Molecular events involved in ochratoxin A induced mitochondrial pathway of apoptosis,modulation by Bcl‐2 family members

In this study, we looked for the role of the mitochondrion in the cytotoxicity of ochratoxin A (OTA), which is one of the most abundant food‐contaminating mycotoxins in the world. In different human carcinoma cell lines, OTA triggered a mitochondria‐dependent apoptotic process, which is characterized by opening of the mitochondrial permeability transition pore (PTPC), loss of mitochondrial transmembrane potential (ΔΨ_m), increase in O₂[chemp]^– production, mitochondrial relocalization of Bax, release of cytochrome c, and caspase activation. However, studies performed on purified organelles suggested that OTA does not directly target the mitochondrion. In addition, we showed that mitochondrial alterations induced by this mycotoxin are favored by the proapoptotic protein Bax, but not Bak. These alterations are prevented by the antiapoptotic proteins, Bcl‐2 and to a lesser degree by Bcl‐X_L. Taken together, these data indicate that although mitochondria, PTPC members and proteins of Bcl‐2 family play a pivotal role in OTA‐induced apoptosis, they do not constitute real targets to overcome its toxicity. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2011.     

Late anti‐apoptotic effect of KATP channel opening in skeletal muscle

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Evidence that apoptotic signalling in hypertrophic cardiomyocytes is determined by mitochondrial pathways involving protein kinase Cδ

1. Cardiomyocyte apoptosis plays an important role in the transition from cardiac hypertrophy to heart failure. Hyper-trophic cardiomyocytes show an increased susceptibility to apoptotic stimuli, but the mechanisms remain unclear. 2. We hypothesized that activated protein kinase Cδ (PKCδ) associated with cardiomyocyte hypertrophy could move from the cytoplasm to mitochondria, and subsequently trigger the apoptotic signalling pathway. 3. Hypertrophy was induced in cultured neonatal rat cardiomyocytes using endothelin-1 (ET-1), insulin-like growth factor-1 (IGF-1), thyroid hormone (T(3) ) or angiotensin-II (AngII). AngII at high concentrations (1 and 10 nmol/L) also induced apoptosis. Hypertrophic cells were then treated with AngII with or without specific inhibitors of the angiotensin receptors AT(1) and AT(2) (losartan and PD123319, respectively), endothelin receptor A (BQ-123) and PKCδ (rottlerin). ET-1 plus AngII had a threefold and significant increase in apoptosis in the hypertrophic cultures compared with AngII alone. In association with the increase in apoptosis, this treatment also promoted mitochondrial translocation of PKCδ, and increased expression of cleaved caspase 9 and activity of caspase 3. All of these increases were modulated by concurrent use of the PKCδ inhibitor, rottlerin. 4. The results suggest that apoptotic signalling in hypertrophic cardiomyocytes is determined by mitochondrial pathways involving PKCδ.     

Synergism of simvastatin with losartan prevents angiotensin II‐induced cardiomyocyte apoptosis in vitro

Objectives Increasing evidence suggests that cardiomyocyte apoptosis has an important role in the transition from compensatory cardiac remodelling to heart failure. The synergistic effect of statins (3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase inhibitors) and angiotensin II (Ang II) type 1 receptor antagonists reduces the incidence of cardiovascular events. However, the anti‐apoptotic potential of the synergism between losartan and simvastatin in heart failure remains unexplored. Here, we demonstrate that Ang II‐induced apoptosis is prevented by losartan and simvastatin in neonatal cardiomyocytes. Methods The in‐vitro cardiomyocyte apoptosis model was established by co‐culturing neonate rat cardiomyocytes with Ang II. Cell viability was analysed by the MTT assay. Cell apoptosis was evaluated using fluorescence microscopy and flow cytometry. Apoptosis‐related proteins Bax and Bcl‐2 expressions were measured by flow cytometry detection. Key findings Incubation with 10^?7 m Ang II for 48 h increased cardiomyocyte apoptosis and decreased cell viability. Losartan (10^?5 m ) and simvastatin (10^?5 m ), either alone or in combination, significantly decreased Ang II‐induced cardiomyocyte apoptosis and increased cell viability. The q values calculated by the probability sum test were 1.31 for cardiomyocyte apoptosis and 1.21 for cell viability. Ang II induced a significant increase in Bax protein expression, whereas Bcl‐2 protein expression was decreased. Losartan alone or in combination with simvastatin blocked the increased Bax expression and increased Bcl‐2 expression. However, simvastatin had no such effect. Conclusions Our data provide the first evidence that synergism of simvastatin with losartan prevents angiotensin II‐induced cardiomyocyte apoptosis in vitro. Synergism between simvastatin and losartan may provide a new therapeutic approach to the prevention of cardiac remodelling.     

Curcumin alleviates glucocorticoid‐induced osteoporosis by protecting osteoblasts from apoptosis in vivo and in vitro

Curcumin, an active component of the rhizomes of Curcumin longa L., possesses broad anti‐inflammation and anti‐cancer properties. Curcumin was previously reported to be capable of protecting ovariectomized rats against osteoporosis. However, the effect of curcumin on glucocorticoid‐induced osteoporosis (GIO) is not yet clear. The present study investigated the effects of curcumin on dexamethasone (Dex)‐induced osteoporosis in vivo and Dex‐induced osteoblast apoptosis in vivo and in vitro. The GIO rat model was induced by subcutaneous injection of Dex for 60 days and verified to be successful as evidenced by the significantly decreased bone mineral density (BMD) determined using dual X‐ray absorptiometry. Subsequently, curcumin administration (100 mg/kg) for 60 days obviously increased BMD and bone‐alkaline phosphatase, decreased carboxy‐terminal collagen cross links, enhanced bone mechanical strength, and improved trabecular microstructure, thereby alleviating Dex‐induced osteoporosis. Mechanically, curcumin remarkably reversed Dex‐induced femoral osteoblast apoptosis in vivo. In cultured primary osteoblasts, pretreatment with curcumin concentration‐dependently decreased the number of Dex‐induced apoptotic osteoblasts by down‐regulating the ratio of Bax/Bcl‐2 as well as the levels of cleaved caspase‐3 and cleaved poly ADP‐ribose polymerase (PARP). Moreover, curcumin pretreatment activated extracellular signal regulated kinase (ERK) signalling in Dex‐induced osteoblasts by up‐regulating the expression level of p‐ERK1/2. Taken together, our study demonstrated that curcumin could ameliorate GIO by protecting osteoblasts from apoptosis, which was possibly related to the activation of the ERK pathway. The results suggest that curcumin may be a promising drug for prevention and treatment of GIO.     

Genistein induces apoptosis in vitro and has antitumor activity against human leukemia HL‐60 cancer cell xenograft growth in vivo

Genistein, a major isoflavone compound in soybeans, has been shown to have biological activities including anti‐cancer activates. In the present, we investigated the anti‐leukemia activity of genistein on HL‐60 cells in vitro. The percentage of viable cell, cell cycle distribution, apoptotic cell death, reactive oxygen species (ROS), and Ca²⁺ production and the level of ΔΨ_m were measured by flow cytometric assay. Cell apoptosis and endoplasmic reticulum (ER) stress associated protein expressions were examined by Western blotting assay. Calpain 1, GRP78, and GADD153 expression were measured by confocal laser microscopy. Results indicated that genistein‐induced cell morphological changes, decreased the total viable cells, induced G₂/M phase arrest and DNA damage and fragmentation (cell apoptosis) in HL‐60 cells. Genistein promoted ROS and Ca²⁺ productions and decreased the level of ΔΨ_m in HL‐60 cells. Western blotting assay demonstrated that genistein increased ER stress‐associated protein expression such as IRE‐1α, Calpain 1, GRP78, GADD153, caspase‐7, caspase‐4, and ATF‐6α at 20‐50 μM treatment and increased apoptosis associated protein expression such as pro‐apoptotic protein Bax, PARP‐cleavage, caspase‐9, and ‐3, but decreased anti‐apoptotic protein such as Bcl‐2 and Bid in HL‐60 cells. Calpain 1, GRP78, and GADD153 were increased in HL‐60 cells after exposure to 40 μM of genistein. In animal xenografted model, mice were intraperitoneally injected with genistein (0, 0.2, and 0.4 mg/kg) for 28 days and the body weight and tumor volume were recorded. Results showed that genistein did not affect the body weights but significantly reduced the tumor weight in 0.4 mg/kg genistein‐treated group. Genistein also increased the expressions of ATF‐6α, GRP78, Bax, Bad, and Bak in tumor. In conclusion, genistein decreased cell number through G₂/M phase arrest and the induction of cell apoptosis through ER stress‐ and mitochondria‐dependent pathways in HL‐60 cells and suppressed tumor properties in vivo.     

Apoptotic and antiproliferative properties of 3β‐hydroxy‐Δ5‐steroidal congeners from a partially purified column fraction of Dendronephthya gigantea against HL‐60 and MCF‐7 cancer cells

Organisms belonging to the genus Dendronephthya are among a group of marine invertebrates that produce a variety of terpenoids with biofunctional properties. Many of these terpenoids have been proven effective as anticancer drugs. Here, we report the antiproliferative effect of 3β‐hydroxy‐Δ5‐steroidal congeners against the proliferation of HL‐60 human leukemia cells and MCF‐7 human breast cancer cells. The sterol‐rich fraction (DGEHF2‐1) inhibited the growth of HL‐60 and MCF‐7 cells with IC₅₀ values of 13.59 ± 1.40 and 29.41 ± 0.87 μg ml^–1 respectively. Treatment with DGEHF2‐1 caused a dose‐dependent increase in apoptotic body formation, DNA damage and the sub‐G₁ apoptotic cell population. Moreover, DGEHF2‐1 downregulated the expression of Bcl‐xL while upregulating Bax, caspase‐9, and PARP cleavage in both HL‐60 and MCF‐7 cells. The steroid fraction was found to act via the mitochondria‐mediated apoptosis pathway. Identification of the sterols was performed via gas chromatography–tandem mass spectrometry analysis. Studying the mechanism of the anticancer effect caused by these sterol derivatives could lead to the identification of other natural products with anticancer properties.     

Inhibition of protein kinase (PK) Cδ attenuates methamphetamine‐induced dopaminergic toxicity via upregulation of phosphorylation of tyrosine hydroxylase at Ser40 by modulation of protein phosphatase 2A and PKA

Recently, we proposed that inhibition of protein kinase (PK) Cδ may be a useful target for protection against methamphetamine (MA)‐induced dopaminergic toxicity. We demonstrated that treatment with MA resulted in a significant decrease in phosphorylation of tyrosine hydroxylase (TH) at Ser⁴⁰ in the striatum, but not in the phosphorylation of TH at Ser³¹. In the present study, treatment with rottlerin (1.5 or 3.0 μg, i.c.v, once a day for 5 days), a PKCδ inhibitor, or a PKCδ antisense oligonucleotide (ASO; 2.5 μg/μl, i.c.v., 3 times) significantly attenuated MA‐induced reductions in the phosphorylation of TH at Ser⁴⁰ and in the expression of PKA in the striatum of mice. This attenuation was significantly counteracted by H89 (10 or 30 ng, i.c.v., 1 h after the last MA administration), a PKA inhibitor. Treatment with rottlerin or ASO significantly attenuated the MA‐induced increase in protein phosphatase (PP) 2A activity. FTY720 (1 or 5 mg/kg, i.p., 1 h after the last MA administration), a PP2A activator, significantly reversed the recovery in TH phosphorylation mediated by inhibition of PKCδ after MA treatment. Both H89 and FTY720 counteracted the recovery of MA‐induced behavioural impairments induced by PKCδ inhibition. The effects, mediated by rottlerin or ASO in MA‐treated wild‐type mice were comparable with those in MA‐treated PKCδ^?/? mice. However, neither inhibition of the mitogen‐activated protein kinase subfamily (extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase, p38) nor inhibition of calcium calmodulin kinase II significantly altered PKCδ inhibition‐mediated attenuation of MA‐induced impairment of TH phosphorylation. The results suggest that genetic or pharmacological inhibition of PKCδ requires modulation of PKA expression and/or PP2A activity to attenuate the impairment of TH phosphorylation at Ser⁴⁰ and behavioural activity induced by MA.     

Benzyl isothiocyanate (BITC) induces apoptosis of GBM 8401 human brain glioblastoma multiforms cells via activation of caspase‐8/Bid and the reactive oxygen species‐dependent mitochondrial pathway

Benzyl isothiocyanate (BITC) is one of member of the isothiocyanate family which has been shown to induce cancer cell apoptosis in many human cancer cells. In the present study, we investigated the effects of BITC on the growth of GBM 8401 human brain glioblastoma multiforms cells. Results indicated that BITC‐induced cell morphological changes decreased in the percentage of viable GBM8401 cells and these effects are dose‐dependent manners. Results from flow cytometric assay indicated that BITC induced sub‐G1 phase and induction of apoptosis of GBM 8401 cells. Furthermore, results also showed that BITC promoted the production of reactive oxygen species (ROS) and Ca²⁺ release, but decreased the mitochondrial membrane potential (ΔΨ_m) and promoted caspase‐8, ‐9, and ‐3 activates. After cells were pretreated with Z‐IETD‐FMK, Z‐LEHD‐FMK, and Z‐DEVD‐FMK (caspase‐8, ‐9, and ‐3 inhibitors, respectively) led to decrease in the activities of caspase‐8, ‐9, and ‐3 and increased the percentage of viable GBM 8401 cells that indicated which BITC induced cell apoptosis through caspase‐dependent pathways. Western blotting indicated that BITC induced Fas, Fas‐L, FADD, caspase‐8, caspase ‐3, and pro‐apoptotic protein (Bax, Bid, and Bak), but inhibited the ant‐apoptotic proteins (Bcl‐2 and Bcl‐x) in GBM 8401 cells. Furthermore, BITC increased the release of cytochrome c, AIF, and Endo G from mitochondria that led to cell apoptosis. Results also showed that BITC increased GADD153, GRP 78, XBP‐1, and ATF‐6β, IRE‐1α, IRE‐1β, Calpain 1 and 2 in GBM 8401 cells, which is associated with ER stress. Based on these observations, we may suggest that BITC‐induced apoptosis might be through Fas receptor, ROS induced ER stress, caspase‐3, and mitochondrial signaling pathways. Taken together, these molecular alterations and signaling pathways offer an insight into BITC‐caused growth inhibition and induced apoptotic cell death of GBM 8401 cells. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1751–1760, 2016.     

Role of 5‐HT2B receptors in cardiomyocyte apoptosis in noradrenaline‐induced cardiomyopathy in rats

1. Serotonin (5‐hydroxytryptamine; 5‐HT) plays important roles in the development of cardiac hypertrophy via activation of 5‐HT receptors. The aim of the present study was to investigate the role of 5‐HT_2B receptors in the development of cardiomyocyte apoptosis and hypertrophy associated with noradrenaline (NA) overload. 2. Cardiac hypertrophy was induced in rats by intraperitoneal injection of 1.5 mg/kg NA for 4 weeks. Starting from Day 15, 5‐HT2B receptor antagonist SB 204741 (i.p., 0.5 or 2 mg/kg) or SDZ SER 082 (i.p., 1 mg/kg) was injected twice daily for another 14 days. Whole‐cell patch‐clamp techniques were used to record ionic currents in freshly isolated ventricular cardiomyocytes. Western blot and terminal deoxyribonucleotidyl transferase‐mediated dUTP–digoxigenin nick end‐labelling (TUNEL) assays were used to assess myocardial apoptosis. 3. Expression of 5‐HT_2B receptors was enhanced in the hypertrophic left ventricle induced by NE overload in vivo. The 5‐HT_2B receptor antagonist SB 204741 partially reversed cardiac hypertrophy induced by NE overload (P < 0.05) and decreased L‐type calcium currents in ventricular cardiomyocytes (P < 0.05). In addition, SB 204741 notably attenuated myocardial apoptosis, as evidenced by downregulation of Bax and caspase 3 (P < 0.05) and upregulation of the anti‐apoptotic Bcl‐2 protein (P < 0.05). 4. In conclusion, the data suggest an involvement of 5‐HT_2B receptors in the generation of apoptotic events associated with cardiac remodelling during increased adrenergic stimulation.     

Angiotensin‐(1–7) treatment ameliorates angiotensin II‐induced apoptosis of human umbilical vein endothelial cells

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Lanthanum chloride promotes mitochondrial apoptotic pathway in primary cultured rat astrocytes

Population surveys and animal experiments have shown that rare earth elements (REEs) cause neurological defects. However, the detailed mechanisms underlying these effects are still unclear. Given that lanthanum is commonly used for investigating into REEs‐induced neurological defects, this study chose lanthanum chloride (LaCl₃) to show that LaCl₃ promotes mitochondrial apoptotic pathway in primary cultured rat astrocytes by regulating expression of Bcl‐2 family proteins. The main findings of this study are (1) LaCl₃ treatment (0.25, 0.5, and 1.0 mM for 12–48 h) induced the astrocytes damages with a concentration‐dependent manner, which were confirmed with methyl thiazolyl tetrazolium and lactate dehydrogenase release assays, and morphological examination. (2) A 24 h treatment of LaCl₃ concentration‐dependently decreased mitochondrial membrane potential, increased cytochrome c release from mitochondria into cytosol, elevated caspase 9 and 3 expression, and promoted astrocyte apoptosis. (3) LaCl₃ treatment increased the ratio of pro‐apoptotic Bax and antiapoptotic Bcl‐2 proteins, which in turn broke the balance among pro‐apoptotic and antiapoptotic Bcl‐2 family proteins, leading to astrocyte apoptosis. Our results indicate that LaCl₃ alters Bcl‐2 family protein expressions, which in turn promote mitochondrial apoptotic pathway, and thus astrocytic damage. © 2011 Wiley Periodicals, Inc. Environ Toxicol 28: 489–497, 2013.     

Antitumor effects of deguelin on H460 human lung cancer cells in vitro and in vivo: Roles of apoptotic cell death and H460 tumor xenografts model

Deguelin, a naturally occurring rotenoid of the flavonoid family, is known to be an Akt inhibitor, to have chemopreventive activities and anti‐tumor effect on several cancers. In this study, investigation to elucidate the effect of deguelin on apoptotic pathways in human lung cancer cells and on the anti‐tumor effect in lung cancer xenograft nu/nu mice was performed. In vitro studies, found that deguelin induced cell morphological changes, and decreased the percentage of viability through the induction of apoptosis in H460 lung cancer cells. Deguelin triggered apoptosis in H460 cells was also confirmed by DAPI staining, DNA gel electrophoresis, and Annexin V‐FITC staining and these effects are dose‐dependent manners. It was also found that deguelin promoted the Ca²⁺ production and activation of caspase‐3 but decreased the level of ΔΨ _m in H460 cells. Western blots indicated that the protein levels of cytochrome c , AIF, and pro‐apoptotic Bax and Bak protein were increased, but the anti‐apoptotic Bcl‐2 and Bcl‐x were decreased that may have led to apoptosis in H460 cells after exposure to deguelin. It was also confirmed by confocal laser microscope examination that deguelin promoted the release of AIF from mitochondria to cytosol. In vivo studies, found that in immunodeficient nu/nu mice bearing H460 tumor xenografts showed that the deguelin significantly suppressed tumor growth. Deguelin might be a potential therapeutic agent for the treatment of lung cancer in the future. This finding might fully support a critical event for deguelin via induction of apoptotic cell death and H460 tumor xenografts model against human lung cancer. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 84–98, 2017.     

Fluoxetine Induces Apoptosis in Ovarian Carcinoma Cell Line OVCAR‐3 Through Reactive Oxygen Species‐Dependent Activation of Nuclear Factor‐κB

Abstract: The apoptotic effect of fluoxetine (FLX), an antidepressant, against human epithelial ovarian cancer cell lines OVCAR‐3 and SK‐OV‐3 was investigated in relation to the mitochondria‐mediated cell death process and nuclear factor (NF)‐κB activation. FLX‐induced mitochondrial membrane permeability change and formation of reactive oxygen species, leading to cell death. FLX‐induced increase in mitochondrial Bax levels, decrease in cytosolic Bid and Bcl‐2 levels, loss of the mitochondrial transmembrane potential, cytochrome c release, caspase‐3 activation and up‐regulation of p53. Oxidant scavengers and Bay 11‐7085 [an inhibitor of nuclear factor kappaB (NF‐κB) activation] prevented the FLX‐induced cell death, increase in phosphorylated inhibitory κB‐α and NF‐κB p65 levels, and binding of NF‐κB p65 to DNA. Results from this study suggest that FLX may exhibit apoptotic effect against ovarian cancer cell lines by inducing the mitochondrial membrane permeability change, which leads to cytochrome c release and subsequent caspase‐3 activation, through reactive oxygen species‐dependent activation of NF‐κB.     

Different mechanisms of acute versus long‐term antihypertensive effects of soluble epoxide hydrolase inhibition: Studies in Cyp1a1‐Ren‐2 transgenic rats

Recent studies have shown that the long‐term antihypertensive action of soluble epoxide hydrolase inhibition (sEH) in angiotensin‐II (AngII)‐dependent hypertension might be mediated by the suppression of intrarenal AngII levels. To test this hypothesis, we examined the effects of acute (2 days) and chronic (14 days) sEH inhibition on blood pressure (BP) in transgenic rats with inducible AngII‐dependent hypertension. AngII‐dependent malignant hypertension was induced by 10 days’ dietary administration of indole‐3‐carbinol (I3C), a natural xenobiotic that activates the mouse renin gene in Cyp1a1‐Ren‐2 transgenic rats. BP was monitored by radiotelemetry. Acute and chronic sEH inhibition was achieved using cis‐4‐(4‐(3‐adamantan‐1‐yl‐ureido)cyclohexyloxy) benzoic acid, given at doses of 0.3, 3, 13, 26, 60 and 130 mg/L in drinking water. At the end of experiments, renal concentrations of epoxyeicosatrienoic acids, their inactive metabolites dihydroxyeicosatrienoic acids and AngII were measured. Acute BP‐lowering effects of sEH inhibition in I3C‐induced rats was associated with a marked increase in renal epoxyeicosatrienoic acids to dihydroxyeicosatrienoic acids ratio and acute natriuresis. Chronic treatment with cis‐4‐(4‐(3‐adamantan‐1‐yl‐ureido)cyclohexyloxy) benzoic acid in I3C‐induced rats elicited dose‐dependent persistent BP lowering associated with a significant reduction of plasma and kidney AngII levels. Our findings show that the acute BP‐lowering effect of sEH inhibition in I3C‐induced Cyp1a1‐Ren‐2 transgenic rats is mediated by a substantial increase in intrarenal epoxyeicosatrienoic acids and their natriuretic action without altering intrarenal renin–angiotensin system activity. Long‐term antihypertensive action of cis‐4‐(4‐(3‐adamantan‐1‐yl‐ureido)cyclohexyloxy) benzoic acid in I3C‐induced Cyp1a1‐Ren‐2 transgenic rats is mediated mostly by suppression of intrarenal AngII concentration.     

Cytotoxic evaluation and induction of mitochondria-mediated apoptosis in human leukaemia HL-60 cells by Carissa spinarum stem isolate

Objective To evaluate Carissa spinarum stem isolate for its anti‐cancer therapeutic potential. Methods The n‐butanol fraction of aqueous extract from Carissa spinarum stem was assessed for its cytotoxic and pro‐apoptotic activity. Key findings We report for the first time the anti‐cancer potential of C. spinarum stem aqueous extract (CSE) and its n‐butanol fraction (CSF). Both inhibited cell proliferation of various human cancer cell lines in which leukaemia HL‐60 cells treated with CSF showed maximum growth inhibition having an inhibitory concentration (IC₅₀) value of 34.58 ± 0.91 µg/ml. In addition, CSF induced concentration‐dependent apoptosis in HL‐60 cells as measured by various end‐points (e.g. Annexin V binding, DNA laddering, apoptotic body formation and an increase in hypodiploid subG0 DNA content). Moreover, persistent levels of reactive oxygen species caused translocation of Bax to mitochondria and Bcl‐2 degradation, which led to loss of mitochondrial membrane potential and release of cytochrome c to the cytosol. These events were associated with significant activation of caspase‐3, caspase‐6 and caspase‐9 leading to poly (ADP‐ribose) polymerase cleavage. Conclusion All the above parameters revealed that CSF induced apoptosis through the mitochondrial dependent pathway in HL‐60 cells.     

p,p'-DDE induces apoptosis and mRNA expression of apoptosis-associated genes in testes of pubertal rats

One,1‐dichloro‐2,2 bis(p‐chlorophenyl) ethylene (p,p'‐DDE), the major metabolite of 2,2‐bis(4‐chlorophenyl)‐1,1,1‐trichloroethane (DDT), is a known persistent organic pollutant and male reproductive toxicant. It has antiandrogenic effect. However, the mechanism by which p,p'‐DDE exposure causes male reproductive toxicity remains unknown. To elucidate the mechanism underpinning the testicular effects of p,p'‐DDE, we sought to investigate apoptotic effects and mRNA expression of apoptosis‐associated genes in the testis of pubertal rats, including Fas, FasL, calpain‐1, cytochrome c, Bax, Bcl‐w, Bak, and caspase‐3, ‐8, ‐9, ‐12. Animals were administered with different doses of p,p'‐DDE (0, 20, 60, 100 mg/kg body weight) every other day by intraperitoneal injection for 10 days. The results indicated that p,p'‐DDE exposure at over 20 mg/kg body weight showed the induction of apoptotic cell death. p,p'‐DDE could induce decrease in SOD and GSH‐Px activity of serum in 60 mg/kg body weight group. Significant elevations in the mRNA levels of Fas, FasL, calpain‐1, cytochrome c, Bax, Bak, and caspase‐3, ‐8, ‐9, ‐12 were observed in testis of rat treated with p,p'‐DDE. Taken together, these results lead us to speculate that in vivo exposure to p,p'‐DDE might induce testicular apoptosis in pubertal rats through the involvement of Fas/FasL, mitochondria and endoplasmic reticulum‐mediated pathways. © 2011 Wiley Periodicals, Inc. Environ Toxicol 2013.     

Polygalasaponin F inhibits neuronal apoptosis induced by oxygen‐glucose deprivation and reoxygenation through the PI3K/Akt pathway

Cerebral ischaemia is a common cerebrovascular disease and often induces neuronal apoptosis, leading to brain damage. Polygalasaponin F (PGSF) is one of the components in Polygala japonica Houtt, and it is a triterpenoid saponin monomer. This research focused on anti‐apoptotic effect of PGSF during oxygen‐glucose deprivation and reoxygenation (OGD/R) injury in rat adrenal pheochromocytoma cells (PC12) and primary rat cortical neurons. OGD/R treatment reduced viability of PC12 cells and primary neurons. This reduced viability was prevented by PGSF, as shown by MTT assay. OGD/R insult decreased expression of Bcl‐2/Bax both in PC12 cells and primary neurons but elevated levels of caspase‐3 in primary neurons. However, PGSF may up‐regulate expression of Bcl‐2/Bax and down‐regulate caspase‐3 in these particular cells. Furthermore, Bcl‐2/Bax and the ratio between phosphorylated Akt and total Akt were decreased in PC12 cells treated with OGD/R, and both were increased by PGSF. Moreover, increase in the ratios of Bcl‐2/Bax and phosphorylated Akt/total Akt in PC12 cells was suppressed by phosphatidylinositol 3‐kinase (PI3K) inhibitor. Data suggest PGSF might prevent OGD/R‐induced injury via activation of PI3K/Akt signalling. The ability of PGSF to block the effects of OGD/R appears to involve regulation of Bcl‐2, Bax and caspase‐3, which are related to apoptosis.     

Synthesis and Biological Evaluation of Benzo[d][1,3]Dioxol‐5‐yl Chalcones as Antiproliferating Agents

A series of chalcone derivatives were designed, synthesized, and evaluated for their cytotoxic potential. These molecules have showed promising cytotoxic activity with IC₅₀ values ranging from 5.24 to 63.12 μm . Among them, conjugates 16k , 16m and 16t showed significant antiproliferative activity with IC₅₀ values ranging from 5.24 to 10.39  μ m in MDA‐MB‐231 cell line. These compounds were further investigated for their effect on cell membrane blebbing, chromatin condensation, DNA fragmentation, Hoechst staining, annexin V, and cell cycle arrest (G2/M). The Western blot experiments revealed up regulation of pro‐apoptotic Bax and downregulation of antiapoptotic Bcl‐2. The studies also indicated reduction of mitochondrial membrane potential and increase in the levels of caspase‐3 and caspase‐7.