Microcystin-RR induced apoptosis in tobacco BY-2 suspension cells is mediated by reactive oxygen species and mitochondrial permeability transition pore status

When tobacco BY-2 cells were treated with 60 mu g/mL MC-RR for 5 d, time-dependent effects of MC-RR on the cells were observed. Morphological changes such as abnormal elongation, evident chromatin condensation and margination, fragmentation of nucleus and formation of apoptotic-like bodies suggest that 60 mu g/mL MC-RR induced rapid apoptosis in tobacco BY-2 cells. Moreover, there was a significant and rapid increase of ROS level before the loss of mitochondrial membrane potential (Delta Psi(m)) and the onset of cell apoptosis. Ascorbic acid (AsA), a major primary antioxidant, prevented the increase of ROS generation, blocked the decrease in Delta Psi(m) and subsequent cell apoptosis, indicating a critical role of ROS in serving as an important signaling molecule by causing a reduction of Delta Psi(m) and MC-RR-induced tobacco BY-2 cell apoptosis. In addition, a specific mitochondrial permeability transition pores (PTP) inhibitor, cyclosporin A (CsA), significantly blocked the MC-RR-induced ROS formation, loss of Delta Psi(m), as well as cell apoptosis when the cells were MC-RR stressed for 3 d, suggesting that PTP is involved in 60 mu g/mL MC-RR-induced tobacco cell apoptosis signalling process. Thus, we concluded that the mechanism of MC-RR-induced apoptosis signalling pathways in tobacco BY-2 cells involves not only the excess generation of ROS and oxidative stress, but also the opening of PTP inducing loss of mitochondrial membrane potential. (C) 2007 Published by Elsevier Ltd.

Microcystin-RR-induced apoptosis in tobacco BY-2 cells

Our previous studies showed that microcystin-RR could induce oxidative damage in plant cells as they do with animal cells. However, whether microcystin can induce plant cell apoptosis is still unknown. In this study, the morphological changes of tobacco BY-2 suspension cells exposed to microcystin-RR were observed under light microscopy and transmission electron microscopy, and apoptosis was clearly distinguished by intense perinuclear chromatin margination, condensation of nuclear chromatin after 6d exposure of 50 mg/L (about 50 mu M) microcystin-RR. We also found that microcystin-RR can induce tobacco cell apoptosis in a dose- and time-dependent manner with flow cytometry analysis. Our study provides the first evidence that microcystins can induce plant cell apoptosis. (c) 2006 Elsevier Ltd. All rights reserved.

potential mechanisms involved in resistant phenotype of MCF-7breast carcinoma cells to ionizing radiation induced apoptosis

In the present study, we investigated the mechanisms of apoptosis resistance and the roles of the phosphorylation of BRCA1, p21, the Bax/Bcl-2 protein ratio and cell cycle arrest in IR-induced apoptosis in MCF-7 cells. X-irradiation, in particular at low dose (1 Gy), but not carbon ion irradiation, had a significant antiproliferative effect on the growth of MCF-7 cells. 1 Gy X-irradiation resulted in G1 and G2 phase arrest, but 4 Gy induced a significant G1 block. In contrast, carbon ion irradiation resulted in a significant accumulation in the G2 phase. Concomitant with the phosphorylation of H2AX induced by DNA damage,carbon ion irradiation resulted in an approximately 1.9–2.8-fold increase in the phosphorylation of BRCA1 on serine residue 1524, significantly greater than that detected for X-irradiation. Carbon ion irradiation caused a dramatic increase in p21 expression and drastic decrease in Bax expression compared with X-irradiation. The data implicated that phosphorylation of BRCA1 on serine residue 1524 might,at least partially, induce p21 expression but repress Bax expression. Together, our results suggested that the phosphorylation of BRCA1 at Ser-1524 might contribute to the G2 phase arrest and might be an upstream signal involved in preventing apoptosis signal via upregulation of p21 and downregulation of the Bax/Bcl-2 ratio.

Emodin-induced apoptosis in human breast cancer BCap-37 cells through the mitochondrial signaling pathway

Emodin, a natural anthraquinone compound isolated from the rhizome of rhubarb, is reported to suppress the growth of tumor in many clinical situations. In this study, we focused on the effect of emodin in human breast cancer BCap-37 cells and further understand the underlying molecular mechanism in treating breast cancer. Using MTT assay and flow cytometry, we demonstrated the critical role of emodin in the suppression of the proliferation of BCap-37 cells based on a concentration- and time-dependent manner. The increase of apoptotic rate was also observed after incubation of BCap-37 cells on emodin at 20 mu M and 50 mu M for 48 h. The cells exhibited typical apoptotic features including cellular morphological change, chromatin condensation and membrane blebbing. The results of the study further showed that Bcl-2 level decreased, while Bax and cytosolic cytochrome c levels in sample cells increased after the emodin treatment by using Western blot. The decline in the Bcl-2/Bax ratio and the increase of cytosolic cytochrome c concentration were consistent with the increase of the apoptotic ratio. The results strongly suggest that the disruption of the mitochondrial signaling pathway was involved in emodin-induced apoptosis in BCap-37 cells.

Mammalian cell cultures as a model system for the determination of cytotoxicity induced by cholesterol oxidation products

Oxysterols are products of cholesterol oxidation, which may be produced endogenously or may be absorbed from the diet where they are commonly found in foods of animal origin. Oxysterols are known to be cyctotoxic to cells in culture and mode of toxicity has been identified as apoptosis in certain cell lines. The cytotoxicity of the oxysterols 25-hydroxycholesterol (25-OH) and 7β-hydroxycholesterol (7β-OH) was examined in two human cell lines, HepG2, a hepatoma cell line, and U937, a monocytic cell line. Both 25-OH and 7β-OH were cytotoxic to the HepG2 cell line but apoptotic cells were not detected and it was concluded that cells underwent necrosis. 25-OH was not cytotoxic to the U937 cell line but it was found to have a cytostatic effect. 7β-OH was shown to induce apoptosis in the U937 line. The mechanism of oxysterol-induced apoptosis has not yet been fully elucidated, however the generation of an oxidative stress and the depletion of glutathione have been associated with the initial stages of the apoptotic process. The concentration of cellular antioxidant enzyme, superoxide dismutase (SOD) was increased in association with 7β-OH induced apoptosis in the U937 cell line. There was no change in the glutathione concentration or the SOD activity of HepG2 cells, which underwent necrosis in the presence of 7β-OH. Many apoptotic pathways center on the activation of caspase-3, which is the key executioner protease of apoptosis. Caspase-3 activity was also shown to increase in association with 7β-OH-induced apoptosis in U937 cells but there was no significant increase in caspase-3 activity in HepG2 cells. DNA fragmentation is regarded as the biochemical hallmark of apoptosis, therefore the comet assay as a measure of DNA fragmentation was assessed as a measure of apoptosis. The level of DNA fragmentation induced by 7β-OH, as measured using the comet assay, was similar for both cell lines. Therefore, it was concluded that the comet assay could not be used to distinguish between 7β-OH-induced apoptosis in U937 cells and 7β-OH-induced necrosis in HepG2 cells. The cytotoxicity and apoptotic potency of oxysterols 25-OH, 7β-OH, cholesterol- 5a,6a-epoxide (a-epoxide), cholesterol-5β,6β-epoxide (β-epoxide), 19-hydroxy-cholesterol (19-OH), and 7-ketocholesterol (7-keto) was compared in the U937 cell line. 7 β-OH, β-epoxide and 7-keto were found to induce apoptosis in U937 cells. 7β-OH-induced apoptosis was associated with a decrease in the cellular glutathione concentration and an increase in SOD activity, 7-keto and β-epoxide did not affect the glutathione concentration or the SOD activity of the cells.a-Epoxide, 19-OH and 25-OH were not cytotoxic to the U937 cell line.

APOLIPROTEIN(A)-INDUCED APOPTOSIS IN VASCULAR ENDOTHELIAL CELLS

Elevated plasma concentrations of lipoprotein(a) (Lp(a)) are a risk factor for a variety of atherosclerotic disorders including coronary heart disease. In the current study, the investigators report that incubation of cultured human umbilical vein endothelial cells (HUVECs) with high concentrations of apolipoprotein(a)(apo(a)/Lp(a)) induces apoptosis and endothelial dysfunction in a dose dependent manner. Apo(a), the component of Lp(a) mediates these effects by inducing externalization of Annexin V, DNA condensation and fragmentation which are the hallmarks of death by apoptosis. The pathway of apo(a)-induced apoptosis is associated with overexpression of Bax, caspase-9, p53 phosphorylation, decreased in Bcl-2 expression and activation of caspase-3. Taken together, the data suggest that elevated concentration of apo(a) induces apoptosis in endothelial cells probably by activating the intrinsic pathway. The data also showed that apo(a) induces increased expression of the growth arrest protein (Gas1), which has been known to induce apoptosis and growth arrest in vitro. In addition the data showed that elevated apo(a)/Lp(a) attenuates endothelial nitric oxide (eNOS) activity and endothelin-1 (ET-1) in a dose and time-dependent manner, particularly with small apo(a) isoforms. In summary, the authors proposed a new signaling pathway by which apo(a)/Lp(a) induce apoptosis and this finding could help explain how apo(a)/Lp(a) mediate atherosclerosis related diseases.

Staurosporine-induced apoptosis and hydrogen peroxide-induced necrosis in two human breast cell lines.

The use of apoptosis-inducing agents in the treatment of malignant cancer is increasingly being considered as a therapeutic approach. In this study, the induction of apoptosis and necrosis was examined in terms of temporal dose responses, comparing a malignant and nonmalignant breast cell line. Staurosporine (SSP)-induced apoptosis and H2O2-induced necrosis were evaluated by two cytotoxicity assays, neutral red (NR) and methyl-thiazolyl tertrazolium (MTT), in comparison with a differential dye uptake assay, using Hoechst33342/propidium iodide (Hoechst/PI). Confirmatory morphological assessment was also performed by routine resin histology and transmission electron microscopy. Cell viability was assessed over a 0.5-48 h time course. In nonmalignant HBL-100 cells, 50 nM SSP induced 100% apoptosis after a 48 h exposure, while the same exposure to SSP caused only 4% apoptosis in metastatic T47D cells. Although complete apoptosis of both cell lines was induced by 50 M SSP, this effect was delayed in T47D (24 h) compared with HBL-100 (4 h). Results also showed that neither MTT or NR can distinguish between the modes of cell death, nor detect the early onset of apoptosis revealed by Hoechst/PI.

BRCA1 Functions as a Differential Modulator of Chemotherapy-induced Apoptosis

We have evaluated the role played by BRCA1 in mediating the phenotypic response to a range of chemotherapeutic agents commonly used in cancer treatment. Here we provide evidence that BRCA1 functions as a differential mediator of chemotherapy-induced apoptosis. Specifically, we demonstrate that BRCA1 mediates sensitivity to apoptosis induced by antimicrotubule agents but conversely induces resistance to DNA-damaging agents. These data are supported by a variety of experimental models including cells with inducible expression of BRCA1, siRNA-mediated inactivation of endogenous BRCA1, and reconstitution of BRCA1-deficient cells with wild-type BRCA1. Most notably we demonstrate that BRCA1 induces a 10–1000-fold increase in resistance to a range of DNA-damaging agents, in particular those that give rise to double-strand breaks such as etoposide or bleomycin. In contrast, BRCA1 induces a >1000-fold increase in sensitivity to the spindle poisons, paclitaxel and vinorelbine. Fluorescence-activated cell sorter analysis demonstrated that BRCA1 mediates G2/M arrest in response to both antimicrotubule and DNA-damaging agents. However, poly(ADP-ribose) polymerase and caspase-3 cleavage assays indicate that the differential effect mediated by BRCA1 in response to these agents occurs through the inhibition or induction of apoptosis. Therefore, our data suggest that BRCA1 acts as a differential modulator of apoptosis depending on the nature of the cellular insult.

Cellular FLICE-inhibitory protein regulates chemotherapy-induced apoptosis in breast cancer cells

Combination treatment regimens that include topoisomerase-II-targeted drugs, such as doxorubicin, are widely used in the treatment of breast cancer. Previously, we demonstrated that IFN-� and doxorubicin co-treatment synergistically induced apoptosis in MDA435 breast cancer cells in a STAT1-dependent manner. In this study, we found that this synergy was caspase 8-dependent. In addition, we found that IFN-γ down-regulated the expression of the caspase 8 inhibitor c-FLIP. Furthermore, IFN-� down-regulated c-FLIP in a manner that was dependent on the transcription factors STAT1 and IRF1. However, IFN-� had no effect on c-FLIP mRNA levels, indicating that c-FLIP was down-regulated at a post-transcriptional level following IFN-� treatment. Characterisation of the functional significance of c-FLIP modulation by siRNA gene silencing and stable over-expression studies, revealed it to be a key regulator of IFN-γ- and doxorubicin-induced apoptosis in MDA435 cells. Analysis of a panel of breast cancer cell lines indicated that c-FLIP was an important general determinant of doxorubicin- and IFN-�-induced apoptosis in breast cancer cells. Furthermore, c-FLIP gene silencing sensitised MDA435 cells to other chemotherapies, including etoposide, mitoxantrone and SN-38. These results suggest that c-FLIP plays a pivotal role in modulating drug-induced apoptosis in breast cancer cells.

Interleukin-8 signaling attenuates TRAIL- and chemotherapy-induced apoptosis through transcriptional regulation of c-FLIP in prostate cancer cells

Chemotherapy-induced interleukin-8 (IL-8) signaling reduces the sensitivity of prostate cancer cells to undergo apoptosis. In this study, we investigated how endogenous and drug-induced IL-8 signaling altered the extrinsic apoptosis pathway by determining the sensitivity of LNCaP and PC3 cells to administration of the death receptor agonist tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL induced concentration-dependent decreases in LNCaP and PC3 cell viability, coincident with increased levels of apoptosis and the potentiation of IL-8 secretion. Administration of recombinant human IL-8 was shown to increase the mRNA transcript levels and expression of c+FLIPL and c-FLIPS, two isoforms of the endogenous caspase-8 inhibitor. Pretreatment with the CXCR2 antagonist AZ10397767 significantly attenuated IL-8-induced c-FLIP mRNA up-regulation whereas inhibition of androgen receptor- and/or nuclear factor-kappa B-mediated transcription attenuated IL-8-induced c-FLIP expression in LNCaP and PC3 cells, respectively. Inhibition of c-FLIP expression was shown to induce spontaneous apoptosis in both cell lines and to sensitize these prostate cancer cells to treatment with TRAIL, oxaliplatin, and docetaxel. Coadministration of AZ10397767 also increased the sensitivity of PC3 cells to the apoptosis-inducing effects of recombinant TRAIL, most likely due to the ability of this antagonist to block TRAIL- and IL-8-induced up-regulation of c-FLIP in these cells. We conclude that endogenous and TRAIL-induced IL-8 signaling can modulate the extrinsic apoptosis pathway in prostate cancer cells through direct transcriptional regulation of c-FLIP. Therefore, targeted inhibition of IL-8 signaling or c-FLIP expression in prostate cancer may be an attractive therapeutic strategy to sensitize this stage of disease to chemotherapy.