Apoptotic DNA Fragmentation Research Articles

Reactive Oxygen Species-Mediated Apoptosis and Cytotoxicity of Newly Synthesized Pyridazin-3-Ones In P815 (Murin Mastocytoma) Cell Line.

In cancer cells, the intracellular antioxidant capacity and the redox homeostasis are mainly maintained by the glutathione- and thioredoxin-dependent systems which are considered as promising targets for anticancer drugs. Pyridazinones constitute an interesting source of heterocyclic compounds for drug discovery. The present investigation focused on studying the in-vitro antitumor activity of newly synthesized Pyridazin-3(2h)-ones derivatives against P815 (Murin mastocytoma) cell line. The in-vitro cytotoxic activities were investigated toward the P815 cell line using tetrazolium-based MTT assay. Lipid peroxidation and the specific activities of antioxidant enzymes were also determined. The newly compounds had a selective dose-dependent cytotoxic effect without affecting normal cells (PBMCs). Apoptosis was further confirmed through the characteristic apoptotic morphological changes and DNA fragmentation. Two compounds (6F: and 7H: ) were highly cytotoxic and were submitted to extend biological testing to determine the likely mechanisms of their cytotoxicity. Results showed that these molecules may induce cytotoxicity via disturbing the redox homeostasis. Importantly, the anticancer activity of 6F: and 7H: could be due to the intracellular reactive oxygen species hypergeneration through significant loss of glutathione reductase and thioredoxin reductase activities. This eventually leads to oxidative stress-mediated P815 cell apoptosis. Furthermore, the co-administration of 6F: or 7H: with Methotrexate exhibited a synergistic cytotoxic effect. considering their significant anticancer activity and chemosensitivity, 6F: and 7H: may improve the therapeutic efficacy of the current treatment for cancer.

Possible use of a Nicotiana tabacum ‘Bright Yellow 2’ cell suspension as a model to assess phytotoxicity of pharmaceuticals (diclofenac)

Growth and developmental changes in plants induced by pharmaceuticals reflect changes in processes at the cellular and subcellular levels. Due to their growth and cellular characteristics, plant cell suspension cultures can be a suitable model for assessing toxicity. In this study, 10–1000 μg/L of the non-steroidal anti-inflammatory drug diclofenac (DCF) decreased the viability of Nicotiana tabacum BY-2 cells after 24 h of treatment. Further, 0.1–10 mg/L DCF diminished the density of the cell suspension by 9–46% after 96 h of treatment, but at 1 and 10 μg/L, DCF increased the density by 13% and 5%, respectively, after 120 h. These changes were accompanied by increased production of total reactive oxygen species (ROS) and mitochondrial superoxide (up to 17-fold and 5-fold, respectively), and a decrease in the mitochondrial membrane potential (by ∼64%) especially at 1000 μg/L DCF. The increased ROS production was accompanied by decrease in level of reactive nitrogen species (RNS; by 36%) and total thiols (by 61%). Damage to BY-2 cells was evidenced by accumulation of neutral red in acidic compartments (up to 10-fold at 1000 μg/L DCF), and increase of autophagic vacuole formation (up to 8-fold at 1000 μg/L DCF). Furthermore, irregular or stretched nuclei were observed in nearly 27% and 50% of cells at 100 and 1000 μg/L DCF, respectively. Highest levels of chromatin condensation (11% of cells) and apoptotic DNA fragmentation (7%) were found at 10 μg/L DCF. The results revealed a significant effect of DCF on BY-2 cells after 24 h of exposure. Changes in the growth and viability parameters were indisputably related to ROS and RNS production, changes in mitochondrial function, and possible activation of processes leading to cell death.

Investigation of chemical profile, biological properties of Lotus corniculatus L. extracts and their apoptotic-autophagic effects on breast cancer cells

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, MTT 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.

Synthesis and Anticancer Activity of Novel 9-O-Substituted Berberine Derivatives.

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 µM for HL-60 cells and 36 to >200 µM 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 µM 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.

Auranofin, an Anti-rheumatic Gold Drug, Aggravates the Radiation-Induced Acute Intestinal Injury in Mice.

Pelvic and abdominal radiotherapy plays an important role in eradication of malignant cells; however, it also results in slight intestinal injury. The apoptosis of cells in the intestinal epithelium is a primary pathological factor that initiates radiation-induced intestinal injury. Auranofin, a gold-containing triethylphosphine, was approved for the treatment of rheumatoid arthritis, and its therapeutic application has been expanded to a number of other diseases, such as parasitic infections, neurodegenerative disorders, AIDS, and bacterial infections. Recently, a treatment strategy combining the use of auranofin and ionizing radiation aimed at increasing the radiosensitivity of cancer cells was proposed for improving the control of local cancers. In this study, we evaluated the effect of auranofin on the radiosensitivity of intestinal epithelial cells. The treatment with a combination of 1 μM auranofin and 5 Gy ionizing radiation showed clear additive effects on caspase 3 cleavage and apoptotic DNA fragmentation in IEC-6 cells, and auranofin administration significantly aggravated the radiation-induced intestinal injury in mice. Auranofin treatment also resulted in the activation of the unfolded protein response and in the inhibition of thioredoxin reductase, which is a key component of the cellular antioxidant system. Pre-treatment with N-acetyl cysteine, a well-known scavenger of reactive oxygen species, but not with a chemical chaperone, which inhibits endoplasmic reticulum stress and the ensuing unfolded protein response, significantly reduced the radiosensitizing effects of auranofin in the IEC-6 cells. In addition, transfection of IEC-6 cells with a small interfering RNA targeted against thioredoxin reductase significantly enhanced the radiosensitivity of these cells. These results suggest that auranofin-induced radiosensitization of intestinal epithelial cells is mediated through oxidative stress caused by the deregulation of thioredoxin redox system, and auranofin treatment can be an independent risk factor for the development of acute pelvic radiation disease.

Anticancer activities of chalcone flavokawain B from Alpinia pricei Hayata in human lung adenocarcinoma (A549) cells via induction of reactive oxygen species-mediated apoptotic and autophagic cell death.

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 decreasedmammalian 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.

Histone H1 quantity determines the efficiencies of apoptotic DNA fragmentation and chromatin condensation.

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.

Induction of 3-hydroxy-3-methylglutaryl-CoA reductase mediates statin resistance in breast cancer cells

The mevalonate pathway has emerged as a promising target for several solid tumors. Statins are inhibitors of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the rate-limiting enzyme of this pathway, and are commonly used to treat patients with hypercholesterolemia. Pleiotropic antitumor mechanisms of statins have been demonstrated for several human cancer types. However, cancer cells differ in their individual statin sensitivity and some cell lines have shown relative resistance. In this study we demonstrate, that the human breast cancer cell lines MDA-MB-231, MDA-MB-468, MCF-7, and T47D are differentially affected by statins. Whereas the vitality of MDA-MB-231 and MDA-MB-468 cells was reduced by up to 60% using atorvastatin, simvastatin, or rosuvastatin (p < 0.001), only marginal effects were seen in T47D and MCF-7 cells following exposure to statins. Statin treatment led to an upregulation of HMGCR mRNA and protein expression by up to sixfolds in the statin-resistant cells lines (p < 0.001), but no alterations of HMGCR were observed in the statin-sensitive MDA-MB-231 and MDA-MB-468 cells. The knockdown of HMGCR prior to statin treatment sensitized the resistant cell lines, reflected by a 70% reduction in vitality, increased apoptotic DNA fragmentation (sixfold) and by accumulation of the apoptosis marker cleaved poly-ADP ribose polymerase. Statins induced a cleavage of the sterol-regulatory element-binding protein (SREBP)-2, a transcriptional activator of the HMGCR, in T47D and MCF-7 cells. The inhibition of SREBP-2 activation by co-administration of dipyridamole sensitized MCF-7 and T47D cells for statins (loss of vitality by 80%; p < 0.001). Furthermore, assessment of a statin-resistant MDA-MB-231 clone, generated by long-term sublethal statin exposure, revealed a significant induction of HMGCR expression by up to 12-folds (p < 0.001). Knockdown of HMGCR restored statin sensitivity back to levels of the parental cells. In conclusion, these results indicate a resistance of cancer cells against statins, which is in part due to the induction of HMGCR.

Design, synthesis and biological evaluation of certain CDK2 inhibitors based on pyrazole and pyrazolo[1,5-a] pyrimidine scaffold with apoptotic activity

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.

DNA laddering to evaluate cytogenetic damage in patients with periodontitis

Background:Inflammatory conditions show cytogenetic damage in peripheral blood leukocytes and this can be assessed using various tests. Cytogenetic damage as observed in the peripheral blood cells, is a marker of periodontal disease. DNA laddering is a sensitive assay which evaluates the cytogenetic damage. DNA laddering is a feature that can be observed when DNA fragments, resulting from apoptotic DNA fragmentation, are visualised after separation by gel electrophoresis which results in a characteristic “ladder” pattern.Aim:The aim of the present study is to investigate the cytogenetic damage in different forms of periodontitis in comparison with healthy controls.Materials and Methods:In this cross-sectional study, 15 systemically healthy subjects with moderate to severe chronic periodontitis (CGP), 15 systemically healthy subjects with generalised aggressive periodontitis(GAP) and 15 systemically healthy control subjects were recruited. Blood samples of the patients were drawn and evaluated for the cytogenetic damage by DNA laddering.Results:Apoptotic DNA fragmentation was observed as a “ladder” pattern at 180-200 BP intervals in both CGP and GAP groups indicating the DNA damage, in contrast with the healthy group where the ladder pattern was not observed suggesting of the healthy DNA.Conclusion:The results indicated that there are cytogenetic damages in both the chronic and aggressive periodontitis groups incontrast to the healthy controls.

Crystal structure and mutation analysis revealed that DREP2 CIDE forms a filament-like structure with features differing from those of DREP4 CIDE

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.

Taurine ameliorates oxidative stress induced inflammation and ER stress mediated testicular damage in STZ-induced diabetic Wistar rats

One of the major consequences of diabetes is reproductive dysfunction but the fundamental mechanisms are still poorly known. The objective of the present study was to explore the beneficial role of taurine against streptozotocin induced testicular dysfunctions in diabetic male Wister rats and understanding the underlying intricate molecular mechanisms. Exposure to streptozotocin (50 mg kg−1 body weight, i.p., once) elevated blood glucose level, induced testicular histological alterations and reduced testis-to-body weight ratio, serum testosterone, testicular markers and activity of antioxidant enzymes. Generation of ER stress (increased expression of calpain-1, caspase-12 and upregulation of CHOP, GRP78 via eIF2α signaling), translocation of NF κB in the nucleus (leading to the upregulation in the levels of inflammatory cytokines), activation of mitochondria dependent apoptotic pathway and DNA fragmentation were revealed from this study. However, administration of taurine at a dose of 100 mg kg−1 body weight for 6 weeks post diabetic induction, successfully ameliorated all these adverse effects. Thus, taurine, as a potential therapeutic agent, may hold promise in preventing oxidative and ER stress mediated diabetic testicular complications in rats.

Cytoprotective effect of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone (CMEP-NQ) is mediated by the inhibition of BAK-dependent mitochondrial apoptosis pathway.

The inhibitory mechanism of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone (CMEP-NQ) against apoptosis induced by the microtubule-damaging agents (MDAs), nocodazole (NOC) and 2-methoxyestradiol (2-MeO-E2), or a DNA-damaging agent (DDA), camptothecin (CPT) were investigated in human Jurkat T cell clones (J/Neo and J/BCL-XL cells). Treatment of J/Neo cells with NOC, 2-MeO-E2, or CPT caused cytotoxicity and apoptotic DNA fragmentation but these events were significantly attenuated in the presence of CMEP-NQ. Although not only MDA (NOC or 2-MeO-E2)-induced mitotic arrest, CDK1 activation, and BCL-2, BCL-XL and BIM phosphorylation, but also DDA (CPT)-induced S-phase arrest and ATM-CHK1/CHK2-p53 pathway activation were not or were barely affected in the presence of CMEP-NQ, the levels of anti-apoptotic BAG3 and MCL-1, which were markedly downregulated after MDA- or DDA-treatment, were rather elevated by CMEP-NQ. Under the same conditions, MDA- or DDA-induced mitochondrial apoptotic events including BAK activation, mitochondrial membrane potential (Δψm) loss, caspase-9 activation, and PARP cleavage were significantly inhibited by CMEP-NQ. While MDA- or DDA-induced sub-G1 peak and Δψm loss were abrogated in J/BCL-XL cells, MDA-induced mitotic arrest and DDA-induced S-arrest were more apparent in J/BCL-XL cells than in J/Neo cells. Simultaneously, the induced cell cycle arrest in J/BCL-XL cells was not significantly disturbed by CMEP-NQ. MDA- or DDA-treatment caused intracellular reactive oxygen species (ROS) production; however, MDA- or DDA-induced ROS production was almost completely abrogated in J/BCL-XL cells. MDA- or DDA-induced ROS production in J/Neo cells was significantly suppressed by CMEP-NQ, but the suppressive effect was hardly observed in J/BCL-XL cells. Together, these results show that CMEP-NQ efficiently protects Jurkat T cells from apoptotic cell death via the elevation of BAG3 and MCL-1 levels, which results in the inhibition of intrinsic BAK-dependent mitochondrial apoptosis pathway, as does the overexpression of BCL-XL.

PlGF knockdown induced apoptosis through Wnt signaling pathway in gastric cancer stem cells.

Despite the fact that much research has focused on gastric cancer, it is still a worldwide concern, because of the difficulties with factors such as signaling pathway crosstalk and gastric cancer stem cell (GCSC). Placental growth factor (PlGF) is one of these factors, and its tumorigenicity potential still remains a question. As a result, we have investigated the effect of PlGF knockdown on apoptosis and genes involved in the Wnt signaling pathway, and apoptosis in cancer stem cells derived from AGS an MKN-45 gastric cancer cell lines. We isolated GCSCs from MKN-45 and AGS cell lines on a nonadherent surface. Then the cell viability, the real-time reverse transcription-polymerase chain reaction data of the genes involved in the Wnt signaling pathway, and apoptosis were evaluated. Furthermore, DNA laddering was used to show the apoptotic effect and DNA fragmentation caused by the PlGF knockdown. Our investigation revealed that the PlGF knockdown with PlGF-specific small interfering RNA at 40 pmol for GCSCs derived from MKN-45 and AGS at 24 hours can significantly affect the cell viability, the Wnt signaling pathway, and the apoptosis-related genes expression. In conclusion, we showed the PlGF knockdown may induce apoptosis via the Wnt signaling pathway in GCSCs.

Benzyl isothiocyanate (BITC) triggers mitochondria-mediated apoptotic machinery in human cisplatin-resistant oral cancer CAR cells.

Benzyl isothiocyanate (BITC), a component of dietary food, possesses a powerful anticancer activity. Previous studies have shown that BITC produces a large number of intracellular reactive oxygen species (ROS) and increases intracellular Ca2+ release from endoplasmic reticulum (ER), leading to the activation of the apoptotic mechanism in tumor cells. However, there is not much known regarding the inhibitory effect of BITC on cisplatin-resistant oral cancer cells. The purpose of this study was to examine the anticancer effect and molecular mechanism of BITC on human cisplatin-resistant oral cancer CAR cells. Our results demonstrated that BITC significantly reduced cell viability of CAR cells in a concentration- and time-dependent manner. BITC was found to cause apoptotic cell shrinkage and DNA fragmentation by morphologic observation and TUNEL/DAPI staining. Pretreatment of cells with a specific inhibitor of pan-caspase significantly reduced cell death caused by BITC. Colorimetric assay analyses also showed that the activities of caspase-3 and caspase-9 were elevated in BITC-treated CAR cells. An increase in ROS production and loss of mitochondria membrane potential (ΔΨm) occurred due to BITC exposure and was observed via flow cytometric analysis. Western blotting analyses demonstrated that the protein levels of Bax, Bad, cytochrome c, and cleaved caspase-3 were up-regulated, while those of Bcl-2, Bcl-xL and pro-caspase-9 were down-regulated in CAR cells after BITC challenge. In sum, the mitochondria-dependent pathway might contribute to BITC-induced apoptosis in human cisplatin-resistant oral cancer CAR cells.

Sodium fluoride causes oxidative stress and apoptosis in cementoblasts

ObjectivesToothpaste with fluoride concentration up to 5000 ppm are recommended to the patients who are susceptible to root caries; however, the effects of fluoride on cementoblasts have received less attention. MethodsThe OCCM-30 cells were exposed to 0,0.5, 5, 10 mM NaF respectively. A TUNEL (TdT-mediated dUTP-biotin nick end labeling) assay kit was used to detect the DNA fragmentation. Hoechst staining was used to determine changes of nuclear morphology. Real-time quantitative RT-PCR and Western blotting were performed to investigate the mRNA and protein expression of caspase-3,-8,-9, cleaved Poly (ADP-ribose) polymerase (PARP) and Fas-ligand (Fas-L), a ligand of death receptor. CA-DCF-DA [5 (6)-Carboxy-2′,7′-dichlorofluorescein diacetate] was used to measure the generation of reactive oxygen species (ROS) in OCCM-30 cells after the NaF stimulation. ResultsThe results showed apoptotic morphological changes and DNA fragmentation in OCCM-30 cells exposed to high concentration of NaF. 10 mM NaF induced the expression of cleaved caspase-3,-8,-9 and cleaved Poly (ADP-ribose) polymerase (PARP). The mRNA expression of the Fas-L was also increased in cells exposed to 5 mM NaF. Furthermore, 10 mM NaF stimulation resulted in a significant generation of ROS in the OCCM-30 cells. ConclusionsOur research demonstrated that apoptosis is activated by NaF in OCCM-30 cells through both of the extrinsic death receptor-dependent and oxidative stress-related intrinsic apoptotic pathway. Clinical significanceMore consideration should be given about the fluoride concentration and the frequency of dental products when used to prevent the root caries for patients with gingival recession.

The effects of benzophenone-3 on apoptosis and the expression of sex hormone receptors in the frontal cortex and hippocampus of rats

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.

177 Lu-DOTMP induces G2/M cell cycle arrest and apoptosis in MG63 cell line.

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 3h time period which was almost constant up to 7days. MG63 cells were incubated with 3.7 and 37MBq of 177 Lu-DOTMP for 48h 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.

Investigations into the therapeutic potential of Asphodeline liburnica roots: In vitro and in silico biochemical and toxicological perspectives

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.

Crystal structure of human Acinus RNA recognition motif domain.

Acinus is an abundant nuclear protein involved in apoptosis and splicing. It has been implicated in inducing apoptotic chromatin condensation and DNA fragmentation during programmed cell death. Acinus undergoes activation by proteolytic cleavage that produces a truncated p17 form that comprises only the RNA recognition motif (RRM) domain. We have determined the crystal structure of the human Acinus RRM domain (AcRRM) at 1.65 Å resolution. It shows a classical four-stranded antiparallel β-sheet fold with two flanking α-helices and an additional, non-classical α-helix at the C-terminus, which harbors the caspase-3 target sequence that is cleaved during Acinus activation. In the structure, the C-terminal α-helix partially occludes the potential ligand binding surface of the β-sheet and hypothetically shields it from non-sequence specific interactions with RNA. Based on the comparison with other RRM-RNA complex structures, it is likely that the C-terminal α-helix changes its conformation with respect to the RRM core in order to enable RNA binding by Acinus.