Nitric oxide (NO) is a short-lived gaseous free radical that predominantly functions as a messenger and effector molecule. It affects a variety of physiological processes, including programmed cell death (PCD) through cyclic guanosine monophosphate (cGMP)-dependent and - independent pathways. In this field, dominant discoveries are the diverse apoptosis networks in mammalian cells, which involve signals primarily via death receptors (extrinsic pathway) or the mitochondria (intrinsic pathway) that recruit caspases as effector molecules. In plants, PCD shares some similarities with animal cells, but NO is involved in PCD induction via interacting with pathways of phytohormones. NO has both promoting and suppressing effects on cell death, depending on a variety of factors, such as cell type, cellular redox status, and the flux and dose of local NO. In this article, we focus on how NO regulates the apoptotic signal cascade through protein S-nitrosylation and review the recent progress on mechanisms of PCD in both mammalian and plant cells.
Animals ; Apoptosis ; physiology ; Caspases ; metabolism ; Caspases, Effector ; metabolism ; Cyclic GMP ; metabolism ; Mitochondria ; metabolism ; physiology ; Nitric Oxide ; metabolism ; physiology ; Plant Cells ; Plant Physiological Phenomena ; Signal Transduction ; physiology

OBJECTIVETo explore the inhibition of Jumi (traditional Chinese medicine) extraction on the growth of human cervical cancer cell line HeLa.

METHODSNude mouse model of human cervical cancer HeLa cell transplantation was established. The nude mice bearing cancer were randomly divided into control group and Jumi treated groups with different concentration (0.001, 0.002, 0.005, 0.01 mg/ml). The growth of cervical cancer cell in experimental mice were measured. Cultured HeLa cells were incubated in culture media with or without Jumi extract for 48 hours. Cell proliferation rate, cell apoptosis, caspase-3/7 and caspase-6 activity were determined by MTT colorimetric assay, flow cytometry analysis and spectrophotometric detection, respectively.

RESULTSWith the increase of the concentration of Jumi extract, tumor-bearing mice tumor inhibition rate gradually increased. The proliferation of cultured HeLa cells were significantly inhibited by Jumi extract in a dose-dependent manner. IC50 was 0.004 mg/ml. Apoptosis rates in the cells treated with Jumi extract were higher than those of the control group. Compared with the control group, except for lower Jumi treated group (0.001 mg/ml), caspase-3/7 and caspase-6 activity were significantly increased in the all Jumi treated groups.

CONCLUSIONJumi extract can inhibit the proliferation of human cervical cancer cell line HeLa in vitro in a dose-dependent manner and promote cell apoptosis through caspase-3, caspase-7 and caspase-6 pathway.

Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 6 ; metabolism ; Caspase 7 ; metabolism ; Cell Proliferation ; drug effects ; Chrysanthemum ; Female ; HeLa Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Plant Extracts ; pharmacology ; Xenograft Model Antitumor Assays

Several studies have shown that curcumin, which is derived from the rhizomes of turmeric, possesses antimicrobial, antioxidant and anti-inflammatory properties. The antitumor properties of curcumin have also now been demonstrated more recently in different cancers. This study was undertaken to investigate the modulation of cell cycle-related proteins and the mechanisms underlying apoptosis induction by curcumin in the SCC25 human tongue squamous cell carcinoma cell line. Curcumin treatment of the SCC25 cells resulted in a time- and dose-dependent reduction in cell viability and cell growth, and onset of apoptotic cell death. The curcumin-treated SCC25 cells showed several types of apoptotic manifestations, such as nuclear condensation, DNA fragmentation, reduced MMP and proteasome activity, and a decreased DNA content. In addition, the treated SCC25 cells showed a release of cytochrome c into the cytosol, translocation of AIF and DFF40/CAD into the nuclei, a significant shift in the Bax/Bcl-2 ratio, and the activation of caspase-9, caspase-7, caspase-6, caspase-3, PARP, lamin A/C, and DFF45/ICAD. Furthermore, curcumin exposure resulted in a downregulation of G1 cell cycle-related proteins and upregulation of p27KIP1. Taken together, our findings demonstrate that curcumin strongly inhibits cell proliferation by modulating the expression of G1 cell cycle-related proteins and inducing apoptosis via proteasomal, mitochondrial, and caspase cascades in SCC25 cells.
Apoptosis* ; Carcinoma, Squamous Cell* ; Caspase 3 ; Caspase 6 ; Caspase 7 ; Caspase 9 ; Cell Cycle Checkpoints* ; Cell Death ; Cell Line* ; Cell Proliferation ; Cell Survival ; Curcuma ; Curcumin ; Cytochromes c ; Cytosol ; DNA ; DNA Fragmentation ; Down-Regulation ; Humans ; Proteasome Endopeptidase Complex ; Rhizome ; Tongue* ; Up-Regulation

Fluoride is widely used in dentistry to prevent dental caries, even though the safety of fluoride is a controversial issue. There are no known adverse effects of long-term fluoride ingestion for caries prevention, but an overdose can cause serious acute toxicity. Nevertheless it is accepted that fluoride is an important material for oral health. This study was undertaken to investigate the modulation of cell cycle-related proteins and apoptosis induction underlying mechanism by NaF treatment on G361 human melanoma cell line. The viability of G361 cells and the growth inhibition of G361 cells were assessed by MTT assay and clonogenic assay respectively. Hoechst staining, DNA electrophoresis and TUNEL staining were conducted to observe G361 cells undergoing apoptosis. G361 cells were treated with NaF, and Western blotting, immunocytochemistry, confocal microscopy, FACScan flow cytometry, MMP activity and proteasome activity were performed. NaF treatment in G361 cells resulted in a time- and does-dependent decrease of cell viability and a does-dependent inhibition of cell growth, and induced apoptotic cell death. And tested G361 cells showed several lines of apoptotic manifestation such as nuclear condensation, DNA fragmentation, the reduction of MMP and proteasome activity, the decrease of DNA contents, the release of cytochrome c into cytosol, the translocation of AIF and DFF40 (CAD) onto nuclei, a significant shift of Bax/Bcl-2 ratio, and the activation of caspase-9, caspase-7, caspase-6, caspase-3, PARP, Lamin A/C and DFF45 (ICAD). Furthermore, NaF resulted in down-regulation of the G1 cell cycle-related proteins, and up-regulation of p53. Taken collectively, our present findings demonstrate that NaF strongly inhibits cell proliferation by modulating the expression of the G1 cell cycle-related proteins and induces apoptosis via proteasome, mitochondria and caspase cascades in G361 cells.
Apoptosis ; Blotting, Western ; Caspase 3 ; Caspase 6 ; Caspase 7 ; Caspase 9 ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Death ; Cell Line ; Cell Proliferation ; Cell Survival ; Cytochromes c ; Cytosol ; Dental Caries ; Dentistry ; DNA ; DNA Fragmentation ; Down-Regulation ; Eating ; Electrophoresis ; Flow Cytometry ; Fluorides ; Humans ; Immunohistochemistry ; In Situ Nick-End Labeling ; Melanoma ; Microscopy, Confocal ; Mitochondria ; Oral Health ; Proteasome Endopeptidase Complex ; Proteins ; Up-Regulation

In the present study, a newly synthesized benzofuran lignan 4-formyl-2-(4-hydroxy-3methoxyphenyl)-5-(2-methoxycarbonyethyl)-7-methoxy-benzo [b] furan (ERJT-12) was tested for its antiproliferative activity on human tumor cells. The related mechanisms were also investigated. In vitro growth inhibitory effects of ERJT-12 on various cancer cell lines were determined by MTT assay. Cell cycle distribution and apoptosis were detected by flow cytometry. The integrity of DNA was assessed by agarose gel electrophoresis. Activation of Caspase-3/7 and Caspase-6 was measured by colorimetric assay. The expressions of cell cycle proteins cell divide cycle 25c (Cdc25c), cyclin dependent kinase 1 (CDK1), CyclinB1 and apoptosis-related proteins Bax and Bcl-2 were detected by Western blotting. MTT assay showed that ERJT-12 inhibited the proliferation of several cancer cell lines including multidrug resistant cells. MCF-7 cells were markedly arrested at gap2/mitosis (G2/M) phase after treatment with ERJT-12 and progressed into apoptosis. The increased activities of Caspase-3/7 and Caspase-6 in MCF-7 cells were observed. The expression of CyclinB1 was down-regulated. The activities of Cdc25c and CDK1 protein were suppressed and Bcl-2 protein was phosphorylated. ERJT-12 displays potent antiproliferative activity towards cancer cells through suppressing cell cycle proteins, arresting cell cycle at G2/M phase and inducing apoptosis. It might be a novel candidate for cancer therapy.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Benzofurans ; pharmacology ; CDC2 Protein Kinase ; metabolism ; Caspase 3 ; metabolism ; Caspase 6 ; metabolism ; Caspase 7 ; metabolism ; Cell Cycle Proteins ; metabolism ; Cell Division ; drug effects ; Cell Line, Tumor ; Cyclin B ; metabolism ; Cyclin B1 ; G2 Phase ; drug effects ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; metabolism ; cdc25 Phosphatases ; metabolism

In a preliminary study, we found that benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD- fmk), unlike Boc-aspartyl(OMe)-fluoromethylketone (BocD-fmk), at usual dosage could not prevent genistein-induced apoptosis of p815 mastocytoma cells. This study was undertaken to reveal the mechanism underlying the incapability of zVAD-fmk in preventing this type of apoptosis. We observed that 14-3-3 protein level was reduced in genistein-treated cells and that BocD-fmk but not zVAD-fmk prevented the reduction of 14-3-3 protein level and the release of Bad from 14-3-3. We also demonstrated that truncated Bad to Bcl-xL interaction in genistein- treated cells was prevented by BocD-fmk but not by zVAD-fmk treatment. Our data indicate that BocD- fmk, compared to zVAD-fmk, has a certain preference for inhibiting 14-3-3/Bad signalling pathway. We also elucidated that this differential efficacy of BocD-fmk and zVAD-fmk resulted from the different effect in inhibiting caspase-6 and that co-treatment of zVAD-fmk and caspase-6 specific inhibitor substantially prevented genistein-induced apoptosis. Our data shows that caspase-6 plays a role on Bad/14-3-3 pathway in genistein-induced apoptosis of p815 cells, and that the usual dose of zVAD-fmk, in contrast to BocD-fmk, did not prevent caspase-6 acting on 14-3-3/Bad-mediated event.
bcl-Associated Death Protein/*metabolism ; *Signal Transduction/drug effects ; Mitochondria/drug effects ; Mice ; Mastocytoma ; Hydrocarbons, Fluorinated/*pharmacology ; Genistein/pharmacology ; Enzyme Inhibitors/*pharmacology ; Cell Line, Tumor ; Caspase 6/antagonists & inhibitors/*metabolism ; Benzyl Compounds/*pharmacology ; Apoptosis/*drug effects ; Animals ; Amino Acid Chloromethyl Ketones/pharmacology ; 14-3-3 Proteins/*metabolism

OBJECTIVETo investigate the apoptosis of J774A.1 cells induced by Leptospira interrogans and the effect of caspase-3, -6 activation on the apoptosis.

METHODSMouse monocyte-macrophage like cell line J774A.1 was infected by L.interrogans serogroup Icterohaemorrhagiae serovar icterohaemorrhagiae Lai strain 56601. The apoptosis or necrosis of infected cells was examined by flow cytometry using fluorescein labeling FITC-Annexin V/PI. The activity of caspase-3, -6, and their cleaved substrates PARP and Lamin A/C were measured by fluorometry and Western Blotting, respectively.

RESULTL. interrogans strain Lai was able to induce apoptosis of J774A.1 cells and the maximal apoptotic rate was(48.81+/-5.95)% when microbe: cell ratio was 100: 1. The maximal activities of caspase-3 and -6 in the infected J774A.1 cells were (1453.41+/-36.07) and (618.65+/-39.82) FU, respectively, which were 16.38- and 9.98-fold of those uninfected cells. PARP and Lamin A/C in the infected cells were detected. Caspase-3 and -6 inhibitors remarkably blocked the L. interrogans-induced apoptosis in J774A.1 cells.

CONCLUSIONL. interrogans is able to induce the apoptosis of J774A.1 cells and intracellular caspase-3 and -6 are closely associated with the apoptosis.

Animals ; Apoptosis ; Caspase 3 ; metabolism ; Caspase 6 ; metabolism ; Cell Line ; Leptospira interrogans ; pathogenicity ; Macrophages ; enzymology ; microbiology ; pathology ; Mice

OBJECTIVETo investigate the effects of snakegourd root polysaccharide on apoptosis of human breast cancer cells (MCF-7 cells).

METHODSColorimetric MTT assay was used to measure the inhibition of snakegourd root polysaccharide on MCF-7 cells. The morphological changes of MCF-7 cells were observed by fluorescence microscope after DAPI staining and transmission electron microscope. The apoptosis of MCF-7 cells was examined by DNA agarose gel electrophoresis analysis of DNA fragmentation amd flow cytometry. The activity of Caspase-3 and Caspase-8 was detected by colorimetric assay.

RESULTSPolysaccharide of snakegourd root significantly inhibited MCF-7 cells in a dose-and time-dependent manner. The nuclear condensation and marginalization were observed by DAPI staining and transmission electron microscope. The characteristic ladder of apoptosis in DNA electrophoresis was detected in MCF-7 cells treated with 10.0 μmol/L polysaccharide of snakegourd root at d 2. The activities of Caspase-3 and Caspase-8 were increased in a time-dependent manner. The rates of apoptosis in MCF-7 cells were (5.2 ±1.3)%, (13.1 ±4.7)%, (27.6 ±6.8)% and (43.8 ±9.8)% treated with 1.0,5.0,10.0 and 20.0 μmol/L snakegourd root polysaccharide at d 2,respectively. The maximal activities of intracellular Caspase-3 and Caspase-8 were (2.32 ±0.12)U/μg and (1.92 ±0.11)U/μg at d 2 and d 1, respectively when MCF-7 cells were treated with 10.0 μmol/L.

CONCLUSIONThe polysaccharide of snakegourd root can induce the apoptosis of MCF-7 cells,which is associated with the activation of intracellular Caspase-3 and Caspase-8.

Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Humans ; MCF-7 Cells ; Plant Roots ; chemistry ; Polysaccharides ; pharmacology ; Trichosanthes ; chemistry

6-Gingerol exerts anti-tumor effects in various cancer cell models. We evaluated the effect of 6-gingerol on the growth of MCF-7 breast cancer cells and MCF-10A breast epithelial cells to determine whether any growth-inhibitory effects found were attributable to apoptosis, and to elucidate the underlying mechanism of action. 6-Gingerol inhibited the viability of both cell lines in a dose- and time-dependent manner; however, the degree of inhibition was greater in MCF-7 than MCF-10A cells. By flow cytometry, induction of dose- and time-dependent apoptosis was found, and the magnitude of apoptosis was also markedly greater in MCF-7 than MCF-10A cells. Expression of caspase-3 and poly (ADP-ribose) polymerase (PARP) was observed in MCF-7 cells treated with 6-gingerol, and further cleavage of PARP occurred in these cells. We suggest that 6-gingerol induces apoptosis in human breast cancer cells mainly by promoting caspase-3 expression and subsequent degradation of PARP.
Apoptosis ; Breast Neoplasms* ; Breast* ; Caspase 3 ; Cell Line ; Epithelial Cells ; Flow Cytometry ; Humans* ; MCF-7 Cells

Mammalian cell is critically dependent on a continuous supply of oxygen. Even brief periods of oxygen deprivation can result in profound cellular damage. The aim of this study was to examine the possible mechanism of apoptosis in response to hypoxia in MC3T3E1 osteoblasts. MC3T3E1 osteoblasts under hypoxic conditions (2% oxygen) resulted in apoptosis in a time-dependent manner, determined by DNA fragmentation assay and nuclear morphology, stained with fluorescent dye (Hoechst 33258) Pretreatment with Z-VAD-FMK, a pan-caspase inhibitor, or Z-DEVD-CHO, a specific caspase-3 inhibitor, suppressed the DNA ladder in response to hypoxia in a concentration-dependent manner. An increase in caspase-3-like protease (DEVDase) activity was observed during apoptosis, but no caspase-1 activity (YVADase) was detected. To confirm what caspases were involved in apoptosis, western blot analysis was performed using an anti-caspase-3 or 6 antibody. The 17-kDa protein, that corresponds to the active products of caspase-3 and the 20-kDa protein of the active protein of caspase-6 were generated in hypoxia-challenged lysates, in which the full length forms of caspase-3 and 6 were evident. With a time course similar to caspase-3 and 6 activation, hypoxic stress also caused the cleavage of Lamin A, typical of caspase-6 activity. In addition, the hypoxic stress elicited the release of cytochrome c into the cytosol during apoptosis. These findings suggested that the activation of caspases accompanied by a cytochrome c release in response to hypoxia was involved in apoptotic cell death in MC3T3E1 osteoblasts.
Anoxia* ; Apoptosis* ; Blotting, Western ; Caspase 3 ; Caspase 6 ; Caspases ; Cell Death ; Cytochromes c ; Cytosol ; DNA ; DNA Fragmentation ; Lamin Type A ; Osteoblasts* ; Oxygen ; Signal Transduction*