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Aim: To investigate the molecular mechanisms of nitric oxide (NO)-induced cytotoxic effect in human gingival fibroblast (HGF) cells., Methodology: After sodium nitroprusside (SNP), as NO donor, was treated to HGF, viability was measured by MTT assay and apoptosis was determined by TUNEL and DNA fragmentation assay. Mitochondrial membrane potential was detected using confocal microscopy, and caspase activity assay was measured by spectrophotometer. Mitogen-activated protein kinases (MAPK) activation, Bax/Bcl-2 ratio and cytochrome c release were analysed by Western blot analyses. Cells were exposed to MAPK inhibitors (U0126, SB203580 and SP600125) before SNP treatment to investigate the effects of MAPK kinases on the NO-induced apoptosis in HGF. Statistical analysis was performed using one-way analysis of variance with the Student-Newman-Keuls post hoc test for multiple group comparison., Results: Apoptosis was significantly increased (P = 0.011 and 0.0004, respectively) in the presence of SNP (1 and 3 mmol L(-1) ) after 12 h in HGF. However, 1H-[1,2,4] oxadiatolo [4, 3-a] cluinoxaline-1-one (ODQ), a soluble guanylate cyclase inhibitor, did not block the decrement of cell viability by NO. SNP treatment induced the loss of mitochondrial membrane potential, release of cytochrome c, increased Bax/Bcl-2 ratio and activation of caspases in HGF. Also, SNP treatment increased phosphorylation of MAPKinases and c-Jun N-terminal kinase (JNK) inhibitor (5 and 10 μmol L(-1) ) rescued cell viability decreased by SNP in HGF (P = 0.024 and 0.0149, respectively)., Conclusion: Nitric oxide induced apoptosis in human gingival fibroblast through the mitochondria-mediated pathway by regulation of Bcl-2 family and JNK activation., (© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: Cigarette smoke is a complex mixture of more than 4700 chemical compounds including free radicals and oxidants and it is a world widely known problem to health. Nicotine is the major compound of tobacco and known as the cause of gingivitis and periodontitis. It induces intracellular oxidative stress recognized as the important agent in the damage of biological molecules. The aim of this study is to clarify the cytotoxic pathway of nicotine in human gingival fibroblasts (HGFs)., Methods: Human gingival fibroblasts stimulated by nicotine were used as an in vitro model. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect cell viability and reactive oxygen species (ROS) generation was assessed with 2,7-dichlorofluoroscein diacetate (DCF-DA). Morphological change was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) assay, stained with 4,6-diamidino-2-phenylindole (DAPI). To delineate the roles of extracellular signal-regulated kinase (ERK), P38 and c-Jun N-terminal kinase (JNK), Western blot and caspase-3 (CASP3) activity assay were performed., Results: Exposure of the human gingival fibroblasts to nicotine reduced cell viability by time and dose dependent and increased the generation of ROS. It also showed morphological evidence of increased apoptosis, resulted in transient activation of JNK and ERK concomitant with activation of P38, and stimulated apoptosis as evidenced by CASP3 activation and Poly ADP ribose polymerase (PARP) cleavage., Conclusion: These results suggest that nicotine induces apoptosis through the ROS generation and CASP3 dependent pathways in HGFs., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Hesperidin, a known flavonoid constituent of citrus, reduces the proliferation of many cancer cells. The apoptotic effects of hesperidin on human colon cancer cells, SNU-C4, were determined at concentrations of 1-100 microM. At 100 microM, hesperidin reduced cell viability to 65.00+/-0.05% of control values in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell death induced by hesperidin showed apoptotic features in 4,6-diamidino-2-phenylindole (DAPI) staining and in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. Examination of the expression of apoptosis-regulating genes indicated that hesperidin treatment decreased the expression of B-cell CLL/lymphoma 2 (BCL2) mRNA, and increased the expression of BCL2-associated X protein (BAX). The expression and activity of the major apoptotic factor caspase3 (CASP3) was increased significantly with hesperidin treatment. Hesperidin down-regulated the protein expression of pro-CASP3, and up-regulated the level of active CASP3. Thus, these results suggest that hesperidin could induce apoptosis in human colon cancer cells through CASP3 activation.

Polyphenols in fruits, soybean, vegetables, herbs, roots and leaves act as bioactive components related with prevention of cancer, heart diseases and diabetes. We investigated the apoptotic effects of polyphenols from red wine on human colon cancer cells SNU-C4 using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 4,6-diamidino-2-phenylindole (DAPI) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, reverse transcription-polymerase chain reaction (RT-PCR) expressions of Bcl-2, Bax and Caspase-3 genes, and Caspase-3 enzyme activity. Polyphenols (100 microg/ml) increased the apoptosis of SNU-C4 cells with apparent apoptotic characteristics including morphological changes of chromatin condensation and apoptotic body formation from DAPI staining and TUNEL assay. Compared with untreated control group, polyphenols (100 microg/ml) reduced the expression of Bcl-2 whereas those of Bax and Caspase-3 were increased. The Caspase-3 activity in the polyphenols treated group was significantly increased compared to those in control group (P<0.05). These results suggest that polyphenols have a strong potential for development as an anti-colon cancer agent.

This paper investigated the induction of apoptosis and perturbation of cell cycle progression caused by carboplatin (CPt) and hyperthermia alone or combined in WERI human retinoblastoma cells in vitro. An incubation of the cells with 25 or 50 microm of CPt at 37 degrees C caused apoptosis, which progressively increased during the 24-72 h treatment. Hyperthermia at 42.5 degrees C for 1 h induced apoptosis, which became significant from 24 h after the heating. Heating the cells in the presence of CPt and subsequent incubation with CPt was far more effective than treating the cells with hyperthermia or CPt treatment alone in inducing apoptosis in the WERI cells, indicating that the combination of these two modalities is potentially useful for the treatment of retinoblastoma. It appeared that the apoptosis in WERI cells caused by hyperthermia and CPt occurs during G1 phase. An interesting observation was that caspase 9 activation preceded the release of cytochrome C from mitochondria during apoptosis in WERI cells, contrary to the general notion that caspase 9 is activated by cytochrome C.

Cytotoxic effects of six isoflavonoids, tectorigenin, glycitein, tectoridin, glycitin, 6''-O-xylosyltectoridin, and 6''-O-xylosylglycitin isolated from the flower of Pueraria thunbergiana Benth. together with genistein, a known differentiation and apoptosis inducer, were examined. Among these isoflavonoids, tectorigenin and genistein exhibited cytotoxicity against various human cancer cells; glycitein showed only mild cytotoxicity. These results suggest that the isoflavone structure and 5-hydroxyl group are crucial for the cytotoxic properties and that glycosides are inactive. Moreover, tectorigenin induced differentiation of human promyelocytic leukemia HL-60 cells to granulocytes and monocytes/macrophages, and caused apoptotic changes of DNA in the cells, as did genistein. Tectorigenin also inhibited autophosphorylation of epidermal growth factor (EGF) receptor by EGF and decreased the expression of Bcl-2 protein, with less activity than genistein. From these results, tectorigenin may be a possible therapeutic agent for leukemia.

Bcl-2 has been reported to inhibit neurotoxicity induced by cisplatin. However, neither the mechanism of cisplatin-induced neurotoxicity nor the mechanism by which Bcl-2 confers neuroprotection is clear. In this study, the signaling pathways involved in cisplatin-induced neurotoxicity were examined using a rat neuroblastoma cell line, B104. Treatment of B104 cells with cisplatin induced apoptosis, accompanying the accumulation of p53 and Bax protein. Interestingly, extracellular signal-regulated kinase 1/2 (ERK1/2) activities of MAP kinases were markedly enhanced prior to cisplatin-induced accumulation of p53 and Bax. Inhibition of ERK1/2 activities using PD98059, a selective MEK inhibitor, blocked the apoptotic cell death preventing cisplatin-induced accumulation of p53 and Bax. These results suggest that ERK mediates cisplatin-induced p53 activation to trigger apoptosis in B104 cells. Overexpression of Bcl-2 in B104 cells resulted in the complete resistance to cisplatin-induced apoptosis blocking ERK activation and the subsequent signaling pathway of p53. Our study clearly demonstrates that the action site of Bcl-2 localizes upstream of ERK in cisplatin-induced apoptotic signaling pathway.

In a course of obtaining more amount of bioactive costunolide and successive phytochemical isolation from Magnolia sieboldii (Magnoliaceae), a novel acyclic monoterpene 1 named deoxygeraniol [2,6(E)-dimethyl-2,6-octadiene] was isolated along with beta-sitosterol 3-O-linoleate (2), trilinolein (3) and high amount of costunolide (4) in the pure state. The structure of compound 1 was determined on the basis of spectroscopic data. Costunolide was found to induce apoptotic cell death in a dose-dependent manner by nucleosomal DNA ladder and flow cytometric analysis. Immunoblot analysis showed that the level of the anti-apoptotic protein, Bcl-2, was decreased, whereas the cleavage of poly-(ADP-ribose) polymerase was activated. Furthermore, the N-acetyl-L-cysteine antioxidant effectively prevented costunolide-induced cytotoxicity. These results suggest that costunolide-induced cell death is mediated by reactive oxygen species

Purpose: The effects of hyperthermia or irradiation on cell killing and induction of apoptosis were evaluated using human maxillary carcinoma IMC-3 cells and low pH (pH 6.8) adapted cells (IMC-3-pH)., Methods and Materials: Cellular heat-sensitivity or radiosensitivity was determined using the clonogenic assay. Apoptosis was assessed on the basis of a flow cytometric determination of the DNA content, DNA fragmentation, and poly(ADP-ribose)polymerase cleavage., Results: When IMC-3 cells or IMC-3-pH cells were exposed to heat at 44 degrees C in pH 6.8 medium, an increase in thermosensitivity was observed compared with when the IMC-3 cells were exposed to heat at 44 degrees C in pH 7.4 medium. However, the selective reduction in survival was not observed after irradiation. In IMC-3 cells, apoptosis after heating at 44 degrees C for 60 min in pH 7.4 medium occurred earlier than that after 8 Gy irradiation, although both thermal and irradiated doses decreased the cell count to 10%. The degree of apoptosis after heating at pH 6.8 in IMC-3 cells or IMC-3-pH cells was greater than that at pH 7.4 in IMC-3 cells. However, the degree of apoptosis after 8 Gy irradiation at pH 6.8 in IMC-3 cells or IMC-3-pH cells was smaller than that at pH 7.4 in IMC-3 cells., Conclusion: Hyperthermia treatment is more effective at inducing apoptosis than radiation is in tumors that contain a population of low pH adapted cells.

The effect of environmental acidity on the induction of apoptosis by heat was investigated. Human colorectal tumour RKO.C cells, carrying wild-type p53 and isogenic RC10.1 cells deficient in p53, were heated at 42.0 degrees C for 1 h in pH 7.5 or pH 6.6 medium and the apoptosis was assessed based on the flow cytometic determination of DNA content, DNA fragmentation, and PARP cleavage. The degree of apoptosis after heating in pH6.6 medium was greater than that in pH 7.5 medium in both RKO.C cells and RC10.1 cells. When heated in the same pH medium, more apoptosis occurred in the RC10.1 cells than in the RKO.C cells. Heating increased the expression of p53 protein and p21 protein markedly in RKO.C cells and slightly in RC10.1 cells. Expression of these proteins was slightly greater in pH 7.5 medium than in pH 6.6 medium. The expressions of Bax protein and Bcl-2 protein, which are known to control apoptosis, were not altered by heating. It was concluded that an acidic environment enhances heat-induced apoptosis. It was also concluded that heat-induced apoptosis is lessened by p53 and that Bcl-2 and Bax are not involved in the induction of apoptosis by hyperthermia.

We investigated the effect of an acidic environment on the radiation-induced G2/M arrest and apoptosis using RKO.C human colorectal cancer cells expressing wild-type p53 and RC10.1 cells, a subline of RKO.C cells deficient in p53 as well as p53+/+ MEFs and p53-/- MEFs (mouse embryonic fibroblasts). The cells were irradiated with 4 Gy or 12 Gy of gamma-rays in pH 7.5 medium or pH 6.6 medium. p53 accentuated the progression of cells from radiation-induced G2/M arrest to apoptosis and the pH 6.6 environment suppressed the progression of cells through G2/M-phase to apoptosis after irradiation. Further analysis indicated that the radiation-induced G2/M arrest was due mainly to G2 arrest in both pH 7.5 and pH 6.6. Therefore, it was concluded that p53 enhances, and an acidic environment suppresses, the exit of cells from radiation-induced G2 arrest by altering cyclin B1-Cdc2 kinase activity.

Hypericin is a unique photosensitizing plant pigment and has been separately reported to induce differentiation and apoptosis in neoplastic cells. In this study, we examined the relationship between activities to induce differentiation and apoptosis in human promyelocytic leukemia HL-60 cells, at a concentration range of 0.15 to 0.2 microM. When treated with hypericin, the cell ratio reducible of nitroblue tetrazolium was significantly increased and the cell size was enlarged by flow cytometry analysis. Hypericin also significantly increased the ratio of the cells, which were of positive alpha-naphthyl acetate esterase activity and phagocytic activity, whereas it hardly influenced the naphthol AS-D chloroacetate esterase activity in the cells, as well as 1 alpha, 25(OH)2D3 (10 nM). In addition, hypericin increased hypodiploid nuclei and caused a nucleosomal ladder. These results indicate that hypericin induces both differentiation toward monocyte/macrophage lineage and apoptosis in HL-60 cells.

An exposure of HL-60 human promyelocytic leukaemia cells to acidic media with pH 6.2-6.6 caused an up-regulation of Bax protein expression within 2 h, which lasted for longer than 6 h. On the other hand, the apoptosis, as judged from PARP cleavage, DNA fragmentation and flow cytometric determination of cell population with sub-G1 DNA content, occurred after the cells were incubated in the acidic media for longer than 4 h. The PARP cleavage and DNA fragmentation in the cells exposed to an acidic environment could be effectively suppressed by inhibitors specific for ICE or CPP32, indicating that activation of these caspases is an essential step in acidic stress-induced apoptosis. It has been known that Bax is involved in the activation of caspases. Taken together, it appears that acidic stress first up-regulates Bax protein thereby activating caspases followed by PARP cleavage and DNA fragmentation. The observation that inhibition of either ICE or CPP32 could suppress acidic stress-induced apoptosis suggested that ICE activates pro-CPP32, which then cleaves PARP. Flow cytometric analysis indicated that acidic stress-induced apoptosis occurs mainly in G1 cells. The finding in the present study demonstrated that acidic intra-tumour environment may markedly perturb the tumour cell proliferation and tumour growth.

The effects of an acidic environment on the induction of apoptosis by 42 degrees C hyperthermia were investigated. An acidic environment (pH 6.6) enhanced the hyperthermia-induced apoptosis in HL-60 human promyelocytic leukemia cells as judged by the DNA fragmentation, flow cytometric analysis of DNA content, and cleavage of poly(ADP-ribose) polymerase. Hyperthermia exerted no effect on the expression of Bcl-2 and Bax, regardless of the environmental acidity during heating. The time of increase in apoptosis after heating coincided with the time of decrease in the G1-phase cell population. It seemed that the increase in heat-induced apoptosis in HL-60 cells in an acidic environment was due to a direct increase in the proteolytic cleavage of poly(ADP-ribose) polymerase by acidic caspases without the involvement of Bcl-2 and Bax, and that heat-induced apoptosis occurred during G1 phase in HL-60 cells.

Purpose: The effect of environmental pH on the radiation-induced apoptosis in tumor cells in vitro was investigated., Methods and Materials: Mammary adenocarcinoma cells of A/J mice (SCK cells) were irradiated with gamma-rays using a 137Cs irradiator and incubated in media of different pHs. After incubation at 37 degrees C for 24-120 h the extent of apoptosis was determined using agarose gel electrophoresis, TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining, flow cytometry, and release of 3H from 3H-thymidine labeled cells. The clonogenicity of the cells irradiated in different pH medium was determined, and the progression of cells through the cell cycle after irradiation in different pHs was also determined with flow cytometry., Results: Irradiation with 2-12 Gy of gamma-rays induced apoptosis in SCK cells in pH 7.5 medium within 48 h as judged from the results of four different assays mentioned. Radiation-induced apoptosis declined as the medium pH was lowered from 7.5 to 6.4. Specifically, the radiation-induced degradation of DNA including the early DNA breaks, as determined with the TUNEL method, progressively declined as the medium pH was lowered so that little DNA fragmentation occurred 48 h after irradiation with 12 Gy in pH 6.6 medium. When the cells were irradiated and incubated for 48 h in pH 6.6 medium and the medium was replaced with pH 7.5 medium, DNA fragmentation promptly occurred. DNA fragmentation also occurred even in pH 6.6 medium when the cells were irradiated and maintained in pH 7.5 medium for 8 h or longer post-irradiation before incubation in pH 6.6 medium. The radiation-induced G2 arrest in pH 6.6 medium lasted markedly longer than that in pH 7.5 medium., Conclusion: Radiation-induced apoptosis in SCK cells in vitro is reversibly suppressed in an acidic environment. Taking the results of four different assays together, it was concluded that early step(s) in the apoptotic pathway, probably the DNA break or upstream of DNA break, is reversibly halted by an acidic environment in irradiated cells. Radiation-induced G2 arrest is prolonged in an acidic environment indicating that the suppression of radiation-induced apoptosis and prolongation of radiation-induced G2 arrest in an acidic environment are related.

The aim was to investigate in detail the influence of intracellular pH (pHi) and intracellular Ca2+ concentration ([Ca2+]i) on apoptosis in HL-60 human promyelocytic leukaemia cells. The pHi was controlled by changing the pH of media as well as by interfering with the pHi regulatory mechanisms with 3-amino-6-chloro-5-(1-homopiperidyl)-N-(diaminomethylene) pyrazincarboxamide (HMA; an inhibitor of Na+/H+ antiport), 4-diiosothiocyanatostilbene-2,2'disulfonic acid, (DIDS; an inhibitor of Na(+)-dependent HCO3-/Cl- exchange) and nigericin (a K+ ionophore). The [Ca2+]i was increased with ionomycin, a Ca2+ ionophore. The apoptosis of HL-60 cells was measured with conventional agarose gel electrophoresis for DNA fragmentation and also with the release of 3H from 3H-thymidine-labelled DNA. Based on the magnitude of DNA fragmentation and 3H release at different pHi, it was shown that apoptosis occurred in HL-60 cells when the pHi was lowered from normal pHi of 7.4 to about 7.2-6.7 with a peak increase at pHi 6.8-6.9. Addition of 4 microM ionomycin to RPMI 1640 medium, which contained 615 microM Ca2+, elevated the apoptosis in the cells. Such an increase in apoptosis by ionomycin in HL-60 cells appeared to result from both an increase in [Ca2+]i and from a decline in pHi. The results indicate that the acidic intratumour environment may greatly affect the response of neoplastic tissues to hyperthermia, radiation and chemotherapeutic drugs which cause apoptosis.

The nature of the endonucleases responsible for DNA fragmentation in apoptosis has not yet been clearly defined. The intracellular acidity has been known to greatly affect apoptosis probably by affecting the activity of the endonucleases. In this study, the implication of pH in the apoptosis was investigated through the use of human HL-60 leukemia cells. The cells were incubated in media with different pH ranging from 3.5 to 7.5 for 4 hrs and the mode of cell death was investigated. The trypan blue exclusion assay showed that close to 25% and 90% of the cells were dead when incubated in pH 6.4 and pH 5.0 media, respectively. The agarose gel electrophoresis of DNA demonstrated that significant DNA fragmentation occurred in the HL-60 cells incubated in the pH 6.2-6.4 media for 4 hr indicating cell death by apoptosis. The electron microscopy study also demonstrated that many of the cells incubated in the pH 6.4 medium were in the process of apoptosis while the cells maintained in the pH 5.0 medium were dying by necrosis. The intracellular pH (pHi) of HL-60 cells was 6.6-6.9 when the extracellular pH (pHe) was 6.2-6.4. These results demonstrated that DNase I which has a maximal endonuclease activity near pH 7.0 may be responsible for the apoptosis accompanied by DNA fragmentation in HL-60 cells in the pH 6.4 medium. This observation is at variance with the previous reports that DNase II mediate the DNA fragmentation in apoptosis. The cell death at extremely low pH (pH 5.0) appeared to be due mainly to necrosis.