Effect of HA14‐1 on Apoptosis‐Regulating Proteins in HeLa Cells

Overexpression of Bcl‐2 has been recognized in various malignancies. Recently, HA14‐1, a Bcl‐2 antagonist, has been identified for its anti‐apoptotic effect. However, mode of action of HA14‐1 still remains to be elucidated. In this study, we examined HA14‐1 binding efficiency with receptor proteins through molecular docking. Cell viability using HeLa cells was evaluated through MTT assay after exposure to different concentration of HA14‐1. Moreover, after HA14‐1 exposure, expressions of tumor suppressor protein (p53), BH3‐only protein (Puma) and apoptosis‐associated proteins were analyzed by Western blotting. From the results, it was found that HA14‐1 occupied all three domains; BH1, BH2, and BH3 within the hydrophobic pocket of Bcl‐2. However, HA14‐1 occupied only BH1 and BH3 of Bcl‐xl, conversely, no such stable bond was observed for Bax and Bak. ARG107 and TYR101 were the amino acids involved in the binding of HA14‐1 to Bcl‐2 and Bcl‐xl, respectively. Additionally, decrease in Bcl‐2 and Bcl‐xl expression along with increase in p53 and Puma expression after exposure to HA14‐1 was observed. The results suggested p53 pathway to be the probable mechanism of action for the induction of apoptosis in HeLa cell by downregulating the effect of anti‐apoptotic proteins suggesting that HA14‐1 may provide therapeutic potential for the treatment of human cervical cancer.     

2.45 GHz microwave radiation induced oxidative and nitrosative stress mediated testicular apoptosis: Involvement of a p53 dependent bax‐caspase‐3 mediated pathway

Deleterious effects of MW radiation on the male reproduction are well studied. Previous reports although suggest that 2.45 GHz MW irradiation induced oxidative and nitrosative stress adversely affects the male reproductive function but the detailed molecular mechanism occurring behind it has yet to be elucidated. The aim of present study was to investigate the underlying detailed pathway of the testicular apoptosis induced by free radical load and redox imbalance due to 2.45 GHz MW radiation exposure and the degree of severity along with the increased exposure duration. Twelve‐week old male mice were exposed to 2.45 GHz MW radiation [continuous‐wave (CW) with overall average Power density of 0.0248 mW/cm² and overall average whole body SAR value of 0.0146 W/kg] for 2 hr/day over a period of 15, 30, and 60 days. Testicular histology, serum testosterone, ROS, NO, MDA level, activity of antioxidant enzymes, expression of pro‐apoptotic proteins (p53 and Bax), anti‐apoptotic proteins (Bcl‐2 and Bcl‐x_L), cytochrome‐c, inactive/active caspase‐3, and uncleaved PARP‐1 were evaluated. Findings suggest that 2.45 GHz MW radiation exposure induced testicular redox imbalance not only leads to enhanced testicular apoptosis via p53 dependent Bax‐caspase‐3 mediated pathway, but also increases the degree of apoptotic severity in a duration dependent manner.     

Apoptotic‐related protein changes induced by hexavalent chromium in mice liver

Although it has been reported that hexavalent chromium (Cr(VI)) could induce apoptosis in a variety of cell types, the molecular mechanisms underlying this process is still largely unknown. This study was undertaken to determine effects of single oral 0, 25, 50, and 100 mg/kg body weight doses of potassium dichromate on the expression level of p53, Bcl‐2, Bax, cytochrome c, and caspase‐3, which are vital regulators of apoptosis, in mice liver. The results showed that Cr(VI) could upregulate the protein expression of p53, Bax, cytochrome c, and caspase‐3 and downregulate the expression of Bcl‐2 in mice liver. All these results suggested that p53, Bcl‐2, Bax, cytochrome c, and caspase‐3 may be involved in the regulation of Cr(VI) induced apoptosis in vivo. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2010.     

Butein,a Plant Polyphenol,Induces Apoptosis Concomitant with Increased Caspase‐3 Activity,Decreased Bcl‐2 Expression and Increased Bax Expression in HL‐60 Cells

Abstract: In the present study we have investigated whether butein could induce apoptosis in human leukaemic HL‐60 cells. The treatment of HL‐60 cells with butein induced apoptotic cell death as determined by morphological and biochemical changes. Apoptotic DNA fragments in the butein‐treated HL‐60 cells were increased gradually as determined by flow cytometric analysis. The caspase‐3 activity was increased during butein‐induced apoptosis. However, caspase‐3 inhibitor abrogated the butein‐induced DNA fragmentation. Furthermore, the treatment of HL‐60 cells with butein decreased the expression of Bcl‐2 protein, but increased the expression of Bax protein. These results suggest that butein‐induced apoptosis is mediated through the activation of caspase‐3 and it is associated with changed expression of Bcl‐2 and Bax proteins.     

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.     

DDTD, an isoflavone derivative, induces cell apoptosis through the reactive oxygen species/apoptosis signal-regulating kinase 1 pathway in human osteosarcoma cells

Osteosarcoma is the most common primary bone tumor associated with childhood and adolescence. In the present study, we investigated the anticancer effect of a new isoflavone derivative, 3',4'-dichloro-3-(3,4-dichlorophenylacetyl)-2,4,6-trihydroxydeoxybenzoin (DDTD) in human osteosarcoma cells. DDTD induced cell apoptosis in human osteosarcoma cell lines (including: U2OS, MG-63, Saos2 and ROS 17/2.8). We found that the accumulation of reactive oxygen species is a critical mediator in DDTD-induced cell death. DDTD induced apoptosis signal-regulating kinase 1 (ASK1) dephosphorylation and its dissociation from 14-3-3. Treatment of osteosarcoma cells with DDTD induced p38 and p53 phosphorylation. Transfection with ASK1, mitogen activated protein kinase (MAPK) kinase (MKK)3/6, and p38 small interfering RNA (siRNA) antagonized the DDTD-induced cell apoptosis. DDTD also triggered the mitochondrial apoptotic pathway, as indicated by a change in Bax/Bcl2 ratio and Caspase-9 activation. Bax knockdown using a Bax siRNA strategy reduced Bax expression and subsequent cell death. In addition, transfection of cells with ASK1, MKK3/6, and p38 siRNA reduced DDTD-induced p38 activation, p53 phosphorylation and Bax expression. These results suggest that DDTD generates reactive oxygen species and activates the ASK1-MKK3/6-p38-p53-Bax pathway to cause osteosarcoma cell death.     

Multisite phosphorylation of Bcl‐2 via protein kinase Cδ facilitates apoptosis of hypertrophic cardiomyocytes

Activated protein kinase Cδ (PKCδ) associated with cardiac hypertrophy moves from the cytoplasm to the mitochondria and subsequently triggers the apoptotic signalling pathway. The underlying mechanisms remain unknown. The aim of the present study was to investigate whether mitochondrial translocation of PKCδ phosphorylates multiple sites of Bcl‐2, resulting in an imbalance between Bcl‐2 and Bak or Bax, thus enhancing the susceptibility of hypertrophic cardiomyocytes to angiotensin II (AngII)‐induced apoptosis. Chronic pressure overload was induced by transverse aortic constriction (TAC) in rats. The apoptotic rate increased in hypertrophied cardiomyocytes. In AngII‐treated hearts (10 nmol/L, 60 min), there was an increase in the number of TERMINAL deoxyribonucleotidyl transferase‐mediated dUTP–digoxigenin nick end‐labelling (TUNEL)‐positive cells; PKCδ inhibition with 500 nmol/L δV1‐1 for 60 min prevented the AngII‐induced increase in apoptosis. In the hypertrophied myocardium, PKCδ expression increased, whereas that of Bcl‐2 decreased compared with the synchronous control. Treatment of hearts with 10 nmol/L AngII for 60 min activated PKCδ and induced translocation of PKCδ to the mitochondria, where activated PKCδ facilitated the phosphorylation of Bcl‐2 at serine‐87 and serine‐70 sites. The multisite phosphorylated Bcl‐2 was released from the mitochondria, and exhibited reduced affinity for Bak and Bax. The imbalance between Bcl‐2 and Bak/Bax induced the release of mitochondrial cytochrome c and then activated the caspase 3 apoptotic pathway during AngII stimulation (10 nmol/L, 60 min) of hypertrophied cardiomyocytes. Inhibition of PKCδ reduced these effects of AngII. The results suggest that PKCδ can counteract the anti‐apoptotic effect of Bcl‐2 and may promote cardiomyocyte apoptosis through multisite phosphorylation of Bcl‐2 in hypertrophied cardiomyocytes.     

Polychlorinated biphenyl quinone induces mitochondrial‐mediated and caspase‐dependent apoptosis in HepG2 cells

Polychlorinated biphenyl (PCB) quinones are known to cause toxic effects, but their mechanisms are quite unclear. In this study, we examined whether 2,3,5‐trichloro‐6‐phenyl‐[1,4]benzoquinone, PCB29‐pQ, induces cell death via apoptosis pathway. Our result showed PCB29‐pQ exposure decreased HepG2 cell viability in a time‐dependent manner. Lactate dehydrogenase leakage assay also implied the cytotoxicity of PCB29‐pQ. 4′,6‐Diamidino‐2‐phenylindole dihydrochloride staining and flow cytometry assays both confirmed PCB29‐pQ caused dose‐dependent apoptotic cell death in HepG2 cells. Furthermore, we found that PCB29‐pQ exposure increased cellular reactive oxygen species (ROS) level, decreased mitochondrial membrane potential and induced the translocation of cytochrome c from mitochondria into cytosol in HepG2 cells. Moreover, PCB29‐pQ exposure induced B‐cell lymphoma 2 (Bcl‐2) downregulation and Bcl‐2‐associated X (Bax) upregulation, poly(ADP‐ribose) polymerase cleavage, accompanied with the increased caspase‐3/9 and p53 expressions. Taking together, these results suggested PCB29‐pQ induced HepG2 cells apoptosis through a ROS‐driven, mitochondrial‐mediated and caspase‐dependent pathway. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1063–1072, 2015.     

Mono(2‐ethylhexyl) phthalate induces apoptosis in p53‐silenced L02 cells via activation of both mitochondrial and death receptor pathways

Mono(2‐ethylhexyl) phthalate (MEHP) is one of the main metabolites of di(2‐ethylhexyl) phthalate. The evidence shows that DEHP may exert its toxic effects primarily via MEHP, which is 10‐fold more potent than its parent compound in toxicity in vitro. MEHP‐induced apoptosis is mediated by either p53‐dependent or ‐independent pathway. However, the detailed mechanism of its toxicity remains unclear. In this study, immortalized normal human liver cell line L02 was chosen, as an in vitro model of nonmalignant liver, to elucidate the role of p53 in MEHP‐induced apoptosis. The cells were treated with MEHP (6.25, 12.50, 25.00, 50.00, and 100.00 μM) for 24 and 36 h, then small interfering RNA (siRNA) was used to specifically silence p53 gene of L02 cells. The results indicated that MEHP caused oxidative DNA damage and apoptosis in L02 cells were associated with the p53 signaling pathway. Further study found that MEHP (50.00 and 100.00 μM) induced apoptosis in p53‐silenced L02 cells, along with the up‐regulations of Fas and FasL proteins as well as increased the Bax/Bcl‐2 ratio and Caspase 3, 8, and 9 activities. Additionally, both FasL inhibitor (AF‐016) and Caspase inhibitor N‐benzyloxycarbonyl‐Val‐Ala‐Asp‐ fluoromethylketone (Z‐VAD‐FMK) could prevent the cell apoptosis induced by MEHP. The findings suggested that MEHP‐induced apoptosis in L02 cells involving a Caspases‐mediated mitochondrial signaling pathway and/or death receptor pathway. p53 was not absolutely necessary for MEHP‐induced L02 cell apoptosis. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1178–1191, 2015.     

Tri‐ortho‐cresyl phosphate (TOCP)‐induced reproductive toxicity involved in placental apoptosis,autophagy and oxidative stress in pregnant mice

Tri‐ortho‐cresyl phosphate (TOCP) has been widely used as plasticizers, and reported causing reproductive toxicity in mammals. However, little is known about the toxic effect on the placenta. In this study, dams were orally administered different doses of TOCP to explore the effect of TOCP on placental development. Results showed that TOCP exposure significantly reduced numbers of implanted embryo, caused atrophy and collapse of ectoplacental cone, and decreased total areas of placenta and numbers of PCNA‐positive cells. Expression levels of placental development genes were prominently downregulated in the TOCP‐treated groups. Moreover, TOCP administration induced placental apoptosis and autophagy by upregulating P53, Bax, Beclin‐1, ratio of LC3 II/LC3 I and Atg5 and downregulating Bcl‐2 protein. In addition, TOCP exposure markedly inhibited activities of catalase and superoxide dismutase and increased the production of H₂O₂ and malondialdehyde. Collectively, these findings suggest that apoptosis, autophagy and oxidative stress may be involved in the TOCP‐induced reproductive toxicity.     

Induction of apoptosis and cell cycle arrest in L‐1210 murine lymphoblastic leukaemia cells by (2E)‐3‐(2‐naphthyl)‐1‐(3′‐methoxy‐4′‐hydroxy‐phenyl)‐2‐propen‐1‐one

Objectives New compounds with biological targets and less cytotoxicity to normal cells are necessary for cancer therapy. In this work ten synthetic chalcones derived from 2‐naphtaldehyde were evaluated for their cytotoxic effect in murine acute lymphoblastic leukemia cells L‐1210. Methods A series of ten chalcones derived from 2‐naphtaldehyde and corresponding acetophenones were prepared by aldolic condensation, using methanol as solvent under basic conditions, at room temperature for 24 h. The cell viability was determined by MTT colorimeter method. The cell cycle phase analysis was carried out by flow cytometry after propidium iodide staining. The apoptosis induction was assessed by exposure to phosphatidylserine (ANNEXIN V‐FITC). Cytometric analysis was performed to evaluate the expression of p53, Bcl‐2 and Bax protein. The caspase‐3 expression was studied by immunoblotting analysis. Key findings A preliminary screening of a series of ten chalcones derived from 2‐naphtaldehyde showed that chalcone 8 , (2E)‐3‐(2‐naphtyl)‐1‐(3′‐methoxy‐4′‐hydroxy‐phenyl)‐2‐propen‐1‐one, had the highest cytotoxic effect (IC50 of 54 µm ), but not in normal human lymphocytes. To better understand the cytotoxic mechanism of chalcone 8 , its effect on cell cycle and apoptosis was assessed. Our results showed that chalcone 8 caused cell cycle arrest in the G2/M phase and a significant increase in the proportion of cells in the subG0/G1 phase. Our results also demonstrated that chalcone 8 promoted a modification in Bax : Bcl‐2 ratio and increased p53 expression and caspase‐3 activation. Conclusions The studied chalcone 8 has cytotoxic effect against L‐1210 lymphoblastic leukaemic cells, and this effect is associated with increase of p‐53 and Bax expression.     

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.     

Effects of SiO2 nanoparticles on HFL‐I activating ROS‐mediated apoptosis via p53 pathway

Silicon dioxide nanoparticles (SiO₂ NPs) have attracted increasing interest as nanovehicles for delivering drugs, genes and bio‐active molecules into cells. However, it is still unknown whether SiO₂ NPs could cause side‐effects to normal cells. In the present study, human lung fibroblasts (HFL‐Is) were directly exposed to two different sizes of SiO₂ NPs. The effect of size and concentration on cell response was studied by analyzing the cell viability, the ratio of apoptosis and the pathway of cell injury. The results demonstrated that a size‐associated and a dose‐dependent toxicity of HFL‐Is was induced by SiO₂ NPs. Meanwhile, the expression of reactive oxygen species in HFL‐I was significantly increased. This activation effect was accompanied by upregulation of p53 expression, release of cytochrome C from chondriosomes, inhibition of Bcl2, and activation of Bax and caspase 9. These findings implied that SiO₂ NPs might induce apoptosis of HFL‐Is by stimulating reactive oxygen species release and subsequently causing the activation of p53 pathway in vitro. Copyright © 2011 John Wiley & Sons, Ltd.     

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.     

The role of GSH in microcystin‐induced apoptosis in rat liver: Involvement of oxidative stress and NF‐κB

Microcystins (MCs) are potent and specific hepatotoxins produced by cyanobacteria in eutrophic waters, representing a health hazard to animals and humans. The objectives of this study are to determine the relationship between oxidative stress and NF‐κB activity in MC‐induced apoptosis in rat liver and the role of glutathione (GSH). Sprague‐Dawley rats were intraperitoneally (i.p.) injected with microcystin‐LR (MC‐LR) at 0.25 and 0.5 LD₅₀ with or without pretreatment of buthionine‐(S,R)‐sulfoximine (BSO), a specific GSH synthesis inhibitor. MC‐LR induced time‐dependent alterations of GSH levels in rat liver. Increased malondialdehyde (MDA) and significant changes of antioxidant enzymes including GSH peroxidase (GPX) and GSH reductase (GR) were also observed, particularly at 24 h post‐exposure. The results indicated that acute exposure to MC‐LR induced oxidative stress, and GSH depletion (BSO pretreatment) enhanced the level of oxidative stress. Furthermore, the modulation of pro‐apoptotic gene p53 and Bax and anti‐apoptotic gene Bcl‐2 was observed in 0.5 LD₅₀ group at 24 h, and the alteration was more pronounced by BSO injection before MC‐LR treatment, suggesting that GSH played a protective role against MC‐induced toxicity. Additionally, electrophoretic mobility shift assay (EMSA) showed that NF‐κB was induced at 0.25 LD₅₀ but inhibited at 0.5 LD₅₀. The above results indicated that the possible crosstalk of oxidative stress and NF‐κB activity was associated with MC‐LR‐induced hepatocytes apoptosis in vivo. Our data will provide a new perspective for understanding the mechanisms of MC‐induced liver injury. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 552–560, 2016.