWE-treated splenocytes. UMWE decreased caspase-3 level at 48 hr and 96 hr. ICAD protein increased at 48 hr culturing time. Hematopoietin IL-2 cytokine was down-regulated, however IL-4 hematopoietin cytokine was up-regulated in UMWE treated cell culture media. Increased IFN-γ levels were verified in the supernatant of UMWEtreated cells in all periods (48 hr and 96 hr). Increased patterns in the production of IL-12 cytokine occurred as compared with control after 48 and 96 hr in UMWE-treated-cell cultures. These results suggested that UMWE can prolong splenocyte life span by controlling various signal factors and cytokines.

Neuroprotective effect of water extract of Panax ginseng on corticosterone-induced apoptosis in PC12 cells and its underlying molecule mechanisms

Ethnopharmacological relevanceThe root of Panax ginseng C.A. Meyer (Family Araliaceae) is an important medicinal plant which has been employed as a panacea for more than 2,000 years in China. It has the actions of invigorating primordial qi, recovering pulse and desertion, engendering liquid, and calming spirit. The water extract of Panax ginseng (WEG) has been used to treat kinds of central nervous system disorders, such as depression, insomnia, Alzheimer׳s disease and Parkinson׳s disease. Our previous work has demonstrated that WEG possessed antidepressant-like activities in both acute and chronic stress models of depression. Nevertheless, there are no studies on the cytoprotection and potential mechanisms of WEG on corticosterone-induced apoptosis. The present study focuses on cytoprotection against corticosterone-induced neurotoxicity in PC12 cells and its underlying molecule mechanisms of the antidepressant-like effect of WEG. Materials and methodsThe PC12 cells were treated with 250μmol/L corticosterone in the absence or presence of WEG for 24h, then 3-(4,5-dimethy thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) detection, Hoechst33342 staining and TUNEL staining were investigated to confirm the neuroprotection of WEG. Then, mitochondrial permeability transition pore (mPTP), mitochondrial membrane potential (MMP), intracellular Ca2+ ([Ca2+]i), reactive oxygen species (ROS) concentration, and the expression level of glucocorticoid receptor (GR), heat shock protein 90 (Hsp90), histone deactylase 6 (HDAC6), glucose-regulated protein 78 (GRP78), growth arrest and DNA damage inducible protein 153 (GADD153), X-box DNA-binding protein-1 (XBP-1), caspase-12, cytochrome C, inhibitor of caspase-activated deoxyribonuclease (ICAD), caspase-3 and caspase-9 were assessed by Western Blot analysis to understand the molecule mechanisms of neuroprotection of WEG. ResultsWEG partly reversed corticosterone-induced damage in PC12 cells, which increased cell viability, decreased LDH release, and attenuated corticosterone-induced apoptosis as compared with the corticosterone-treated group. Mechanistically, compared with the corticosterone-treated group, WEG strongly attenuated [Ca2+]i overload and ROS level, and restored mitochondrial function, including mPTP and MMP. Furthermore, WEG strongly up-regulated the expression of GR and HDAC6, and down-regulated the expression of Hsp90, cytochrome C, ICAD, caspase-3, caspase-9 as well as endoplasmic reticulum (ER) stress-related proteins, such as GADD153, GRP78, XBP-1, and caspase-12. ConclusionWEG possessed neuroprotection against corticosterone-induced damage in PC12 cells, and the underlying molecule mechanisms was depended on the intervening of HDAC6 and HSP90 of the GR-related function proteins, and subsequent restoration of ER and mitochondria functions.

Auxin-induced Rapid Degradation of Inhibitor of Caspase-activated DNase (ICAD) Induces Apoptotic DNA Fragmentation, Caspase Activation, and Cell Death: A Cell Suicide Module

Caspase-activated DNase (CAD) is a major apoptotic nuclease, responsible for DNA fragmentation and chromatin condensation during apoptosis. CAD is normally activated in apoptosis as a result of caspase cleavage of its inhibitory chaperone ICAD. Other aspects of CAD regulation are poorly understood. In particular, it has been unclear whether direct CAD activation in non-apoptotic living cells can trigger cell death. Taking advantage of the auxin-inducible degron (AID) system, we have developed a suicide system with which ICAD is rapidly degraded in living cells in response to the plant hormone auxin. Our studies demonstrate that rapid ICAD depletion is sufficient to activate CAD and induce cell death in DT40 and yeast cells. In the vertebrate cells, ectopic CAD activation triggered caspase activation and subsequent hallmarks of caspase-dependent apoptotic changes, including phosphatidylserine exposure and nuclear fragmentation. These observations not only suggest that CAD activation drives apoptosis through a positive feedback loop, but also identify a unique suicide system that can be used for controlling gene-modified organisms.

Minocycline inhibits ICAD degradation and the NF-κB activation induced by 6-OHDA in PC12 cells

6-Hydroxydopamine (6-OHDA) is a neurotoxin that is commonly employed to induce lesions of the dopaminergic pathways to generating experimental models of Parkinson’s disease (PD) in rodents. Antioxidant and anti-inflammatory therapy approaches have been the focus of attention in the treatment of neurodegenerative. PD and Alzheimer’s diseases, and oxidative stress have been implicated in these diseases. In this study, we investigated the neuroprotective effects of minocycline and the signalling pathway that is possibly involved in a PC12 cell model of PD. The results indicated that 6-OHDA cytotoxicity was accompanied by an increment in lactate dehydrogenase (LDH) release, an increase in caspase-3 protein activity, an increase in ROS generation, MDA content and decrease in the SOD, CAT activities and cell viability. Moreover, treatment with 6-OHDA alone for 24h resulted in ICAD degradation, increased nuclear translocation of NF-κB, and increased p53 expression. However, pretreatment with minocycline (5, 10, 20µM) for 24h significantly reduced LDH release, reduced caspase-3 protein production, reduced ROS production, MDA content and attenuated the decrease in SOD, CAT activities and cell viability. Additionally, minocycline (20µM) markedly decreased the levels of cleaved ICAD protein, down-regulated p53 activity and inhibited the nuclear translocation of NF-κB. The neuroprotective effects of minocycline were attributable to its potent antioxidant activities, which prevented the nuclear translocation of NF-κB and the subsequent promotion of cell death. Therefore, the present study supports the notion that minocycline may be a promising neuroprotective agent for the treatment of Parkinson’s disease.

The endogenous oxindole isatin induces apoptosis of MCF‑7 breast cancer cells through a mitochondrial pathway.

Isatin is an endogenous indole in mammalian tissues and fluids that is expected to have antitumor effects in human breast cancer cells. Human breast cancer cells (MCF-7) were exposed to isatin at various concentrations (0, 50, 100, 200µmol/l) for 48h. Apoptotic features were demonstrated by nuclei staining with Hoechst 33258 and flow cytometry. Bcl-2 and Bax mRNA were analyzed via reverse transcription-polymerase chain reaction. Bcl-2, Bax, the inhibitor of caspase-activated DNase (ICAD), and cytochromec protein were analyzed by western blot analysis. Apoptosis, caspase-9 and -3 activation and mitochondrial depolarization were assayed by flow cytometry. The results showed that isatin induced apoptosis of MCF-7 cells. Furthermore, Bcl-2 expression was decreased and the ratio of Bcl-2 to Bax was significantly decreased by isatin. The mitochondrial transmembrane potential was markedly decreased and the release of cytochrome c into the cytosol was elevated following treatment with isatin. At the same time, caspase-9 and -3 were stimulated, followed by the degradation of ICAD, a caspase-3 substrate.