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The CIDE domain was initially identified in apoptotic nucleases and now forms a highly conserved family with diverse functions ranging from cell death to lipid metabolism. Based on structural determination of the DREP3 domain, it is suggested that the head-to-tail helical filament structure might be a unified mechanism of CIDE-domain assembly and represents a critical higher-order scaffolding structure that is important for the function of CIDE-domain-containing proteins in DNA fragmentation and lipid-droplet fusion., The cell-death-inducing DFF45-like effector (CIDE) domain is a protein-interaction module comprising ∼80 amino acids and was initially identified in several apoptotic nucleases and their regulators. CIDE-domain-containing proteins were subsequently identified among proteins involved in lipid metabolism. Given the involvement of CIDE-domain-containing proteins in cell death and lipid homeostasis, their structure and function have been intensively studied. Here, the head-to-tail helical filament structure of the CIDE domain of DNA fragmentation factor-related protein 3 (DREP3) is presented. The helical filament structure was formed by opposing positively and negatively charged interfaces of the domain and was assembled depending on protein and salt concentrations. Although conserved filament structures are observed in CIDE family members, the structure elucidated in this study and its comparison with previous structures indicated that the size and the number of molecules used in one turn vary. These findings suggest that this charged-surface-based head-to-tail helical filament structure represents a unified mechanism of CIDE-domain assembly and provides insight into the function of various forms of the filament structure of the CIDE domain in higher-order assembly for apoptotic DNA fragmentation and control of lipid-droplet size.

Background: Global trend is moving towards the use of natural phytochemicals to fight against pathogens. Human cervical cancer is directly associated with onco-potent type of Human Papilloma Virus (HPV). There is no known medicine for clearance of HPV type whose persistence is the cause of occurrence and re-occurrence of cervical cancer. The different species of fig fruit and their latex are reported to have HPV associated genital warts clearance capability. Methods: In the current investigation, the effect of the methanol extract of Ficus benghalensis L. fruits on HPV type18 viral load in HeLa cell line was tested by doing PCR using HPV L1 primers (MY09/My011) and the cytotoxicity was also analysed by MTT assay. The induction of apoptotic activity in terms of DNA fragmentation and hyper-chromic effects of DNA was analysed. Results: The PCR results showed a reduction in the HPV18 DNA and also the treatment exhibited a promising cytotoxicity with IC50 value at 211.86 μg/ml. The DNA samples from treated HeLa cells showed DNA shearing and laddering as a mark of apoptotic DNA fragmentation (Fig. 2) and the UV absorbance value at 260 nm was found to be significantly (P

Senescence is an irreversible permanent cell cycle arrest accompanied by changes in cell morphology and physiology. Bioactive compounds including tocotrienols (vitamin E) can affect important biological functions. The aim of this study was to investigate how γ- and δ-tocotrienols can affect stress-induced premature senescence. We established two different models of premature stress senescence by induction of senescence with either hydrogen peroxide or etoposide in human lung fibroblasts MRC-5 (ECACC, England). We observed increased percentage of cells with increased SA-β-galactosidase activity, decreased cell viability/proliferation and increased level of p21 in both models. In addition, γ-tocotrienol or δ-tocotrienol (both at concentrations of 150, 200 and 300 μM) were added to the cells along with the inductor of senescence (cotreatment). We have found that this cotreatment led to the decrease of cell viability/proliferation in both models of premature stress senescence, but did not change the percentage of senescent cells. Moreover, we detected no expression of caspase-3 or apoptotic DNA fragmentation in any models of premature stress senescence after the cotreatment with γ- as well as δ-tocotrienols. However, an increased level of autophagic protein LC-3 II was detected in cells with hydrogen peroxide-induced senescence after the cotreatment with γ-tocotrienol as well as δ-tocotrienol. In case of etoposide-induced senescence only δ-tocotrienol cotreatment led to an increased level of LC-3 II protein in cells. According to our work δ-tocotrienol is more effective compound than γ-tocotrienol.

Briefly depicted are the publications in CYTOMETRY that received the highest frequency of citations. Among them are seminal papers describing application of metachromatic fluorochrome acridine orange to differentially stain DNA versus RNA or to analyze susceptibility of DNA in situ to denaturation; both features being markers of different sections of the cell cycle including identification of noncycling quiescent cells. The papers reviewing detection of cyclins D1, E, A or B1, each in relation to cell cycle phase, were also among the highly cited ones. The highest citation rates received publications describing development of the TUNEL methodology to detect apoptotic DNA fragmentation, and more recently expression of ϒH2AX to reveal DNA damage. © 2020 International Society for Advancement of Cytometry.

The current research was conducted to investigate neuroprotective, anticancer, antiproliferative and apoptotic DNA fragmentation potentials of the extracts obtained from aerial-parts, raw-fruits, and coffee samples (prepared by roasting and boiled-roasting) of kenger (Gundelia tournefortii var. armata). Neuroprotection capacities of the samples were screened against enzymes that are linked to the progression of Alzheimer’s and Parkinson’s diseases. Since the oxidative damage is closely related to the development of neurodegeneration and cancer, the extracts were also analyzed for their antioxidant activities. Anticancer and antiproliferative activities of the samples were evaluated towards A549, MCF-7, and HeLa cells, and they exhibited remarkable anticancer activities in dose-and time-dependent manners. Furthermore, the apoptotic activity regarding DNA fragmentation was also imaged. The extracts obtained from the aerial-parts and raw-fruits of kenger possessed the highest anticancer and antiproliferative activities on HeLa cells with the IC(50) values varied from 290.71 ± 0.69 to 342.46 ± 0.42, whilst kenger coffee samples had the weakest activity on the cell death towards MCF-7. However, the tested extracts exhibited strong antioxidant activities, in which the best reducing antioxidant capacity was defined in the raw-fruits and aerial-parts of the extracts. The roasted-coffee samples had the best inhibition on tyrosinase (88.26 ± 0.15%, p

Osteosarcoma (OS) is the most common tumor of the musculoskeletal system. Recently, cold atmospheric plasma (CAP) has been regarded as a promising anti-oncogenic therapy. Previous experimental studies have demonstrated that CAP treatment results in significant growth inhibition of human sarcoma and is able to induce apoptosis. However, due to device-specific parameters, there is a large variability in the antitumor effects of different CAP sources. In the present study, the cellular effects of CAP treatment from two different CAP devices were investigated and their pro-apoptotic efficacy was characterized. The OS cell lines, U2-OS and MNNG/HOS, were treated with two CAP devices, kINPen MED and MiniJet-R. Control groups were treated with argon. The anti-proliferative effect of each treatment was demonstrated using cell counting and the activation of apoptotic mechanisms was determined using Comet, TUNEL and Caspase-3/Caspase-7 assays. The results revealed that treatment of both OS cell lines with the two CAP sources resulted in significant inhibition of cell growth. Subsequently, the activation of Caspases and the induction of apoptotic DNA fragmentation was demonstrated. The biological effects of each CAP source did not differ significantly. The treatment of OS cells with CAP lead to an induction of apoptosis and a reduction of cell growth. Therefore, the biological effects of CAP appear to be general as the two devices of different design produced highly comparable cell responses. Therefore, the type of device used does not seem to affect the efficacy of CAP-based antitumor therapy.

Different series of novel pyrazole and pyrazolo[1,5-a] pyrimidine derivatives (2a-g), (3a-c), (7a-d) and (10a-e) were designed, synthesized and evaluated for their ability to inhibit CDK2/cyclin A2 enzyme in vitro. In addition, the cytotoxicity of the newly synthesized compounds was screened against four different human cancer cell lines. The CDK2/cyclin A2 enzyme inhibitory activity revealed that compounds (2d) and (2 g) are among the most active with inhibitory activity values of 60% and 40%, respectively, while compounds (7d) and (10b) exhibited the highest activity among the newly synthesized derivatives against four tumor cell lines (HepG2, MCF-7, A549 and Caco2) with IC50 values 24.24, 14.12, 30.03 and 29.27 μM and 17.12, 10.05, 29.95 and 25.24 μM, respectively. Flow cytometry cell cycle assay was carried for compounds (7d) and (10b) to investigate their apoptotic activity. The obtained results revealed that they induced cell-cycle arrest in the G0-G1phase and reinforced apoptotic DNA fragmentation. Molecular modeling studies have been carried out to gain further understanding the binding mode of the target compounds together with field alignment to define the similar field properties.

Chalcones found in fruits and vegetables have promising cancer chemopreventive properties. This study attempts to identify the anticancer efficacies of chalcone flavokawain B (FKB) in the rhizomes of Alpinia pricei Hayata by examining key molecular events in non-small-cell lung cancer (A549) cells. Our results indicated that in human A549 cells, FKB (0-15 μg/ml) decreases cell viability and colony formation, dysregulates the Bax:B-cell lymphoma 2 ratio and increases apoptotic DNA fragmentation. Mitochondrial (caspase-9/-3 and poly ADP ribose polymerase [PARP]) signaling was found to be involved in FKB-induced apoptosis. In addition, FKB-induced reactive oxygen species (ROS) generation, and N-acetylcysteine attenuated FKB-induced apoptotic cell death. Moreover, FKB triggered autophagy, as evidenced by the improved acidic vesicular organelle formation, lipidated light chain 3 (microtubule-related light chain 3) accumulation, and ATG7 expression and the decreased mammalian target of rapamycin phosphorylation. Furthermore, FKB suppressed ROS-mediated ATG4B expression. Inhibiting autophagy using 3-methyladenine/chloroquine diminished FKB-induced cell death, indicating that autophagy is triggered as a death mechanism by FKB. In summary, FKB has a crucial role in the execution and propagation of ROS-mediated apoptotic and autophagic cell death of lung adenocarcinoma cells.

Oligonucleosomal DNA fragmentation and chromatin condensation are two hallmarks of apoptosis. However, their generation mechanisms are not entirely understood. Histone H1, a positively charged nuclear protein located in the linker region of chromatin, is involved in higher-order chromatin structures and tight chromatin packing. On the basis of the physical and biochemical characteristics of histone H1, we hypothesized that histone H1 plays a role in determining the efficiencies of apoptotic DNA fragmentation and chromatin condensation. Therefore, we examined histone H1 quantity in five human leukemia cell lines and compared the efficiencies. The cell lines were categorized into two groups according to their origins: (i) Ramos and Molt-4 cells of lymphoid origin and (ii) U937, ML-1, and HL60 cells of myeloid origin. Compared to the lymphoid-origin group, the myeloid-origin group had lower levels of histone H1 but more open chromatin. Furthermore, the myeloid-origin group showed marked DNA fragmentation but less chromatin condensation during apoptosis. These results suggested that histone H1 determined chromatin structure and that its quantity affected the efficiencies of DNA fragmentation and chromatin condensation in apoptosis.

DNA double strand breaks (DSB) induced by ionizing radiation (IR) are usually measured using γH2AX/53BP1 DNA repair foci, that is considered to be the most sensitive assay for DSB analysis. While fluorescence microscopy (FM) is the gold standard for this analysis, imaging flow cytometry (IFC) may offer number of advantages such as lack of the fluorescence background, higher number of cells analyzed, and higher sensitivity in detection of DNA damage induced by IR at low doses. Along with appearance of γH2AX foci, the variable fraction of the cells exhibits homogeneously stained γH2AX signal resulting in so-called γH2AX pan-staining, which is believed to appear at early stages of apoptosis. Here, we investigated incidence of γH2AX pan-staining at different time points after irradiation with γ-rays using IFC and compared the obtained data with the data from FM. Appearance of γH2AX pan-staining during the apoptotic process was further analyzed by fluorescence-activated cell sorting (FACS) of cells at different stages of apoptosis and subsequent immunofluorescence analysis. Our results show that IFC was able to reveal dose dependence of pan-staining, while FM failed to detect all pan-staining cells. Moreover, we found that γH2AX pan-staining could be induced by therapeutic, but not low doses of γ-rays and correlate well with percentage of apoptotic cells was analyzed using flow cytometric Annexin-V/7-AAD assay. Further investigations showed that γH2AX pan-staining is formed in the early phases of apoptosis and remains until later stages of apoptotic process. Apoptotic DNA fragmentation as detected with comet assay using FM correlated with the percentage of live and late apoptotic/necrotic cells as analyzed by flow cytometry. Lastly, we successfully tested IFC for detection of γH2AX pan-staining and γH2AX/53BP1 DNA repair foci in lymphocyte of breast cancer patients after radiotherapy, which may be useful for assessing individual radiosensitivity in a clinically relevant cohort of patients.

Extrachromosomal circular DNA elements (eccDNAs) have been described in the literature for several decades, and are known for their broad existence across different species1,2. However, their biogenesis and functions are largely unknown. By developing a new circular DNA enrichment method, here we purified and sequenced full-length eccDNAs with Nanopore sequencing. We found that eccDNAs map across the entire genome in a close to random manner, suggesting a biogenesis mechanism of random ligation of genomic DNA fragments. Consistent with this idea, we found that apoptosis inducers can increase eccDNA generation, which is dependent on apoptotic DNA fragmentation followed by ligation by DNA ligase 3. Importantly, we demonstrated that eccDNAs can function as potent innate immunostimulants in a manner that is independent of eccDNA sequence but dependent on eccDNA circularity and the cytosolic DNA sensor Sting. Collectively, our study not only revealed the origin, biogenesis and immunostimulant function of eccDNAs but also uncovered their sensing pathway and potential clinical implications in immune response. By developing a new eccDNA purification and profiling method, the study revealed close-to-random genomic origination, mechanism of biogenesis and function of eccDNAs.

This in vitro study aimed to measure the ameliorative effect of L-carnitine against apoptosis, DNA fragmentation, membrane integrity and lipid peroxidation of spermatozoa from men with asthenoteratozoospermia (ATS). L-carnitine has an impressive effect on boosting the quality and quantity of spermatozoa and also can prevent apoptosis induction. For this purpose, semen samples were collected from 50 ATS men. Semen was divided into control and L-carnitine (0.5 mg/ml) groups at 2, 4, 6 and 24 hr. Concentrating on the reasons for apoptosis is an arduous process, but in the present research for this evaluation, mitochondrial membrane potential (MMP), DNA fragmentation by TUNEL and SCD methods, and lipid peroxidation were carried out. Also, sperm viability was performed. In the control group, MDA levels were increased significantly at 6 hr; however, sperm viability was decreased significantly at 4 and 6 hr. Moreover, in the L-carnitine group, TUNEL, SCD and MDA levels were decreased significantly and MMP and viability were increased significantly compared with the control group. In this writers' view, in vitro L-carnitine treatment can downregulate apoptosis in men with ATS.

Fertility loss, recurrent spontaneous abortion and poor outcome in assisted reproductive techniques (ART) have been associated with DNA fragmentation. This work was achieved to evaluate the protective role of melatonin versus apoptosis, DNA fragmentation, membrane integrity and lipid peroxidation of spermatozoa from men with asthenoteratozoospermia (ATS). Some researchers maintain that melatonin can serve as a remedy for apoptosis induction, and it has an impressive effect on boosting the quality and quantity of spermatozoa. For this purpose, semen samples were collected from 50 ATS men and they were divided into control and melatonin (6 mM) groups at 2, 4, 6 and 24 hr. Concentrating on the reasons for apoptosis is an arduous process, but in the present study for this evaluation mitochondrial membrane potential (MMP), DNA fragmentation by TUNEL and sperm chromatin dispersion (SCD) methods and lipid peroxidation were carried out. Also, sperm viability was performed. In the control group, MDA, TUNEL-positive and SCD were significantly increased but viability and MMP of spermatozoa were significantly decreased. Moreover, in the melatonin group, TUNEL-positive, SCD and MDA levels were significantly decreased and viability and MMP significantly increased, compared to the control group. In outcome, melatonin prescription paves the way for apoptosis down-regulating in the ATS men.

Progressive Multifocal Leukoencephalopathy (PML) is a fatal demyelinating disease of the CNS, resulting from the lytic infection of oligodendrocytes by the human neurotropic polyomavirus JC (JCPyV), typically associated with severe immunocompromised states and, in recent years, with the use of immunotherapies. Apoptosis is a homeostatic mechanism to dispose of senescent or damaged cells, including virally infected cells, triggered in the vast majority of viral infections of the brain. Previously, we showed upregulation of the normally dormant anti-apoptotic protein Survivin in cases of PML, which&mdash, in vitro&mdash, resulted in protection from apoptosis in JCPyV-infected primary cultures of astrocytes and oligodendrocytes. In the present study, we first demonstrate the absence of apoptotic DNA fragmentation and the lack of caspase activity in 16 cases of PML. We also identified the viral protein large T-Antigen as being responsible for the activation of the Survivin promoter. Chromatin Immunoprecipitation assay shows a direct binding between T-Antigen and the Survivin promoter DNA. Finally, we have identified the specific region of T-Antigen, spanning from amino acids 266 and 688, which binds to Survivin and translocates it to the nucleus, providing evidence of a mechanism that results in the efficient replication of JCPyV and a potential target for novel therapies.

Lactoferrin has gained extensive attention due to its ample biological properties. In this study, recombinant human lactoferrin carrying humanized glycosylation (rhLf-h-glycan) expressed in the yeast Pichia pastoris SuperMan5, which is genetically glycoengineered to efficiently produce functional humanized glycoproteins inclosing (Man)(5)(GlcNAc)(2) Asn-linked glycans, was analyzed, inspecting its potential toxicity against cancer cells. The live-cell differential nuclear staining assay was used to quantify the rhLf-h-glycan cytotoxicity, which was examined in four human cell lines: acute lymphoblastic leukemia (ALL) CCRF-CEM, T-cell lymphoblastic lymphoma SUP-T1, cervical adenocarcinoma HeLa, and as control, non-cancerous Hs27 cells. The defined CC(50) values of rhLf-h-glycan in CCRF-CEM, SUP-T1, HeLa, and Hs27 cells were 144.45 ± 4.44, 548.47 ± 64.41, 350 ± 14.82, and 3359.07 ±164 μg/mL, respectively. The rhLf-h-glycan exhibited a favorable selective cytotoxicity index (SCI), preferentially killing cancer cells: 23.25 for CCRF-CEM, 9.59 for HeLa, and 6.12 for SUP-T1, as compared with Hs27 cells. Also, rhLf-h-glycan showed significant antiproliferative activity (P < 0.0001) at 24, 48, and 72 h of incubation on CCRF-CEM cells. Additionally, it was observed via fluorescent staining and confocal microscopy that rhLf-h-glycan elicited apoptosis-associated morphological changes, such as blebbing, nuclear fragmentation, chromatin condensation, and apoptotic bodies in ALL cells. Furthermore, rhLf-h-glycan-treated HeLa cells revealed shrinkage of the microfilament structures, generating a speckled/punctuated pattern and also caused PARP-1 cleavage, a hallmark of apoptosis. Moreover, in ALL cells, rhLf-h-glycan altered cell cycle progression inducing the G2/M phase arrest, and caused apoptotic DNA fragmentation. Overall, our findings revealed that rhLf-h-glycan has potential as an anticancer agent and therefore deserves further in vivo evaluation.

Increasing evidence suggests that apoptosis of tubular cells and renal inflammation mainly determine the outcome of sepsis-associated acute kidney injury (AKI). The study aim was to investigate the molecular mechanism involved in the renoprotective effects of simvastatin in endotoxin (lipopolysaccharide, LSP)-induced AKI. A sepsis model was established by intraperitoneal injection of a single non-lethal LPS dose after short-term simvastatin pretreatment. The severity of the inflammatory injury was expressed as renal damage scores (RDS). Apoptosis of tubular cells was detected by Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling (TUNEL assay) (apoptotic DNA fragmentation, expressed as an apoptotic index, AI) and immunohistochemical staining for cleaved caspase-3, cytochrome C, and anti-apoptotic Bcl-xL and survivin. We found that endotoxin induced severe renal inflammatory injury (RDS = 3.58 &plusmn, 0.50), whereas simvastatin dose-dependently prevented structural changes induced by LPS. Furthermore, simvastatin 40 mg/kg most profoundly attenuated tubular apoptosis, determined as a decrease of cytochrome C, caspase-3 expression, and AIs (p&thinsp, &lt, &thinsp, 0.01 vs. LPS). Conversely, simvastatin induced a significant increase of Bcl-XL and survivin, both in the strong inverse correlations with cleaved caspase-3 and cytochrome C. Our study indicates that simvastatin has cytoprotective effects against LPS-induced tubular apoptosis, seemingly mediated by upregulation of cell-survival molecules, such as Bcl-XL and survivin, and inhibition of the mitochondrial cytochrome C and downstream caspase-3 activation.

The current increase in the use of multi-herbal remedies coupled with loose regulation on public access to these products underscore research efforts to evaluate their biochemical effect, noting that many of the herbal medicines lack scientific evidence to support their medicinal claims. Objective: We therefore investigated the potential genotoxicity and hematotoxicity of commonly consumed multi-herbal formulations (YoyoBitters™, Ogidiga™ and BabyOku™) in Lagos, Nigeria, in experimental mice. Methods: Fifty (50) adult female albino mice were randomly selected and distributed into 5 groups of 10 mice each. Two mL/kg body weight of distilled water were orally administered to the control groups while BabyOku™, YoyoBitters™ and Ogidiga™ herbal formulations were administered to the experimental groups at doses of 2 mL/kg body weights. Results: A dose- and tissue-dependent increase in induction of apoptotic DNA fragmentation was observed in the triherbal groups relative to control groups. Also, an increase in micronucleated polychromatic erythrocytes was formed in a dose-dependent manner in the multi-herbal groups when compared with the control groups. Conclusion: From our findings, multi-herbal formulations may possess hematotoxic and genotoxic potentials in mice.

A large number of plant extracts have demonstrated to provide health benefits and mitigate several disease conditions. However, at the molecular and cellular levels, few studies have been conducted. The present work was designed to study the effect of Myrtus communis leaf extract (ME) (300 mg/kg bw) against hepatotoxicity induced by monosodium glutamate (MSG) (100 mg/kg bw), and acrylamide (ACR) (20 mg/kg bw) in male rats and determining its molecular and cellular mechanisms. The data showed that the treatment with MSG and/or ACR induced significant changes in numerous biomarkers (Bcl-2 and the programmed cell death protein-1) related to liver damage, as recorded by genotoxicity, apoptosis, and histopathological changes. On the other side, the oral administration of ME (300 mg/kg bw) improved the hepatic conditions as confirmed by the improvement in cell viability, programmed cell death, and histopathological alterations. It can be concluded that the consumption of ME might be useful for minimizing the occurred hepatotoxicity through up-regulation of the key apoptotic regulators as well as the improvement of DNA content and cell cycle restoration. Graphical abstract.

Type I/II interferons (IFNα,β/IFNɣ) are cytokines that activate signal-transducer-and-activator-of-transcription-1 (STAT1). The STAT1 N-terminal domain (NTD) mediates dimerization and cooperative DNA-binding. The STAT1 DNA-binding domain (DBD) confers sequence-specific DNA-recognition. STAT1 has been connected to growth inhibition, replication stress and DNA-damage. We investigated how STAT1 and NTD/DBD mutants thereof affect fibrosarcoma cells. STAT1 and indicated mutants do not affect proliferation of resting and IFNα-treated cells as well as checkpoint kinase signaling, and phosphorylation of the tumor-suppressive transcription factor p53 ensuing ɣ-irradiation. Of the STAT1 reconstituted U3A cells those with STAT1 NTD mutants accumulate the highest levels of the replication stress/DNA-damage marker S139-phosphorylated histone H2AX (ɣH2AX). This is similarly seen with a STAT1 NTD/DBD double mutant, indicating transcription-independent effects. Furthermore, U3A cells with STAT1 NTD mutants are most susceptible to apoptotic DNA fragmentation and cleavage of the DNA repair protein PARP1. These data provide novel insights into the relevance of the STAT1 NTD.

This study aims to establish the biological and chemical profile of Asphodeline liburnica (Scop.) Rchb. root. The antioxidant, antimicrobial, enzyme inhibitory, DNA protection, apoptotic DNA ladder fragmentation analysis, and anti-proliferative of A. liburnica were established using standard assays. In silico study was also performed to understand interactions between quantified anthraquinones and key enzymes of clinical relevance. Total phenolic and flavonoid contents were found to be 9.67 mgGAE/g and 1.48 mgRE/g extract, respectively. Chrysophanol was detected as a major anthraquinone. The extract exhibited radical scavenging ability against DPPH and ABTS with values of 13.23 and 66.99 mgTE/g extract, respectively. Good inhibitory activity against tyrosinase was recorded. In silico experiments showed that the anthraquinones were able to establish coordinative bonds with the copper atoms present in the enzymatic cavity of tyrosinase. MTT cell viability test on MDA-MB-231 cells showed that at 0.1 and 1 μg of extracts induced anti-proliferative effect. Apoptotic DNA fragmentation analysis indicated nuclear condensation resulting in DNA fragmentation, which exhibited apoptotic cell death in the presence of A. liburnica. This study has provided insights on the potential usage of A. liburnica which could open new avenues for research and stimulate future interest for the development of safe novel biopharmaceuticals.

Bone pain is the major manifestation of skeletal metastases. Although various treatment modalities are available for bone pain palliation, use of radiolabeled phosphonates is documented to be more effective. Among radionuclides available for this purpose, lutetium-177 is gaining popularity due to its moderate beta energy, theranostic capability, favorable half-life and convenient production logistics. 177 Lu-DOTMP has shown considerable promise as a metastatic bone pain palliating agent in preliminary evaluations and recent clinical studies. Therefore, an attempt was made to elucidate the possible mechanism of in vitro cell death induced by 177 Lu-DOTMP in MG63 cells. 177 Lu-DOTMP binding studies were carried out in mineralized bone of MG63 cells and around 50% binding was observed. Skeletons of Wistar rats showed 1.78 ± 0.5% IA/g at a 3 h time period which was almost constant up to 7 days. MG63 cells were incubated with 3.7 and 37 MBq of 177 Lu-DOTMP for 48 h prior to perform assays. An increase in the magnitude of cell toxicity and apoptotic DNA fragmentation was observed. Enhancement of G2/M phase cell cycle arrest and apoptosis were documented which were dose-dependent. Thus, 177 Lu-DOTMP induced apoptotic cell death in MG63 cells, which might be one of the primary causes of pain relief in osseous metastases.

Canine osteosarcoma is an aggressive primary bone tumor that shows metastasis to distal regions and is associated with a high mortality rate. However, the pathophysiological mechanisms of canine osteosarcoma are not well characterized. In addition, development of prognostic factors and novel therapeutic agents is necessary to efficiently treat osteosarcoma. Therefore, we studied the effects of myricetin, an antioxidant found in berries, nuts, teas, wine, and vegetables, on apoptosis and signal transduction in the canine osteosarcoma cell lines, D-17 and DSN. Results of the present study demonstrated that treatment with myricetin decreased cell proliferation and DNA replication, while it increased apoptotic DNA fragmentation in D-17 and DSN cells. In addition, it increased generation of ROS, lipid peroxidation, and depolarization of MMP in both D-17 and DSN cells. Myricetin treatment activated phosphorylation of AKT, p70S6K, ERK1/2, JNK, and p90RSK in canine osteosarcoma cells. Moreover, inhibition of PI3K and MAPK using LY294002, U0126, or SP600125, in addition to myricetin treatment, effectively suppressed cell proliferation compared to treatment with myricetin or each inhibitor alone. Therefore, we concluded that myricetin may be a potentially effective and less toxic therapeutic agent to prevent and control progression of canine osteosarcoma.

One hallmark of cancer is its ability to recruit a vascular supply to support rapid growth. Suppression of angiogenesis holds potential as a second-line or adjuvant therapy to stunt cancer growth, progression, metastasis, and post-resection regeneration. To begin to test the hypothesis that semaphorin 3A and 3F together, will induce endothelial cell apoptosis by inducing DNA damage, mixed primary cultures isolated from normal adult mouse skeletal muscle were treated for 48 hr with Sema3A ± Sema3F (100ng/mL). Changes in surviving-cell density, DNA synthesis, DNA repair (gamma-Histone 2AX, γH2AX, an indirect measure for DNA damage), and apoptotic DNA fragmentation (TUNEL staining) were assayed in cultures of CD31+ endothelial and desmin+ muscle cells. Sema3F increased DNA damage-associated DNA repair in both cell types. Co-treatment with Sema3A+3F increased γH2AX staining ~25-fold over control levels, and further increased apoptosis compared to control and Sema3A alone. Results were negated by treatment with neutralizing anti-semaphorin antibodies and are interpreted as suggesting that Sema3A may sensitize endothelial but not muscle cells to Sema3F-induced DNA damage. These preliminary findings on a complex system of interacting cells may contribute to developing applications that could target angiogenic regulatory mechanisms for their therapeutic potential against cancer progression and metastasis.

Significance Cell death-inducing DFF45-like effector (CIDE) domains, initially identified in apoptotic nucleases, form a highly conserved family with diverse functions ranging from cell death to lipid homeostasis and synaptic regulation. Through structural determination of two CIDE family proteins, Drep2 and Drep4, we found that CIDE domains can form helical oligomers. Our results reveal that such higher-order structures not only are conserved in the CIDE family, but also are critically important for both DNA fragmentation and lipid droplet fusion. Therefore, our findings identify the CIDE domain as a scaffolding component for higher-order structure assembly. Our results expand the importance of higher-order structures from the established field of immune signaling to broader biological functions.

Purpose 4-[4′′-(2′′, 2′′, 6′′, 6′′-tetramethyl-l′′-piperidinyloxy) amino]–4′-demethyl-epipodophyllotoxin (GP7) is a new semi-synthesized nitroxyl spin-labeled derivative of podophyllotoxin with anti-leukemic and anti-osteosarcoma effects. The purpose of the present study is to investigate the anti-gastric cancer (GC) effects of GP7 and the possible involvement of caspase pathway in GP7-induced apoptotic DNA fragmentation in human GC cells. Materials and methods Effects of GP7 on the proliferation of human GC cell lines MKN28, AGS, BGC-823 and HGC-27 in different degrees of differentiation and normal human gastric epithelial cell line GES-1 were studied by MTT assay and compared with the effects of etoposide. Effects of GP7 on cell viability and heat shock protein 90 expression of BGC-823 and HGC-27 cells were analyzed by trypan blue exclusion test and western blotting, respectively. Effects of GP7 on apoptotic DNA fragmentation and caspase pathway of BGC-823 and HGC-27 cells were detected by agarose gel electrophoresis, colorimetric assay and western blotting. Caspase-3 inhibitor was used to manipulate the activity of caspase-3. Results GP7 inhibited concentration- and time-dependently the proliferation of human GC cells, and the inhibitory effect of GP7 on the proliferation of BGC-823 or HGC-27 cells was 1.15- or 1.21-fold higher than that of etoposide. GP7 downregulated heat shock protein 90, improved the anti-GC effects of adriamycin, cisplatin, 5-fluorouracil and their combinations, induced apoptotic DNA fragmentation, activations of caspase-9 and -3 but not -8, cytochrome-c release and BID cleavage in BGC-823 and HGC-27 cells. Caspase-3 inhibitor abrogated GP7-induced BID cleavage, decreased cytochrome-c release, caspase-9 and -3 activities and apoptotic DNA fragmentation but increased cell viability in BGC-823 and HGC-27 cells. Conclusion Our findings indicate that GP7 is a promising anti-GC derivative of podophyllotoxin, and GP7-induced apoptosis in human GC cells may be mediated by mitochondrial pathway with caspase-3-dependent BID cleavage.

Graphene, a crystalline allotrope or carbon, presents numerous useful properties; however, its toxicity is yet to be determined. One of the most dramatic and irreversible toxic abilities of carbon nanomaterials is the induction of DNA fragmentation produced by endogenous cellular endonucleases. This study demonstrated that pristine graphene exposed to cultured kidney tubular epithelial cells is capable of inducing DNA fragmentation measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, which is usually associated with cell death. TUNEL (cell death) and endonuclease activity measured using a near infrared fluorescence probe was significantly higher in cells containing graphene aggregates detected by Raman spectroscopy. The elevation of TUNEL coincided with the increased abundance of heme oxygenase 1 (HO-1), heat shock protein 90 (HSP90), active caspase-3 and endonucleases (deoxyribonuclease I [DNase I] and endonuclease G [EndoG]), as measured by quantitative immunocytochemistry. Specific inhibitors for HO-1, HSP90, caspase-3, DNase I and EndoG almost completely blocked the DNA fragmentation induced by graphene exposure. Therefore, graphene induces cell death through oxidative injury, caspase-mediated and caspase-independent pathways; and endonucleases DNase I and EndoG are important for graphene toxicity. Inhibition of these pathways may ameliorate cell injury produced by graphene. Copyright © 2017 John Wiley & Sons, Ltd.

Background: Apoptosis results in specific and stage-dependent morphological alterations of the cell nucleus, including pyknosis and cell shrinking. The experimental investigation of apoptotic processes is still challenging and routinely based on the assessment of molecular events like chromatin fragmentation and caspase enzyme activity. Alternatively, the establishment of a fluorescence microscopy nuclear morphology assay would provide a simple and robust low-cost method for detection and quantification of apoptotic cascades. Materials and Methods: Model cell lines LNCaP and MDA-MB-231 were incubated in the presence of the apoptosis-inducer cycloheximide (CHX). After evaluation of apoptotic cascades by terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL) assay, stained cell nuclei were analyzed regarding area, perimeter, major and minor axis, as well as brightness of nuclear fluorescence signal. Results: When compared to vehicle-treated control cells, administration of CHX led to significantly reduced cell growth and elevated rates of chromatin fragmentation of both cell lines as shown by cell counting and TUNEL assay, respectively. These apoptotic effects were accompanied by apoptosis-specific modulations of the nuclei demonstrated by diminished nuclear morphology parameters, such as area, perimeter, major and minor axis, as well as elevated levels of nuclear staining intensity. Conclusion: We present a computerized method for apoptosis detection and quantification using images of fluorescent dye-stained cell nuclei. The advantages of this nuclear morphology assay include the (i) ability to routinely assess apoptosis by a fast, highly reproducible low-cost technique, (ii) applicability of an experimental approach analyzing high numbers of single nuclei and (iii) detection of apoptosis in early, as well as late, stages of the apoptotic cascade.

Lectins are proteins that show a variety of biological activities. Nevertheless, information on lectin from Gluttonous beauts and their anticancer activities are very limited. In this study, we purified a lectin from hemolymph of G. beauts and identified its molecular weight to be 66 kDa. The effect of lectin at different concentrations (μg/mL) on the cell growth and apoptosis were evaluated against MCF-7 and MCF-10A cells, whereas cytotoxicity to the MCF-7 cells mediated by lectin was observed and the mechanism of action of the lectin in including apoptosis in cancer cells via the intrinsic pathway was also proposed. The MCF-7 cells were employed for in vitro studies on cytotoxicity, induction of apoptosis and apoptotic DNA fragmentation. In MCF-10A cells lectin did not show any adverse effect even at higher concentration. Cell cycle analysis also showed a significant cell cycle arrest on selected cells after lectin treatment. Western blotting suggested that lectin up regulates the apoptotic protein expression in MCF-7 cells while it down regulates the level of Bcl-2 expression.

Patients with a relapse of idiopathic nephrotic syndrome have significantly increased levels of serum complement component 5a (C5a), and proteinuria has been noted in mice treated with C5a via changes in permeability of kidney endothelial cells (KECs) in established animal models. However, the apoptosis of KECs treated with high concentrations of C5a has also been observed. As mitochondrial damage is known to be important in cell apoptosis, the aim of this study was to examine the association between C5a-induced mouse KEC apoptosis and mitochondrial damage. Mouse KECs were isolated and treated with different concentrations of C5a. Cell viability assays showed that a high-concentration mouse recombinant protein C5a (rmC5a) treatment reduced mouse KEC growth. Cell cycle phase analysis, including apoptosis (sub-G1 phase) showed an increased percentage of the subG1 phase with a high-concentration rmC5a treatment. Cytochrome c and caspase 3/9 activities were significantly induced in the mouse KECs after a high-dose rmC5a (50 ng/mL) treatment, and this was rescued by pretreatment with the C5a receptor (C5aR) inhibitor (W-54011) and N-acetylcysteine (NAC). Reactive oxygen species (ROS) formation was detected in C5a-treated mouse KECs, however, W-54011 or NAC pretreatment inhibited high-dose rmC5a-induced ROS formation and also reduced cytochrome c release, apoptotic cell formation, and apoptotic DNA fragmentation. These factors determined the apoptosis of mouse KECs treated with high-dose C5a through C5aR and subsequently led to apoptosis via ROS regeneration and cytochrome c release. The results showed that high concentrations of C5a induced mouse KEC apoptosis via a C5aR/ROS/mitochondria-dependent pathway. These findings may shed light on the potential mechanism of glomerular sclerosis, a process in idiopathic nephrotic syndrome causing renal function impairment.

Background and Objectives: The antitumor activities of capsaicin on various types of cancer cell lines have been reported but the effect of capsaicin on oral cancer, which is prevalent among Asians, are very limited. Thus, this study aimed to investigate the effects of capsaicin on ORL-48, an oral cancer cell line of Asian origin. Materials and Methods: Morphological changes of the ORL-48 cells treated with capsaicin were analyzed using fluorescence microscopy. The apoptotic-inducing activity of capsaicin was further confirmed by Annexin V-Fluorescein isothiocyanate / Propidium iodide (V-FITC/PI) staining using flow cytometry. In order to establish the pathway of apoptosis triggered by the compound on ORL-48 cells, caspase activity was determined and the mitochondrial pathway was verified by mitochondrial membrane potential (MMP) assay. Cell cycle analysis was also performed to identify the cell cycle phase of ORL-48 cells being inhibited by the capsaicin compound. Results: Fluorescence microscopy exhibited the presence of apoptotic features in capsaicin-treated ORL-48 cells. Apoptosis of capsaicin-treated ORL-48 cells revealed disruption of the mitochondrial-membrane potential, activation of caspase-3, -7 and -9 through an intrinsic apoptotic pathway and subsequently, apoptotic DNA fragmentation. The cell cycle arrest occurred in the G1-phase, confirming antiproliferative effect of capsaicin in a time-dependent manner. Conclusion: This study demonstrated that capsaicin is cytotoxic against ORL-48 cells and induces apoptosis in ORL-48 cells possibly through mitochondria mediated intrinsic pathway resulting in cell cycle arrest.

WOS: 000479328000035, PubMed: 31185340, This study aimed to reveal chemical profiles and biological activities of ethyl acetate (EA), methanol (MeOH), and water extracts of Lotus corniculatus. Ethnobotanical reports have indicated the importance of phytochemical properties of the genus Lotus. In this study, the effects of medicinal plant extracts on antioxidant (DPPH, ABTS, CUPRAC, FRAP, phosphomolybdenum, and metal chelating assays), enzyme inhibitory (on cholinesterase, tyrosinase, a-amylase and a-glucosidase), DNA protection and anticancer properties (including anti-proliferative, cell death and telomerase activity marker gene analysis, apoptotic DNA fragmentation analysis, cell migration test) were evaluated. According to chemical analysis, quercetin derivatives geraldol, isorhamnetin and kaempferol-O-coumaroylhexoside-O-deoxyhexoside isomers were dominant in the extracts. MeOH extracts showed the highest total flavonoids capacity with 21.13 mg RE/g. EA extract showed the strongest anti-amylase activity among the tested extracts. Water extract had the most protective activity against plasmid DNA. To indicate cell survival, MIT test was performed against human MCF-7 and MDA-MB-231 breast cancer cells. Half-maximal inhibitory concentration for cells were calculated and used for detection of mechanisms behind the cancer cell death. EA extract showed up-regulation of Bax proapoptotic gene and apoptotic DNA fragmentation activity on highly invasive MDA-MB-231 cells. Beclin-1 and LC3-II autophagy genes were higly expressed after treatment of MCF-7 cells with EA extracts. EA and MeOH extracts inhibited cell migration ability of both cancer cells. Linoleamide, was dominant component in EA extract and caused apoptosis on MDA-MB-231 breast cancer cells via increasing intranuclear Ca2+. The detailed mechanism behind the anticancer properties should be further investigated. (C) 2019 Elsevier B.V. All rights reserved., Kastamonu University (Kastamonu University Scientific Projects Research Office) [KUBAP-01/2018-7], The authors gratefully acknowledge the generous technical and financial support of Kastamonu University (Kastamonu University Scientific Projects Research Office (Project Number KUBAP-01/2018-7).

Berberine is a bioactive isoquinoline alkaloid derived from many plants. Although berberine has been shown to inhibit growth and induce apoptosis of several tumor cell lines, its poor absorption and moderate activity hamper its full therapeutic potential. Here, we describe the synthesis of a series of 9-O-substituted berberine derivatives with improved antiproliferative and apoptosis-inducing activities. An analysis of novel berberine derivatives by EPR spectroscopy confirmed their similar photosensitivity and analogous behavior upon UVA irradiation as berberine, supporting their potential to generate ROS. Improved antitumor activity of novel berberine derivatives was revealed by MTT assay, by flow cytometry and by detection of apoptotic DNA fragmentation and caspase-3 activation, respectively. We showed that novel berberine derivatives are potent inhibitors of growth of HeLa and HL-60 tumor cell lines with IC50 values ranging from 0.7 to 16.7 &micro, M for HL-60 cells and 36 to &gt, 200 &micro, M for HeLa cells after 48 h treatment. Further cell cycle analysis showed that the observed inhibition of growth of HL-60 cells treated with berberine derivatives was due to arresting these cells in the G2/M and S phases. Most strikingly, we found that berberine derivative 3 (9-(3-bromopropoxy)-10-methoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquino[3,2-a] isoquinolin-7-ylium bromide) possesses 30-fold superior antiproliferative activity with an IC50 value of 0.7 &micro, M and 6-fold higher apoptosis-inducing activity in HL-60 leukemia cells compared to berberine. Therefore, further studies are merited of the antitumor activity in leukemia cells of this berberine derivative.

Cell death-inducing DFF45-like effect (CIDE) domain-containing proteins, DFF40, DFF45, CIDE-A, CIDE-B, and FSP27, play important roles in apoptotic DNA fragmentation and lipid homeostasis. The function of DFF40/45 in apoptotic DNA fragmentation is mediated by CIDE domain filament formation. Although our recent structural study of DREP4 CIDE revealed the first filament-like structure of the CIDE domain and its functional importance, the filament structure of DREP2 CIDE is unclear because this structure was not helical in the asymmetric unit. In this study, we present the crystal structure and mutagenesis analysis of the DREP2 CIDE mutant, which confirmed that DREP2 CIDE also forms a filament-like structure with features differing from those of DREP4 CIDE.

Treatment of Jurkat T cells with the dynamin inhibitor, myristyl trimethyl ammonium bromides (MiTMAB) caused cytokinesis impairment and apoptotic DNA fragmentation along with down-regulation of anti-apoptotic BAG3 and Mcl-1 levels, Bak activation, mitochondrial membrane potential (Δψm) loss, activation of caspase-9 and -3, and PARP cleavage, without accompanying necrosis. Bcl-xL overexpression completely abrogated these MiTMAB-induced mitochondrial damage and resultant caspase cascade activation, except for impaired cytokinesis and down-regulated BAG3 and Mcl-1 levels. Additionally, autophagic responses including Akt-mTOR pathway inhibition, formation of acridine orange-stainable acidic vesicular organelles, LC3-I/II conversion, and p62/SQSTM1 down-regulation were detected regardless of Bcl-xL overexpression. The autophagy inhibitors 3-methyladenine and LY294002 enhanced MiTMAB-induced apoptotic sub-G1 peak, BAG3 and Mcl-1 down-regulation, Bak activation, Δψm loss, and caspase activation. These results indicate that MiTMAB-caused cytokinesis failure leads to concomitant induction of apoptosis and cytoprotective autophagy, and suggest that inhibition of autophagy is a promising strategy to augment antitumor activity of MiTMAB.

Deoxyribonuclease I (DNase I) is an endonuclease responsible for the destruction of chromatin during apoptosis. However, its role in diabetes remains unclear. The aim of the current study was to investigate the role of DNase I combined with high glucose levels in β-cell apoptosis. Human samples were collected and the DNase I activity was examined. High glucose-cultured INS-1 cells were transfected with DNase I small interfering RNA (siRNA) and the cell apoptosis was examined by western blotting and flow cytometry. Cell viability was analyzed by the Cell Counting Kit-8 assay. Cell apoptosis resulting from 50 mU/μl DNase I was also observed by flow cytometry, terminal deoxynucleotidyl transferase dUTP nick-end labeling stain and western blotting. Compared with healthy controls, the serum DNase I activity of patients with diabetes was significantly increased (P

Background Cleistopholine is a natural alkaloid present in plants with numerous biological activities. However, cleistopholine has yet to be isolated using modern techniques and the mechanism by which this alkaloid induces apoptosis in cancer cells remains to be elucidated. Hypothesis/purpose This study aims to isolate cleistopholine from the roots of Enicosanthellum pulchrum by using preparative-HPLC technique and explore the mechanism by which this alkaloid induces apoptosis in human ovarian cancer (CAOV-3) cells in vitro from 24 to 72 h. This compound may be developed as an anticancer agent that induces apoptosis in ovarian cancer cells. Study design/methods Cytotoxicity was assessed via the cell viability assay and changes in cell morphology were observed via the acridine orange/propidium iodide (AO/PI) assay. The involvement of apoptotic pathways was evaluated through caspase analysis and multiple cytotoxicity assays. Meanwhile, early and late apoptotic events via the Annexin V-FITC and DNA laddering assays, respectively. The mechanism of apoptosis was explored at the molecular level by evaluating the expression of specific genes and proteins. In addition, the proliferation of CAOV-3-cells treated with cleistopholine was analysed using the cell cycle arrest assay. Results The IC 50 of cleistopholine (61.4 µM) was comparable with that of the positive control cisplatin (62.8 µM) at 24 h of treatment. Apoptosis was evidenced by cell membrane blebbing, chromatin compression and formation of apoptotic bodies. The initial phase of apoptosis was detected at 24 h by the increase in Annexin V-FITC binding to cell membranes. A DNA ladder was formed at 48 h, indicating DNA fragmentation in the final phase of apoptosis. The mitochondria participated in the process by stimulating the intrinsic pathway via caspase 9 with a reduction in mitochondrial membrane potential (MMP) and an increase in cytochrome c release. Cell death was further validated through the mRNA and protein overexpression of Bax, caspase 3 and caspase 9 in the treated cells compared with the untreated cells. In contrast, Bcl-2, Hsp70 and survivin decreased in expression upon cleistopholine treatment. Cell cycle was arrested at the G0/G1 phase and cell population percentage significantly increased to 43.5%, 45.4% and 54.3% in time-dependent manner in the cleistopholine-treated CAOV-3 cells compared with the untreated cells at 24, 48 and 72 h respectively. Conclusion The current study indicated that cleistopholine can be a potential candidate as a new drug to treat ovarian cancer disease.

Nontypeable Haemophilus influenzae (NTHi) is a common airway commensal and opportunistic pathogen that persists within biofilm communities in vivo. Biofilm studies so far are mainly based on assays on plastic surfaces. The aim of this work was to investigate the capacity of clinical NTHi strains to form biofilm structures on polarized Calu-3 human airway epithelial cells and primary normal human bronchial epithelial cells and to characterize the biofilm architecture. Formation of adherent NTHi biofilms post colonization of host cells at multiple time-points was evaluated using confocal laser scanning microscopy and electron microscopy. NTHi biofilms were analyzed in terms of biofilm height and presence of extracellular matrix components, and their apoptotic effects on epithelial cells were measured by TUNEL assay. Strain Fi176 was observed to form robust biofilms on airway epithelia over time, while disrupting the integrity of Calu-3 monolayer by 72 h of co-culture. NTHi biofilms were observed to induce apoptotic DNA fragmentation in host cells at 24 h post infection. Biofilm formation on cell monolayers by Fi176ΔpilA strain was markedly reduced compared to WT strain. Biofilm inhibition and disruption assays by crystal violet staining indicated that DNA and proteins are part of NTHi biofilms in vitro. Our findings highlight critical stages of NTHi pathogenesis following host colonization and provide useful biofilm models for future antimicrobial drug discovery investigations.

A series of phosphino-thiosemicarbazone gold(I) dinuclear complexes obtained by two different synthetic procedures have been prepared. All the compounds have been spectroscopically characterized including single crystal X ray diffraction analysis in some of cases. [Au2(HL1)Cl2] (1), [Au2(HL2)2]Cl2 (2) and [Au2(HL3)2]Cl2 (3) have been prepared by chemical synthesis using a gold(III) salt as precursor; while [Au2(L1)2] (4), [Au2(L2)2]∙2CH3CN (5) and [Au2(L3)2] (6) have been isolated from an electrochemical synthesis (HLn = 2-[2-(diphenylphosphanyl)-benzylidene]-N-R-thiosemicarbazone; HL1: R = methyl, HL2: R = methoxyphenyl, HL3: R = nitrophenyl). The in vitro cytotoxic activity of these gold(I) complexes was tested against some human tumor cell lines: HeLa 229 (cervical epithelial carcinoma), MCF-7 (ovarian adenocarcinoma), NCI-H460 (non-small-cell lung cancer) and MRC5 (normal human lung fibroblast), and the IC50 values compared with those of cisplatin. The neutral methyl-substituted complexes 1 and 4 and methoxyphenyl 5 displayed significant cytotoxic activities in all investigated cancer cell lines, being 1 and 4 the most effective. The ability of complexes 1 and 4 to induce cell death by apoptosis in Hela 229 was also investigated by fluorescence microscopy using the apoptotic DNA fragmentation as marker. These results indicated that the inhibition of cell proliferation is mainly due to an apoptotic process. In order to obtain more information about the mechanism of action of these metallocompounds, the interactions of complexes 1 and 4 with the thioredoxin reductase (TrxR) enzyme were analyzed. Both complexes exhibited a strong inhibition of the thioredoxin reductase activity.

Possible genotoxic effect of thiacloprid on bovine cultures of whole blood was investigated using chromosomal aberrations (CAs), micronuclei (MN), sister chromatid exchanges (SCEs), DNA damage and apoptotic DNA fragmentation assays. The cells of whole blood were exposed to thiacloprid (30, 60, 120, 240 and 480 μg mL−1) for the last 24 and 48 h of cultivation. Thiacloprid did not induce significant increase in CAs after 24 and 48 h; only the concentration of 120 μg mL−1 caused elevation of CAs (p

PURPOSE: Amongst the epigenetically-targeted therapies, targeting of the histone deacetylases (HDACs) has yielded numerous drugs for clinical use in hematological malignancies, but none as yet for acute lymphocytic leukemia (ALL). Single agent activity of HDAC inhibitors (HDACi) has been elusive in ALL, and has prompted study of combinatorial strategies. Because several HDACi raise levels of intracellular oxidative stress, we evaluated combinations of two structurally distinct HDACi with the redox active compound adaphostin in acute lymphocytic leukemia (ALL). METHODS: The HDACi vorinostat and entinostat were tested in combination with adaphostin in human ALL cell lines. DNA fragmentation, caspase activation, mitochondrial disruption and levels intracellular peroxides, superoxide and glutathione were measured in cells treated with the HDACi/adaphostin combinations. Antioxidant blockade of cell death induction and gene expression profiling of cells treated with vorinostat/adaphostin versus entinostat/adaphostin combinations was evaluated. RESULTS: Both combinations synergistically induced apoptotic DNA fragmentation, which was preceded by an increase in superoxide levels, a reduction in mitochondrial membrane potential, and an increase in caspase-9 activation. The antioxidant N-acetylcysteine (NAC) blocked superoxide generation and prevented reduction of mitochondrial membrane potential. NAC decreased DNA fragmentation and caspase activity in cells treated with adaphostin and vorinostat, but not in those treated with adaphostin and entinostat. Gene expression arrays revealed differential regulation of several redox genes prior to cell death induction. CONCLUSIONS: A redox modulatory agent, adaphostin, enhances efficacy of two HDACi, vorinostat or entinostat, but via different mechanisms indicating a point of divergence in the mechanisms of synergy between the two distinct HDACi and adaphostin.

Benzophenone-3 (BP-3) is the most commonly used chemical UV filter. This compound can easily be absorbed through the skin and the gastrointestinal tract and can disturb sex hormone receptor function. BP-3 is lipophilic and should cross the blood-brain barrier and it may reduce the survival of neurons, although so far, its effects on nerve cells have been studied in only in vitro cultures. The aim of the present study was to determine the effects of BP-3 on apoptosis and the expression of oestrogen, androgen and arylhydrocarbon receptors (AhR) in the rat frontal cortex and hippocampus. This compound was administered dermally to female rats during pregnancy and next to their male offspring through 6 and 7 weeks of age. BP-3 in the frontal cortex induced the mitochondrial apoptosis pathway by increasing the active forms of caspase-3 and caspase-9, inducing the pro-apoptotic proteins Bax and Bak and increasing the number of cells with apoptotic DNA fragmentation. In the hippocampus, an increase in the caspase-9 level and a downward trend in the level of anti-apoptotic proteins were observed. In both brain regions, the contents of ERβ in the nuclear fraction and GPR30 in the membrane fraction were significantly reduced. BP-3 significantly increased AhR in the cytosol of the frontal cortex but had no effect on the content of this receptor in the hippocampus. This is the first study showing that exposure to BP-3 induces the mitochondrial apoptosis pathway in the rat frontal cortex and this effect may result from a weakening of the neuroprotective effects of oestrogen and/or an intensification of AhR-mediated apoptosis.

Cells contain several apoptotic endonucleases, which appear to act simultaneously before and after cell death by destroying the host cell DNA. It is largely unknown how the endonucleases are being induced and whether they can regulate each other. This study was performed to determine whether apoptotic mitochondrial endonuclease G (EndoG) can regulate expression of other apoptotic endonucleases. The study showed that overexpression of mature EndoG in kidney tubular epithelial NRK-52E cells can increase expression of caspase-activated DNase (CAD) and four endonucleases that belong to DNase I group including DNase I, DNase X, DNase IL2, and DNase γ, but not endonucleases of the DNase 2 group. The induction of DNase I-type endonucleases was associated with DNA degradation in promoter/exon 1 regions of the endonuclease genes. These results together with findings on colocalization of immunostained endonucleases and TUNEL suggest that DNA fragmentation after EndoG overexpression was caused by DNase I endonucleases and CAD in addition to EndoG itself. Overall, these data provide first evidence for the existence of the integral network of apoptotic endonucleases regulated by EndoG.

Summary We aimed to determine the protective effects of thymoquinone (TQ), against ischaemia–reperfusion (I/R) injury in the testis tissue of rats. Twenty-seven male Wistar albino rats were randomly divided into three equal groups as follows: Group I, sham group; Group II, torsion group; and Group III, torsion + thymoquinone group. The ischaemia period was 2 h, and orchiectomy was performed after 30 min of detorsion. Testis tissue sections were analysed with the terminal transferase mediated dUTP-nick end labelling (TUNEL) assay to determine in situ apoptotic DNA fragmentation. Additionally, Caspase 3 and Bax proteins were analysed immunohistochemically. The superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) activity levels in the testis tissue were also measured. The superoxide dismutase activity and malondialdehyde levels in the torsion group were significantly higher than those of the sham group (P

The present study sought to determine the correlation between 2-methoxyestradiol (2-MeO-E2)-induced cell cycle arrest and 2-MeO-E2-induced apoptosis. Exposure of Jurkat T cell clone (JT/Neo) to 2-MeO-E2 (0.5-1.0 μM) caused G2/M arrest, Bak activation, Δψm loss, caspase-9 and -3 activation, PARP cleavage, intracellular ROS accumulation, and apoptotic DNA fragmentation, whereas none of these events except for G2/M arrest were induced in Jurkat T cells overexpressing Bcl-2 (JT/Bcl-2). Under these conditions, Cdk1 phosphorylation at Thr-161 and dephosphorylation at Tyr-15, up-regulation of cyclin B1 expression, histone H1 phosphorylation, Cdc25C phosphorylation at Thr-48, Bcl-2 phosphorylation at Thr-56 and Ser-70, Mcl-1 phosphorylation at Ser-159/Thr-163, and Bim phosphorylation were detected irrespective of Bcl-2 overexpression. Concomitant treatment of JT/Neo cells with 2-MeO-E2 and the G1/S blocking agent aphidicolin resulted in G1/S arrest and abrogation of all apoptotic events, including Cdk1 activation, phosphorylation of Bcl-2, Mcl-1 and Bim, and ROS accumulation. The 2-MeO-E2-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by a Cdk1 inhibitor, but not by an Aurora A kinase (AURKA), Aurora B kinase (AURKB), JNK, or p38 MAPK inhibitor. Immunofluorescence microscopic analysis revealed that 2-MeO-E2-induced mitotic arrest was caused by mitotic spindle network impairment and prometaphase arrest. Whereas 10-20 μM 2-MeO-E2 reduced the proportion of intracellular polymeric tubulin to monomeric tubulin, 0.5-5.0 μM 2-MeO-E2 increased it. These results demonstrate that the apoptogenic effect of 2-MeO-E2 (0.5-1.0 μM) was attributable to mitotic spindle defect-mediated prometaphase arrest, Cdk1 activation, phosphorylation of Bcl-2, Mcl-1, and Bim, and activation of Bak and mitochondria-dependent caspase cascade.

Key points Doxorubicin induced functional deteriorations and elevations of USP7-related apoptotic/catabolic signalling in the senescent heart Resveratrol protects against doxorubicin-induced alterations through the restoration of SIRT1 deacetylase activity Abstract A compromised cardiac function is often seen in elderly cancer patients receiving doxorubicin therapy. The present study tested the hypothesis that acute intervention with resveratrol, a natural anti-oxidant found in grapes and red wine, reduces the cardiotoxicity of doxorubicin through restoration of sirtuin 1 (SIRT1) deacetylase activity, and attenuation of the catabolic/apoptotic pathways orchestrated by USP7, a p53 deubiquitinating protein, using young (aged 2 months) and old (aged 10 months) senescence-accelerated mice prone 8 (SAMP8). Animals were randomised to receive saline, doxorubicin, and doxorubicin in combination with resveratrol, in the presence or absence of SIRT1 inhibitors, sirtinol or EX527. Resveratrol alone, but not in combination with either of the SIRT1 inhibitors, suppressed the doxorubicin-induced impairment of cardiac systolic function in aged animals. Doxorubicin reduced SIRT1 deacetylase activity, and elevated proteasomal activity and USP7; it also increased the protein level of p300 and ubiquitinated proteins in hearts from aged SAMP8. These doxorubicin-induced alterations were prevented by resveratrol, whereas the protective action of resveratrol was antagonised by sirtinol and EX527. In young SAMP8 hearts, resveratrol attenuated the doxorubicin-induced increases in acetylation of Foxo1 and transactivation of MuRF-1, whereas these mitigations were not found after treatment with SIRT1 inhibitors. However, the protein contents of acetylated Foxo1 and MuRF-1 were not affected by any of the drugs studied in aged SAMP8 hearts. Resveratrol also ameliorated the augmentation of pro-apoptotic markers including p53, Bax, caspase 3 activity and apoptotic DNA fragmentation induced by doxorubicin in hearts from aged animals, whereas these reductions were diminished by combined treatment with SIRT1 inhibitors. These data demonstrate that resveratrol ameliorates doxorubicin-induced cardiotoxicity in aged hearts through the restoration of SIRT1 activity to attenuate USP7-related catabolic/pro-apoptotic signalling.

Apoptosis mediates cardiac damage in severe forms of myocarditis. In fatal myocarditis, large amounts of cardiomyocytes show apoptotic DNA fragmentation, while in human controls, few apoptotic cardiomyocytes are found. In the present study the frequency of apoptosis in 88 SIDS cases (category 1b according to the San Diego Classification) and 15 control cases was investigated. In every case myocardial samples from 8 standard locations were collected. Detection of apoptotic cardiomyocytes was performed by TUNEL method. Furthermore the myocardial tissue was stained with HE and immunohistochemical methods (LCA, CD68, CD45-R0). More than 90% of the slides did not contain apoptotic cardiomyocytes at all. The detection rate of apoptotic cardiomyocytes was almost equal in control group (26.7%) and SIDS group (23.86%). A quantification of apoptotic cardiomyocytes per mm(2) revealed no significant difference between both groups either. Altogether there is no evidence for a higher rate of apoptosis in SIDS.

Ultraviolet A (UVA) irradiation is toxic to skin as it penetrates deep into the dermis and damages cellular components through excessive reactive oxygen species (ROS) production, which accelerates photoaging and skin cancer. We evaluated the dermato-protective efficacies of zerumbone (natural sesquiterpene of Zingiber zerumbet) in UVA-irradiated human skin keratinocyte (HaCaT) cells and mouse epidermis. Zerumbone pretreatment (2–10 μM) substantially suppressed UVA (15 J/cm2)-induced HaCaT cell death and lactate dehydrogenase release in a dose-dependent manner. UVA-induced excessive ROS production, DNA single-strand breaks, apoptotic DNA fragmentation and a dysregulated Bax/Bcl-2 ratio were remarkably reversed by zerumbone in keratinocytes. Zerumbone-mediated cytoprotective properties were associated with increased nuclear translocation of nuclear factor-E2-related factor-2 (Nrf2) and elevated antioxidant response element (ARE) luciferase activity. Activation of Nrf2/ARE signaling was accompanied by induction of heme oxygenase-1 (HO-1) and γ-glutamyl cysteine ligase (γ-GCLC) genes in zerumbone-treated keratinocytes. Zerumbone-induced Nrf2 transcriptional activation was mediated by the p38 MAPK, PI3K/AKT and PKC signaling cascades. Notably, silencing of Nrf2 (siRNA transfection) significantly diminished zerumbone-mediated cytoprotective effects, as evidenced by impaired antioxidant genes, uncontrolled ROS/apoptotic DNA fragmentation and keratinocytes death, following UVA irradiation. In vivo evidence demonstrated that zerumbone treatment to nude mice (55 and 110 μg/day) significantly ameliorated UVA (15 J/cm2/every 2-day/14-day) cytotoxicity via increased nuclear localization of Nrf2 and Nrf2-dependent antioxidant genes (HO-1 and γ-GCLC) in UVA-treated skin tissues. Our findings emphasized the significance of Nrf2/ARE-signaling in zerumbone-mediated induction of antioxidant genes against UVA-toxicity. The molecular evidence suggests zerumbone can be a natural medicine to treat/prevent UVA-induced skin damage/photoaging.

// Chae Eun Kim 1 , Do Youn Jun 1, 2 , Jong-Sik Kim 3 and Young Ho Kim 1 1 Laboratory of Immunobiology, School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 41566, Korea 2 Leading-edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease Bio-Medical Research Institute, Kyungpook National University Hospital, Daegu 702-201, Korea 3 Department of Biological Sciences, Andong National University, Andong 36729, Korea Correspondence to: Young Ho Kim, email: ykim@knu.ac.kr Keywords: CDK1-dependent mitochondrial apoptosis; mitotic spindle damage; phosphorylation of BCL-2 family proteins; prometaphase arrest; cis-trimethoxy resveratrol Received: August 18, 2017 Accepted: December 05, 2017 Published: December 22, 2017 ABSTRACT Cis-trimethoxy resveratrol (cis-3M-RES) induced dose-dependent cytotoxicity and apoptotic DNA fragmentation in Jurkat T cell clones (JT/Neo); however, it induced only cytostasis in BCL-2-overexpressing cells (JT/BCL-2). Treatment with 0.25 μM cis-3M-RES induced G 2 /M arrest, BAK activation, Δψm loss, caspase-9 and caspase-3 activation, and poly (ADP-ribose) polymerase (PARP) cleavage in JT/Neo cells time-dependently but did not induce these events, except G 2 /M arrest, in JT/BCL-2 cells. Moreover, cis-3M-RES induced CDK1 activation, BCL-2 phosphorylation at Ser-70, MCL-1 phosphorylation at Ser-159/Thr-163, and BIM (BIM EL and BIM L ) phosphorylation irrespective of BCL-2 overexpression. Enforced G 1 /S arrest by using a G 1 /S blocker aphidicolin completely inhibited cis-3M-RES-induced apoptotic events. Cis-3M-RES-induced phosphorylation of BCL-2 family proteins and mitochondrial apoptotic events were suppressed by a validated CDK1 inhibitor RO3306. Immunofluorescence microscopy showed that cis-3M-RES induced mitotic spindle defects and prometaphase arrest. The rate of intracellular polymeric tubulin to monomeric tubulin decreased markedly by cis-3M-RES (0.1-1.0 μM). Wild-type Jurkat clone A3, FADD-deficient Jurkat clone I2.1, and caspase-8-deficient Jurkat clone I9.2 exhibited similar susceptibilities to the cytotoxicity of cis-3M-RES, excluding contribution of the extrinsic death receptor-dependent pathway to the apoptosis. IC 50 values of cis-3M-RES against Jurkat E6.1, U937, HL-60, and HeLa cells were 0.07-0.17 μM, whereas those against unstimulated human peripheral T cells and phytohaemagglutinin A-stimulated peripheral T cells were >10.0 and 0.23 μM, respectively. These results indicate that the antitumor activity of cis-3M-RES is mediated by microtubule damage, and subsequent prometaphase arrest and prolonged CDK1 activation that cause BAK-mediated mitochondrial apoptosis, and suggest that cis-3M-RES is a promising agent to treat leukemia.