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.     

Exploration of intrinsic and extrinsic apoptotic pathways in zearalenone‐treated rat sertoli cells

Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin produced mainly by Fusarium. ZEA causes reproductive disorders and is both cytotoxic and genotoxic in animals; however, little is known regarding the molecular mechanism(s) leading to ZEA toxicity. Sertoli cells are somatic cells that support the development of spermatogenic cells. The objective of this study was to explore the effects of ZEA on the proliferation, apoptosis, and necrosis of rat Sertoli cells to uncover signaling pathways underlying ZEA cytotoxicity. ZEA reduced the proliferation of rat Sertoli cells in a dose‐dependent manner, as indicated by a CCK8 assay, while flow cytometry revealed that ZEA caused both apoptosis and necrosis. Immunoblotting revealed that ZEA treatment increased the ratio of Bax/Bcl‐2, as well as the expression of FasL and caspases‐3, ?8, and ?9, in a dose‐dependent manner. Collectively, these data suggest that ZEA induced apoptosis and necrosis in rat Sertoli cells via extrinsic and intrinsic apoptotic pathways. This study provides new insights into the molecular mechanisms by which ZEA exhibits cytotoxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1731–1739, 2016.     

Imperatorin,a Furanocoumarin from Angelica dahurica (Umbelliferae), Induces Cytochrome c‐Dependent Apoptosis in Human Promyelocytic Leukaemia,HL‐60 Cells

Abstract: Imperatorin, a biologically active furanocoumarin from the roots of Angelica dahurica (Umbelliferae), was found to induce apoptosis in human promyelocytic leukaemia, HL‐60 cells. DNA fragmentation assay, morphology‐based evaluation, and flow cytometric analysis demonstrated that imperatorin at micromolar concentrations was able to trigger apoptosis of HL‐60 cells. Neither necrosis nor differentiation was observed at cytotoxic micromolar concentrations of imperatorin. Further studies showed that the cytochrome c/caspase‐9 pathway was responsible for imperatorin‐induced apoptosis; i.e., mitochondrial membrane was depolarized, Bcl‐2 was down‐regulated, cytochrome c was released from mitochondria, caspase‐9 and caspase‐3 were activated, and poly(ADP‐ribose) polymerase was cleaved. Furthermore, imperatorin‐induced apoptosis was significantly blocked by Z‐VAD‐FMK (a broad spectrum caspase inhibitor), Z‐LEHD‐FMK (a caspase‐9 inhibitor) and Ac‐DMQD‐CHO (a caspase‐3 inhibitor), but not by Z‐IEDT‐FMK (a caspase‐8 inhibitor).     

Heterogeneous role of caspase-8 in fenretinide-induced apoptosis in epithelial ovarian carcinoma cell lines

The mechanism of action of fenretinide, a synthetic retinoid currently undergoing testing as a chemopreventive and chemotherapeutic agent, is incompletely understood. In the present study, fenretinide caused apoptotic changes, including DNA fragmentation and cleavage of caspase substrates, in six low-passage ovarian cancer cell lines. However, the caspase activation pathway used by this agent varied. Transient transfection of cDNA-encoding cytokine response modifier A (CrmA), a caspase-8 inhibitor, diminished fenretinide-induced death in OV177 cells. Likewise, IETD(OMe)-fluoromethylketone (fmk) inhibited fenretinide-induced apoptosis by >80% in OV177 or OV266 cells and by approximately 50% in OV17, OV167, or OV207 cells. Further analysis demonstrated that inhibition of Fas ligand, tumor necrosis factor-alpha, or TRAIL signaling with blocking reagents did not affect fenretinide-induced apoptosis, raising the possibility that fenretinide activates caspase-8 in a death receptor-independent manner. In contrast, CrmA transfection or IETD(OMe)-fmk treatment did not inhibit fenretinide-induced apoptosis in OV202 cells. These divergent behaviors did not correlate with increased levels of procaspase-10, which is relatively resistant to CrmA and IETD(OMe)-fmk, nor with the expression of procaspase-8 and -9, apoptotic protease activating factor-1, or cellular FLICE-like inhibitory protein. Similarly, fenretinide treatment increased ceramide levels equally in cells that do (OV177) and do not (OV202) rely on caspase-8 to initiate apoptosis. These results indicate that synthetic retinoids can use caspase-8 as an initiating caspase, but they also indicate unexpected heterogeneity in caspase activation pathways among closely related cell lines.     

Sulphasalazine accelerates apoptosis in neutrophils exposed to immune complex: Role of caspase pathway

• 1 Neutrophils release several histotoxic molecules that cause tissue injury. Neutrophil apoptosis is a crucial process that governs the persistence of inflammatory disorders and tissue damage. Thus, in the present study, we investigated whether the anti‐inflammatory drug sulphasalazine (SSZ) affects neutrophil apoptosis in the presence of insoluble immune complex (IC).
• 2 Neutrophils were obtained from healthy donors. Neutrophils were resuspended in incubation medium and incubated for 2–12 h with or without 10, 30 or 100 μmol/L SSZ and 25 μg/mL IC. In some experiments, cells were co‐incubated with 20 μmol/L Z‐IETD‐fmk (a caspase 8 inhibitor) or 20 μmol/L Z‐LEHD‐fmk (a caspase 9 inhibitor). Apoptosis was evaluated morphologically on cytological preparations stained with May–Grünwald–Giemsa as well as by flow cytometry analysis of annexin V and propidium iodide staining. Caspase 3 activity was determined spectrophotometrically.
• 3 At 100 μmol/L, SSZ significantly accelerated IC‐induced neutrophil apoptosis. Treatment of neutrophils with 20 μmol/L of the caspase 8 or 9 inhibitors Z‐IETD‐fmk or Z‐LEHD‐fmk, respectively, demonstrated that the SSZ‐induced pro‐apoptotic effect was mediated by a caspase 8‐ but not caspase 9‐dependent pathway. The caspase 3 activity assay showed that treatment with 100 μmol/L SSZ increased caspase 3 activation.
• 4 In conlusion, the results of the present study indicate that it is possible that the molecular mechanism underlying SSZ protection against neutrophil‐mediated tissue injury inflammatory disorders, such as rheumatoid arthritis and inflammatory bowel diseases, involves a caspase 8‐dependent pathway.

     

Loss of mitochondrial transmembrane potential and caspase-9 activation during apoptosis induced by the novel styryl-lactone goniothalamin in HL-60 leukemia cells.

Styryl-lactones such as goniothalamin represent a new class of compounds with potential anti-cancer properties. In this study, we investigated the mechanisms of goniothalamin (GTN), a plant styryl-lactone induced apoptosis in human promyelocytic leukemia HL-60 cells. This plant extract resulted in apoptosis in HL-60 cells as assessed by the externalisation of phosphatidylserine. Using the mitochondrial membrane dye (DIOC(6)) in conjunction with flow cytometry, we found that GTN treated HL-60 cells demonstrated a loss of mitochondrial transmembrane potential (Deltapsi(m)). Further immunoblotting on these cells showed activation of initiator caspase-9 and the executioner caspases-3 and -7. Pretreatment with the pharmacological caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.FMK) abrogated apoptosis as assessed by all of the apoptotic features in this study. In summary, our results demonstrate that goniothalamin-induced apoptosis occurs via the mitochondrial pathway in a caspase dependent manner.     

Ouabain induces apoptotic cell death in human prostate DU 145 cancer cells through DNA damage and TRAIL pathways

Ouabain, a cardiotonic steroid and specific Na⁺/K⁺‐ATPase inhibitor, has a potential to induce cancer cell apoptosis but the mechanisms of apoptosis induced by ouabain are not fully understand. The aim of this study was to investigate the cytotoxic effects of ouabain on human prostate cancer DU 145 cells in vitro. Cell morphological changes were examined by phase contrast microscopy. Cell viability, cell cycle distribution, cell apoptosis, DNA damage, the production of ROS and Ca²⁺, and mitochondrial membrane potential (ΔΨ_m) were measured by flow cytometry assay. Results indicated that ouabain induced cell morphological changes, decreased total cell viability, induced G0/G1 phase arrest, DNA damage, and cell apoptosis, increased ROS and Ca²⁺ production, but decreased the levels of ΔΨ_m in DU 145 cells. Ouabain also increased the activities of caspase‐3, ‐8, and ‐9. Western blotting was used for measuring the alterations of apoptosis‐associated protein expressions in DU 145 cells and results indicated that ouabain increased the expression of DNA damage associated proteins (pATM^Ser1981, p‐H2A.X^Ser139, and p‐p53^Ser15) and ER‐stress‐associated proteins (Grp78, ATF6β, p‐PERK^Thr981, PERK, eIF2A, GADD153, CaMKIIβ, and caspase‐4) in time‐dependently. Furthermore, ouabain increased apoptosis‐associated proteins (DR4, DR5, Fas, Fas Ligand, and FADD), TRAIL pathway, which related to extrinsic pathway, promoted the pro‐apoptotic protein Bax, increased apoptotic‐associated proteins, such as cytochrome c, AIF, Endo G, caspase‐3, ‐8, and ‐9, but reduced anti‐apoptotic protein Bcl‐2 and Bcl‐x in DU 145 cells. In conclusion, we may suggest that ouabain decreased cell viability and induced apoptotic cell death may via caspase‐dependent and mitochondria‐dependent pathways in human prostate cancer DU 145 cells.     

Zinc Protects Human Kidney Cells from Depleted Uranium‐induced Apoptosis

Depleted uranium (DU) is a weak radioactive heavy metal, and zinc (Zn) is an effective antidote to heavy metal poisoning. However, the effect of Zn on DU‐induced cytotoxicity and apoptosis is not completely understood. The purpose of this study was to evaluate the effect of Zn on DU‐induced cell apoptosis in human kidney cells (HK‐2) and explore its molecular mechanism. Pre‐treatment with Zn significantly inhibited DU‐induced apoptosis. It reduced the formation of reactive oxygen species in the cells, increased the catalase (CAT) and glutathione (GSH) concentrations, suppressed the DU‐induced soluble Fas receptor (sFasR) and soluble Fas ligand (sFasL) overexpression, suppressed the release of cytochrome c and apoptosis inhibitor factor (AIF) from mitochondria to cytoplasm, inhibited the activation of caspase‐9, caspase‐8 and caspase‐3, and induced metallothionein (MT) expression. Furthermore, exogenous MT effectively inhibited DU‐induced cell apoptosis. In conclusion, mitochondrial and FasR‐mediated apoptosis pathways contribute to DU‐induced apoptosis in HK‐2 cells. Through independent mechanisms, such as indirect antioxidant effects, inhibition of the activation of caspase‐9, caspase‐8 and caspase‐3, and induction of MT expression, Zn inhibits DU‐induced apoptosis.     

p53‐Dependent pathway and the opening of mPTP mediate the apoptosis of co‐cultured Sertoli‐germ cells induced by microcystin‐LR

Microcystin‐LR (MC‐LR), a potent endotoxin, can induce reproductive toxicity. In order to investigate the role and mechanisms of apoptosis (p53‐dependent and mitochondrial pathways) of germ cells induced by MC‐LR, the co‐cultured primary Sertoli‐germ cells from Sprague‐Dawley rats were used for the experiments. Expression levels of proteins, genes, and mitochondrial membrane potential (MMP) were obtained after exposing co‐cultured Sertoli‐germ cells to MC‐LR with or without the addition of the p53 inhibitor, pifithrin‐α (PFT‐α), and MMP inhibitor, cyclosporin A (CsA). Results indicated that MC‐LR could activate p53‐dependent pathway‐associated proteins in Sertoli‐germ cells, leading to a decrease in MMP (indicating the opening of mitochondrial permeability transition pore [mPTP] and the release of Cytochrome‐c [Cyt‐c]) from the mitochondria into the cytoplasm and eventually the induction of apoptosis. PFT‐α inhibited the expression ofp53, ameliorated the MMP of the co‐cultured Sertoli‐germ cells, and prevented the release of Cyt‐c from the mitochondria into the cytoplasm, which reduces the occurrence of apoptosis. Similarly, the decreased release of Cyt‐c from the mitochondria into the cytoplasm and the declined level of apoptosis in Sertoli‐germ cells induced by MC‐LR were observed after the addition of CsA. These results indicated that the apoptosis of the co‐cultured Sertoli‐germ cells induced by MC‐LR was mediated by the p53‐dependent pathway, with the involvement of the opening of mPTP.     

Neural stem cells and cell death

Neural stem cells (NSC) undergo apoptotic cell death as an essential component of neural development. Here, we present the results of our studies on the mechanisms by which NSC undergo cell death in response to neurotoxic insults. As experimental models we used primary culture of adult NSC from the subventricular zone of the rat brain, and the neural stem cell line C17.2 initially derived from developing mouse cerebellum. NSC undergo apoptosis in response to staurosporine (0.25 microM) as well as agents inducing oxidative stress such as 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). Exposed cells demonstrate an apoptotic morphology, positive TUNEL staining and phosphatidyl serine exposure as labeled with Annexin V. Using an antibody specific for cytochrome c, we found that cells exposed to staurosporine or DMNQ exhibited diffuse fluorescence throughout the cytosol, implying a release of cytochrome c from the mitochondria. In addition to positive immunoreactivity against the active fragment (p17) of caspase-3, the administration of the pan-caspase inhibitor, zVAD-fmk (40 microM), prevents apoptosis. Both NSC and C17.2 express the Fas receptor, and procaspase-8, but exposure to agonistic Fas mAb (250 ng/ml) fails to induce apoptosis. Pretreatment with cycloheximide or actinomycin D does not influence the cell response to Fas mAb, suggesting that the endogenous inhibitor of caspase-8 FLICE-inhibitory protein (FLIP) is not responsible for the inhibition of the Fas pathway. Thus, it appears that the Fas dependent cell death pathway is not operative in these cells, while the mitochondrial pathway is active and caspase-3 serves as an executioner caspase in the apoptotic machinery. It is known that Fas not only induces apoptosis, but can also deliver growth stimulatory signals through activation of the extracellular-signal regulated kinase (ERK) pathway. The Fas-induced ERK phosphorylation that we detect in C17.2 cells suggests that in NSC Fas may function as a mediator of growth rather than death.     

HPMA copolymer-bound doxorubicin induces apoptosis in human ovarian carcinoma cells by a Fas-independent pathway

The mechanism of cell death in A2780 human ovarian carcinoma cells induced by free doxorubicin (DOX) and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound DOX [P-(GFLG)-DOX] was investigated. In particular, the involvement of the Fas receptor system in drug-induced apoptosis was evaluated. P-(GFLG)-DOX was shown to effect apoptosis-induced tumor cell death as manifested by positive Annexin V-FITC staining, cleavage of procaspase 3 and its physiological substrate, poly(ADP-ribose) polymerase (PARP), and cleavage of procaspase 8. Using the fluorochrome-labeled caspase inhibitor assay, it was found that both free DOX and P-(GFLG)-DOX activated caspases 3 and 9, but both forms of DOX did not have an effect on the activity of caspase 8, when compared to untreated cells. It was shown that free DOX and P-(GFLG)-DOX upregulated Fas receptor expression at the cell membrane in a time-dependent manner. Triggering the drug-induced Fas receptor with an exogeneous soluble Fas ligand (sFasL) resulted in an increase in the extent of apoptotic cell death, indicating that the Fas signaling pathway remained functionally active. Also, antagonistic anti-Fas ZB4 antibody blocked the increase in the level of apoptosis following the application of sFasL, but did not interfere with drug-induced apoptosis. The study of the functional activity of the Fas receptor and of the activation of the most proximal effector of the caspase cascade, caspase 8, indicated that the Fas receptor pathway was not decisive in the induction of cell death by free DOX and P-(GFLG)-DOX in A2780 cells. This study suggests further investigation of the involvement of the mitochondrial pathway in A2780 cell apoptotic death, induced by free and HPMA copolymer-bound DOX.     

Critical role of pro-apoptotic Bcl-2 family members in andrographolide-induced apoptosis in human cancer cells

Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess potent anti-inflammatory activity. In this study, Andro induced apoptosis in human cancer cells via activation of caspase 8 in the extrinsic death receptor pathway and subsequently with the participation of mitochondria. Andro triggered a caspase 8-dependent Bid cleavage, followed by a series of sequential events including Bax conformational change and mitochondrial translocation, cytochrome c release from mitochondria, and activation of caspase 9 and 3. Inhibition of caspase 8 blocked Bid cleavage and Bax conformational change. Consistently, knockdown of Bid protein using small interfering RNA (siRNA) technique suppressed Andro-induced Bax conformational change and apoptosis. In conclusion, the pro-apoptotic Bcl-2 family members (Bid and Bax) are the key mediators in relaying the cell death signaling initiated by Andro from caspase 8 to mitochondria and then to downstream effector caspases, and eventually leading to apoptotic cell death.     

Induction of Apoptosis by Lovastatin through Activation of Caspase‐3 and DNase II in Leukaemia HL‐60 Cells

Abstract: Lovastatin, an HMG‐CoA reductase inhibitor, was found to suppress growth and induce apoptosis in culture human promyelocytic leukaemic cell, HL‐60. However, the mechanisms of lovastatin‐induced apoptosis are still unclear. In this study, we attempted to elucidate the signal transduction pathway for lovastatin‐induced apoptosis in HL‐60 cells in a dose‐ and time‐dependent manner. The features of this apoptosis were attenuated by the presence of mevalonate, a metabolic intermediate of cholesterol synthesis. Treatment of lovastatin caused a rapid release of mitochondrial cytochrome c into cytosol and subsequent induction of caspase‐3, but not caspase‐1 activity. Lovastatin also stimulated proteolytic cleavage of poly‐(ADP‐ribose) polymerase (PARP), and followed by the appearance of caspase activity and DNA fragmentation. Pretreatment with caspase‐3 inhibitors, Ac‐DEVD‐CHO and Z‐VAD‐FMK, inhibited lovastatin–induced caspase‐3 activity and DNA fragmentation. Furthermore, we demonstrated that DNase II was involved in the DNA fragmentation induced by lovastatin. These results suggested that the mechanism of lovastatin induced HL‐60 cells apoptosis through activation of caspase‐3 and DNase II activities.     

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.     

Upregulation of Fas/Fas ligand-mediated apoptosis by gossypol in an immortalized human alveolar lung cancer cell line

Gossypol has wide antineoplastic effects in vitro, but its effects on human lung cancer have not been explored. To evaluate the activity of gossypol against alveolar cell lung cancer and to provide information on the mechanism of action, we examined the effects of gossypol on the proliferation of A549 cells indirectly using an XTT assay and on the distribution of cells within the phases of the cell cycle using flow cytometry. We also examined several factors that may affect apoptosis, including p53, p21/WAF1, Fas receptor, Fas ligand (FasL) and caspase 8 activity. The results showed that gossypol inhibited proliferation of A549 cells at a concentration of 0.5 micromol/L after 12 h treatment. The effect was both dose- and time-dependent by the induction of apoptosis without the effect of p53 and p21/WAF1. Upregulation of Fas/FasL, in association with the activation of downstream caspase 8 activity, was observed following treatment with gossypol. The Fas/FasL pathway accounted for 75% of gossypol-mediated apoptosis. We suggest that the Fas/FasL apoptotic system is the major pathway for gossypol-mediated apoptosis of A549 cells. Gossypol had no effect on the distribution of A549 cells within the phases of the cell cycle. In conclusion, gossypol inhibited A549 cells mainly by induction of the Fas/FasL apoptotic pathway, but not the p53 and p21/WAF1 pathway.     

Peltophorum dubium and soybean Kunitz-type trypsin inhibitors induce human Jurkat cell apoptosis

Plants constitute an important source of compounds which can induce apoptosis in a variety of cells. Previously, we reported the isolation of a trypsin inhibitor from Peltophorum dubium seeds (PDTI). This inhibitor, as well as soybean trypsin inhibitor (SBTI), both belonging to the Kunitz family, have lectin-like properties and trigger rat lymphoma cell apoptosis. In the present study, we demonstrate for the first time that PDTI and SBTI induce human leukemia Jurkat cell death. Induction of apoptosis was confirmed by flow cytometry after propidium iodide labeling of apoptotic nuclei, showing a considerable increase of the sub G(0)/G(1) fraction, with no cell cycle arrest. With the purpose of gaining insight into the signaling pathways involved, we investigated the activation of caspases and the effect of caspase inhibitors, and showed caspases-3 and -8-like activation by PDTI or SBTI-treatment. Consistent with these results, pan caspase inhibitor and caspase-8 inhibitor protected Jurkat cells from apoptosis. However, there was no caspase-9 activation, confirmed by the failure of caspase-9 inhibitor to prevent cell death. No significant release of cytochrome c from mitochondria was detected suggesting that the intrinsic mitochondrial pathway is not predominant in the apoptotic process. On the other hand, recruitment of Fas-associated death domain (FADD) to the cell membrane indicates the involvement of this adaptor protein in PDTI- and SBTI-induced apoptosis in Jurkat cells. Furthermore, human peripheral lymphocytes, either stimulated with phytohemagglutinin or not, are also susceptible to viability decrease induced by these inhibitors.     

Synthesis and evaluation of cytotoxic activities of artemisinin derivatives

Artemisinin is a naturally occurring antimalarial agent which has shown potent anticancer activity. In this work, new artemisinin derivatives with the piperazine group were synthesized. The cytotoxic activities of derivatives 5a – 5d were evaluated by MTT assay against ten cell lines. The results showed that 5a – 5d were more effective in inhibiting cancer cell growth than artemisinin. 5d was the most active against HepG2 and PLC‐PRF‐5 cells and presented no cytotoxicity on L‐02 cells. Hoechst 33342 staining and flow cytometry experiment revealed that 5d could induce HepG2 and PLC‐PRF‐5 cell apoptosis. Flow cytometry analysis showed that 5d induced the loss of mitochondrial membrane potential (MMP) and increased the levels of intracellular free calcium and reactive oxygen species. 5d also induced cell cycle arrest in G2/M phase in HepG2 cells. According to the results of Western blotting and caspase‐3 kit, 5d could significantly increase the content of p53, bax, Apaf‐1, and caspase‐3 and decrease the protein level of bcl‐2, pro‐caspase‐9, and pro‐caspase‐3 in HepG2 cells. These findings indicate that 5d activates the mitochondria‐mediated apoptotic pathway in HepG2 cells and may merit further investigation as a potential therapeutic agent for hepatocellular carcinoma.     

Sulforaphane‐induced apoptosis in human leukemia HL‐60 cells through extrinsic and intrinsic signal pathways and altering associated genes expression assayed by cDNA microarray

Sulforaphane (SFN), one of the isothiocyanates, is a biologically active compound extracted from cruciferous vegetables, and has been shown to induce cytotoxic effects on many human cancer cells including human leukemia cells. However, the exact molecular mechanism and altered gene expression associated with apoptosis is unclear. In this study, we investigated SFN‐induced cytotoxic effects and whether or not they went through cell‐cycle arrest and induction of apoptosis and further examined molecular mechanism and altered gene expression in human leukemia HL‐60 cells. Cell viability, cell‐cycle distribution, sub‐G1 (apoptosis), reactive oxygen species (ROS) and Ca²⁺ production, levels of mitochondrial membrane potential (ΔΨ_m), and caspase‐3, ?8, and ?9 activities were assayed by flow cytometry. Apoptosis‐associated proteins levels and gene expressions were examined by Western blotting and cDNA microarray assays, respectively. Results indicated that SFN decreased viable cells, induced G2/M phase arrest and apoptosis based on sub‐G1 phase development. Furthermore, SFN increased ROS and Ca²⁺ production and decreased the levels of ΔΨ_m and activated caspase‐3, ?8, and ?9 activities in HL‐60 cells. SFN significantly upregulated the expression of BAX, Bid, Fas, Fas‐L, caspase‐8, Endo G, AIF, and cytochrome c, and inhibited the antiapoptotic proteins such as Bcl‐x and XIAP, that is associated with apoptosis. We also used cDNA microarray to confirm several gene expressions such as caspase ?8, ?3, ?4, ?6, and ?7 that are affected by SFN. Those results indicated that SFN induced apoptosis in HL‐60 cells via Fas‐ and mitochondria‐dependent pathways. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 311–328, 2017.     

百里醌对人胶质瘤细胞凋亡的作用及其机制研究

目的 分析百里醌对胶质瘤U87细胞生长抑制和凋亡诱导的功能.方法 体外胶质瘤细胞株U87以及人星形胶质细胞株NHA中添加不同浓度百里醌后,CCK-8法检测细胞活力;克隆形成实验观察U87细胞形成细胞克隆的能力;流式细胞术检测细胞周期和ROS含量;Hoechst染色法和流式细胞术测定细胞凋亡;流式细胞术(JC-1荧光染色...     

Diclofenac induces apoptosis in hepatocytes by alteration of mitochondrial function and generation of ROS

Diclofenac is a non-steroidal anti-inflammatory drug that is widely used clinically but side effects associated with the administration of the drug have been reported. The apoptotic effect of the drug has been evaluated in human and rat hepatocytes. Apoptosis was observed after exposure to sub-cytotoxic concentrations of the drug, without overlapping with cell necrosis. Flow cytometric analysis revealed a time- and dose-dependent increase of apoptotic nuclei with sub-diploid DNA content. Caspase 8 and 9 mediate the cell-receptor and the mitochondria-initiated apoptotic pathways, respectively. Inhibition of both caspases prevented activation of downstream caspases, thus indicating that diclofenac at least activates caspase 3 and both effector caspases 8 and 9. The hierarchy of caspase activation by diclofenac was investigated. Analysis of kinetics revealed a simultaneous activation of these caspases that was maximal after 12 hr of exposure to the drug. Inhibitors of MPT, prevented the downstream activation of the caspase cascade, thus showing that diclofenac opened the mitochondrial pore. On the other hand, antioxidants were able to prevent caspase activation by diclofenac, revealing that oxidative stress at the mitochondrial level is in the root of MPT induction and caspase cascade activation. Caspase activation is not mediated by Bid cleavage, suggesting that the cell-receptor pathway seems not to be involved. However, a dose-dependent release of caspase 8 from the mitochondria was observed, indicating that caspase 8 can be processed independently of cell death receptors. Caspases 8 and 9 are very likely the apical caspases in diclofenac-induced apoptosis. In addition, an early dose-dependent increase of bclX(L) expression parallel to the generation of reactive oxygen species in the mitochondria was found. In conclusion, the mitochondrial pathway is very likely the only pathway involved in diclofenac-induced apoptosis, which was related to CYP-mediated metabolism of diclofenac, with the highest apoptotic effect produced by the metabolite 5OH-diclofenac.