Protein tyrosine nitration and poly(ADP-ribose) polymerase activation in N-methyl-N-nitro-N-nitrosoguanidine-treated thymocytes: implication for cytotoxicity

1-Methyl-3-nitro-1-nitrosoguanidine (MNNG) is a DNA alkylating agent. DNA alkylation by MNNG is known to trigger accelerated poly(ADP-ribose) metabolism. Various nitroso compounds release nitric oxide (NO). Therefore, we set out to investigate whether MNNG functions as NO donor and whether MNNG-derived NO or secondary NO metabolites such as peroxynitrite contribute to MNNG-induced cytotoxicity. MNNG in aqueous solutions resulted in time- and concentration-dependent NO release and nitrite/nitrate formation. Moreover, various proteins in MNNG-treated thymocytes were found to be nitrated, indicating that MNNG-derived NO may combine with cellular superoxide to form peroxynitrite, a nitrating agent. MNNG also caused DNA breakage and increased poly(ADP-ribose) polymerase activity and cytotoxicity in thymocytes. MNNG-induced DNA damage (measured by the comet assay) and thymocyte death (measured by propidium iodide uptake) was prevented by the PARP inhibitor PJ-34 and by glutathione (GSH) or N-acetylcysteine (NAC). The cytoprotection provided by PJ-34 against necrotic parameters was paralleled by increased outputs in apoptotic parameters (caspase activity, DNA laddering) indicating that PARP activation diverts apoptotic death toward necrosis. As MNNG-induced cytotoxicity showed many similarities to peroxynitrite-induced cell death, we tested whether peroxynitrite was responsible for at least part of the cytotoxicity induced by MNNG. Cell-permeable enzymic antioxidants (superoxide dismutase and catalase), the NO scavenger cPTIO or the peroxynitrite decomposition catalyst FP15 failed to inhibit MNNG-induced DNA breakage and cytotoxicity. In conclusion, MNNG induces tyrosine nitration in thymocytes. Furthermore, MNNG damages DNA by a radical mechanism that does not involve NO or peroxynitrite.     

Genotoxicity, mitochondrial damage, and apoptosis in human lymphoblastoid cells exposed to peroxynitrite generated from SIN-1

SIN-1 (3-morpholinosydnonimine), the active metabolite of the vasodilator drug molsidomine, decomposes spontaneously in solution. In the presence of oxygen, NO* and O(2)(*-) are released, generating peroxynitrite, a potent oxidizing agent, at a constant rate over a 2 h period. We utilized this system to investigate mechanisms of peroxynitrite-induced cytotoxicity, genotoxicity, apoptosis, and mitochondrial damage in two human lymphoblastoid cell lines carrying either wild-type (TK6 cells) or mutant p53 (WTK-1 cells) genes. Treatment of TK6 cells with 5 mM SIN-1 for 1.5 h resulted in 28 +/- 6% survival 24 h later. Exposure in the presence of different radical scavengers significantly increased survival, as follows: cytochrome c, 96 +/- 3%; Tiron, 69 +/- 0%; SOD plus catalase, 83 +/- 5%; carboxy-PTIO, 87 +/- 3%; and uric acid, 87 +/- 2%. D-mannitol was ineffective in reducing lethality, as were SOD and catalase when added individually or in heat-inactivated form. Spontaneous as well as SIN-1-induced mutant fractions (MF) in both HPRT and TK genes were significantly higher in WTK-1 cells than in TK6 cells (p < 0.05-0.01). Exposure to 2.5 mM SIN-1 induced time-dependent apoptosis in TK6 cells, but not in WTK-1 cells. Mitochondrial membrane depolarization was also observed in both cell lines after SIN-1 treatment. Neutral comet assay demonstrated that SIN-1 treatment resulted in higher levels of DNA double-strand breaks in TK6 cells than in WTK-1 cells. Collectively, these data show that SIN-1 can be used as an effective peroxynitrite generator in cell culture experiments under these experimental conditions, in which it induced a greater apoptotic response but was less potent as a mutagen in TK6 cells compared with WTK-1 cells. Thus, p53 status was an important determinant of SIN-1 induced mutagenesis and apoptosis in these two human lymphoblastoid cell lines.     

Oxidative stress initiates DNA damager MNNG-induced poly(ADP-ribose)polymerase-1-dependent parthanatos cell death

The alkylating agent N-methyl-N′-nitro-N′-nitrosoguanidine (MNNG) can cause excess DNA strand breaks that lead to poly(ADP-ribose)polymerase-1 (PARP-1) overactivation and cell death (parthanatos). However, the detail mechanism of MNNG-induced parthanatos was not well-investigated. In this study, we used MNNG-treated mouse embryonic fibroblasts (MEFs) to elucidate the signaling pathways of MNNG-induced parthanatos. We found that MNNG-induced cell death accompanied by rapid PARP-1 activation, c-Jun N-terminal kinase (JNK) activation, biphasic reactive oxygen species (ROS) production and intracellular calcium increase. The early ROS production occurring at 1 min and peaking at 5–15 min after MNNG treatment partially resulted from NADPH oxidase. In contrast, the late phase of ROS production occurring at 30 min and time-dependently increasing up to 6 h after MNNG treatment was generated by mitochondria. The antioxidant, NAC can abrogate all phenomena caused by MNNG. Results indicate that the calcium rise was downstream of early ROS production, and was involved in PARP-1 and JNK activation. Moreover, the PARP inhibitor was able to reduce MNNG-induced late-phase ROS production, calcium elevation, and cell death. Results further indicated the involvement of RIP1 in sustained ROS production and calcium increase. We characterized the interactive roles of ROS, calcium, JNK, and RIP1 in MNNG-induced cell death. We found that in addition to the alkylating property previously demonstrated, ROS production triggered by MNNG results in enhanced DNA damage and PARP-1 activation. Moreover, intracellular calcium elevation and ROS production have mutual amplification effects and thus contribute to PARP-1-mediated parthanatos.     

Cell cycle was disturbed in the MNNG-induced initiation stage during in vitro two-stage transformation of Balb/3T3 cells

In vitro two-stage transformation, an important method for the screening of carcinogens, is a valuable approach for the mechanistic study of multi-stage carcinogenesis. However, very little is known about the molecular and cellular mechanisms, particularly in terms of cell cycle control during in vitro two-stage transformation. We improved the in vitro two-stage transformation method using N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as an initiator and cadmium as a promoter, and reconfirmed the promotional effect of cadmium (Fang et al., 2001a). To determine the alterations of cell cycle control in the MNNG-induced initiation stage during transformation, we examined the effects of MNNG on Balb/3T3 A31 cell growth, and determined the alterations of the protein and/or mRNA levels of cyclins B1, D1, E, and G, PCNA, GADD45, p27, and wild-type p53. After 4 hour treatment of MNNG, populations of G2/M phase distribution and apoptotic fraction and the cyclin G mRNA level increased, while the cyclin B1 mRNA level decreased in a concentration-dependent manner. Wild-type p53, p27, and GADD45 protein levels also increased as a function of MNNG concentrations. However, cyclin D1, cyclin E, and PCNA expressions remained unchanged. During the initiation stage, PCNA protein expression decreased on the first day after MNNG-treatment, then increased gradually during the following 6 days, and further increased on the first day after cadmium treatment. Although wild-type p53 and p27 protein expressions also showed temporary retardation on the first day after MNNG-treatment, the expressions increased gradually during the following 6 days, but decreased rapidly by the cadmium treatment. These results indicated that during the initiation stage, MNNG induced G2/M arrest and apoptosis with increased expressions of wild-type p53, p27, and GADD45 proteins; and down-regulated mRNA level of cyclin B1 and up-regulated mRNA level of cyclin G. In addition, although a few of the G2/M-arrested cells proliferated gradually, most cells continued to be suppressed and inactivated by the over-expressions of wild-type p53 and p27 until the cadmium treatment.     

地塞米松通过抑制p53蛋白降解诱导成骨细胞凋亡

目的 观察地塞米松对成骨细胞凋亡的影响并探讨其作用机制。方法 分别采CellTiter-Glo?荧光细胞活力检测试剂盒和Annexin V-FITC/PI双染凋亡检测试剂盒测定地塞米松作用后MC3T3-E1细胞的增殖和凋亡。蛋白质印迹法(Western blotting)检测地塞米松作用后MC3T3-E1细胞和hFOB1.19细胞胱天蛋白酶3(caspase-3)/裂解caspase-3和PARP/裂解PARP的表达。用Western blotting和PCR分析地塞米松作用后MC3T3-E1细胞中p53和检控点激酶2(Chk2)的表达。探讨p53敲除和Chk2敲除在地塞米松诱导MC3T3-E1细胞凋亡中的作用。结果 地塞米松能显著抑制MC3T3-E1细胞的生长并诱导其凋亡。地塞米松能通过促进p53的表达诱导成骨细胞凋亡。地塞米松对P53蛋白表达的调节作用是通过上调Chk2来实现的，Chk2与P53相互作用，抑制P53蛋白的降解。P53基因敲除可通过降低裂解caspase-3和裂解PARP的表达来抑制地塞米松诱导的MC3T3-E1细胞凋亡，经Dex处理的MC3T3-E1和hFOB1.1...     

DNA damage-triggered apoptosis: critical role of DNA repair, double-strand breaks, cell proliferation and signaling

Genotoxic DNA damaging agents may activate both membrane death receptors and the endogenous mitochondrial damage pathway leading to cell death via apoptosis. Here, apoptotic responses in cells exhibiting a defect in various DNA repair pathways such as alkyltransferase, base excision repair, nucleotide excision repair and mismatch repair are reviewed. The HSVTk/ganciclovir and VZV/BVDU suicide system will also be discussed. Data are available to show that critical DNA damage triggers apoptosis in a DNA replication dependent way by activating the mitochondrial damage pathway in fibroblasts. It is proposed that DNA double-strand breaks (DSBs) are common ultimate apoptosis-triggering lesions arising from primary DNA lesions during DNA replication. Thus, DNA replication is a necessary component in DNA damage-triggered apoptosis, at least in fibroblasts treated with genotoxins not inducing DSBs themselves. For methylating agents inducing O(6)-methylguanine, an additional requirement is mismatch repair provoking DSB formation that triggers Bcl-2 decline and caspase-9/-3 activation. This occurs independent of p53 since most of the repair deficient cell lines under study were mutated for p53. Moreover, p53 knockout fibroblasts are more sensitive to methylating agents and UV light than p53 wt cells, suggesting p53 to play a protective rather than a pro-apoptotic role in this cell system, probably by its involvement in DNA repair. However, for lymphoblastoid cells p53 wt variants are more sensitive to DNA damage indicating that p53 participates in apoptotic signaling in a cell type-specific fashion. The role of topoisomerase II inhibitors and c-Fos/AP-1 in apoptosis will also be discussed.     

Hydroquinone induces TK6 cell growth arrest and apoptosis through PARP‐1/p53 regulatory pathway

Hydroquinone (HQ), one of the most important metabolites derived from benzene, induces cell cycle arrest and apoptosis. Poly(ADP‐ribose) polymerase‐1 (PARP‐1) participates in various biological processes, including DNA repair and cell cycle regulation. To explore whether PARP‐1 regulatory pathway mediated HQ‐induced cell cycle arrest and apoptosis, we assessed the effect of PARP‐1 suppression on induction of apoptosis analyzed by FACSCalibur flow cytometer in PARP‐1 deficientTK6 cells (TK6‐shPARP‐1). We observed an increase in the fraction of cells in G1 phase by 7.6% and increased apoptosis by 4.5% in PARP‐1‐deficient TK6 cells (TK6‐shPARP‐1) compared to those negative control cells (TK6‐shNC cells) in response to HQ treatment. Furthermore, HQ might activate the extrinsic pathways of apoptosis via up‐regulation of Fas expression, followed by caspase‐3 activation, apoptotic body, and sub G1 accumulation. Enhanced p53 expression was observed in TK6‐shPARP‐1 cells than in TK6‐shNC cells after HQ treatment. In contrast, Fas expression was lower in TK6‐shPARP‐1 cells than in TK6‐shNC cells. Therefore, we conclude that HQ may activate apoptotic signals via Fas up‐regulation and p53‐mediated apoptosis in TK6‐shNC cells. The reduction of PARP‐1 expression further intensified up‐regulation of p53 in TK6‐shPARP‐1 cells, resulting in an increased G1→S phase cell arrest and apoptosis in TK6‐shPARP‐1 cells compared to TK6‐shNC cells.     

MAPK inhibitors and pifithrin-alpha block cinnamaldehyde-induced apoptosis in human PLC/PRF/5 cells

Cinnamaldehyde (Cin) has been shown to be effective in inducing apoptotic cell death in a number of human cancer cells. The aim of this study was to investigate the effect of pifithrin-alpha (PFTalpha; a specific p53 inhibitor) and mitogen-activated protein kinases (MAPKs) inhibitors [namely SP600125 (a specific JNK inhibitor), SB203580 (a specific p38 inhibitor) and PD98059 (a specific ERK inhibitor)] on apoptotic signaling transduction mechanism induced by Cin in human hepatoma PLC/PRF/5 (CD95-negative) cells. Using XTT assay, Cin exhibited a powerful cytotoxic effect and apoptotic induction in PLC/PRF/5 cells. Apoptosis was elicited when cells were treated with 1 microM Cin as characterized by morphological changes and the appearance of phosphatidylserine on the outer surface of the plasma membrane. Cin down-regulated the expression of Bcl-(XL), up-regulated mutant p53 and Bax proteins and promoted caspase-3 to active forms, as well as cleaving poly (ADP-ribose) polymerase (PARP) in a time-dependent pattern. This could be supported by the activation and phosphorylation of MAPKs, including JNK, ERK and p38 kinases. Pre-incubation with PFTalpha and specific MAPK inhibitors significantly diminished the effect of Cin-induced apoptosis. The activities of anti-apoptotic (Bcl-(XL)) and pro-apoptotic (Bax) proteins were remarkably affected by PFTalpha and PD98059 pre-treatment. PFTalpha effectively blocked PARP cleavage in cells treated with Cin, and also markedly prevented the phosphorylation of JNK, p38 and ERK proteins. These results suggest that p53 induction and MAPK signaling pathways are required for Cin-mediated apoptosis in PLC/PRF/5 cells.     

Cadmium affects genes involved in growth regulation during two-stage transformation of Balb/3T3 cells

Cadmium (Cd), a carcinogenic metal in human and rodents, has been shown to transform cells in vitro. However, the carcinogenic mechanisms of Cd as a mutagen and/or promoter are not well clarified. We already reported that CdCl2 in a range of 1.5 approximately 360 ng/ml enhanced transformation of Balb/3T3 A31 cells induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 0.1 microg/ml) in a dose-dependent manner (Fang et al., Toxicol. In Vitro 15(3) (2001a) 51-7). In previous study, we observed that Cd stimulated cell proliferation on MNNG-initiated cells through inactivation of p53 and p27 and increase of proliferating cell nuclear antigen (PCNA) expression after 24 h treatment (Fang et al., Toxicology 163 (2001b) 175-84). The aim of this study is to further elucidate the long-term effect of Cd in terms of cell cycle control gene expressions during the promotion stage of in vitro two-stage transformation. For the purpose, we determined the expression levels of the genes involved in growth regulation, such as p53, p27, c-myc, mdm2, cyclins D1 and B1, CDK4, and PCNA in the cells treated with Cd for 14 days after MNNG-initiation. In MNNG+CdCl2 group, cells apparently expressed cellular tumor antigen p53 mRNA, but did not express the wild-type p53 protein; the protein and mRNA levels of p27 were reduced apparently in the cells of MNNG+CdCl2 group compared to the cells of control and MNNG group. In addition, the protein levels of cyclin D1, CDK4, PCNA, c-myc, and mdm2, and cyclin B1 mRNA level were higher in MNNG+CdCl2 group than control and MNNG group. Together with previous data (Fang et al., Toxicology 163 (2001b) 175-84), our results indicated that during the transformation process of MNNG-treated cells, Cd may activate oncogenes such as c-myc, mdm2, and cellular tumor antigen p53, inhibit the tumor suppressor genes such as wild-type p53 and p27, and consequently accelerate the proliferation of initiated cells. This work firstly demonstrates that Cd affects the genes involved in growth regulation on initiated cells during the promotion stage of in vitro cell transformation.     

Methyl methanesulfonate induces apoptosis in p53-deficient H1299 and Hep3B cells through a caspase 2- and mitochondria-associated pathway

Methyl methanesulfonate (MMS) has been shown to induce apoptosis in various cell types through p53-dependent pathways. Nevertheless, pharmacological and genetic blockade of p53 functions results in similar or delayed sensitivity to MMS treatment, suggesting the presence of p53-independent apoptotic mechanisms. To understand the p53-independent mechanisms that are engaged during MMS-induced apoptosis, we established MMS-induced apoptotic cell models using p53-deficient H1299 and Hep3B cells. Our results demonstrated that MMS at concentrations of 50, 100, 200, 400 and 800 μM induced the formation of gammaH2AX foci, and that at higher concentrations, 400 and 800 μM, MMS treatment led to apoptosis in the two cell lines. This apoptotic cell death was concurrent with the loss of mitochondrial membrane potential, nuclear-cytosolic translocation of active caspase 2, release of cytochrome c from mitochondria, and the cleavage of caspase 9, caspase 3 and PARP. However, MMS-induced DNA damage failed to stabilize the p53 family members TAp73 and DNp73. These results demonstrated a p53- and p73-independent mechanism for MMS-induced apoptosis that involves the nuclear-cytosolic translocation of active caspase 2 as well as the mitochondria-mediated pathway.     

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...     

Capsaicin-induced apoptosis is regulated by endoplasmic reticulum stress- and calpain-mediated mitochondrial cell death pathways

Capsaicin, a pungent compound found in hot chili peppers, induces apoptotic cell death in various cell lines, however, the precise apoptosis signaling pathway is unknown. Here, we investigated capsaicin-induced apoptotic signaling in the human breast cell line MCF10A and found that it involves both endoplasmic reticulum (ER) stress and calpain activation. Capsaicin inhibited growth in a dose-dependent manner and induced apoptotic nuclear changes in MCF10A cells. Capsaicin also induced degradation of tumor suppressor p53; this effect was enhanced by the ER stressor tunicamycin. The proteasome inhibitor MG132 completely blocked capsaicin-induced p53 degradation and enhanced apoptotic cell death. Capsaicin treatment triggered ER stress by increasing levels of IRE1, GADD153/Chop, GRP78/Bip, and activated caspase-4. It led to an increase in cytosolic Ca²⁺, calpain activation, loss of the mitochondrial transmembrane potential, release of mitochondrial cytochrome c, and caspase-9 and -7 activation. Furthermore, capsaicin-induced the mitochondrial apoptotic pathway through calpain-mediated Bid translocation to the mitochondria and nuclear translocation of apoptosis-inducing factor (AIF). Capsaicin-induced caspase-9, Bid cleavage, and AIF translocation were blocked by calpeptin, and BAPTA and calpeptin attenuated calpain activation and Bid cleavage. Thus, both ER stress- and mitochondria-mediated death pathways are involved in capsaicin-induced apoptosis.     

Nitric Oxide-Induced Autophagy in MC3T3-E1 Cells is Associated with Cytoprotection via AMPK Activation

Nitric oxide (NO) is important in the regulation of bone remodeling, whereas high concentration of NO promotes cell death of osteoblast. However, it is not clear yet whether NO-induced autophagy is implicated in cell death or survival of osteoblast. The present study is aimed to examine the role of NO-induced autophagy in the MC3T3-E1 cells and their underlying molecular mechanism. The effect of sodium nitroprusside (SNP), an NO donor, on the cytotoxicity of the MC3T3-E1 cells was determined by MTT assay and expression of apoptosis or autophagy associated molecules was evaluated by western blot analysis. The morphological observation of autophagy and apoptosis by acridine orange stain and TUNEL assay were performed, respectively. Treatment of SNP decreased the cell viability of the MC3T3-E1 cells in dose- and time-dependent manner. SNP increased expression levels of p62, ATG7, Beclin-1 and LC3-II, as typical autophagic markers and augmented acidic autophagolysosomal vacuoles, detected by acridine orange staining. However, pretreatment with 3-methyladenine (3MA), the specific inhibitor for autophagy, decreased cell viability, whereas increased the cleavage of PARP and caspase-3 in the SNP-treated MC3T3-E1 cells. AMP-activated protein kinase (AMPK), a major autophagy regulatory kinase, was activated in SNP-treated MC3T3-E1 cells. In addition, pretreatment with compound C, an inhibitor of AMPK, decreased cell viability, whereas increased the number of apoptotic cells, cleaved PARP and caspase-3 levels compared to those of SNP-treated MC3T3-E1 cells. Taken together, it is speculated that NO-induced autophagy functions as a survival mechanism via AMPK activation against apoptosis in the MC3T3-E1 cells.     

G2 arrest and apoptosis by 2-amino-N-quinoline-8-yl-benzenesulfonamide (QBS), a novel cytotoxic compound

We screened a library of 11,000 small molecular weight chemicals, looking for compounds that affect cell viability. We have identified 2-amino-N-quinoline-8-yl-benzenesulfonamide (QBS) as a potent cytotoxic compound that induces cell cycle arrest and apoptosis. Treatment of Jurkat T cells with QBS increased the levels of cyclin B1 as well as phosphorylated-cdc2, which was accompanied by reduced activity of cdc2 kinase, suggesting that QBS may induce cell cycle arrest at G2 phase. Structural analogues of QBS also exhibited similar effects on cell cycle progression and cell viability. Long-term treatment with QBS resulted in DNA fragmentation, cytochrome C release, and PARP cleavage, and an increase in the number of subdiploidy cells, indicative of cellular apoptosis. Moreover, QBS-induced apoptosis was blocked by z-VAD-fmk, a pan-caspase inhibitor. These results suggest that QBS is a novel and potent compound that induces G2 arrest and subsequent apoptosis, implicating it as a putative candidate for chemotherapy.     

A mutated p53 status did not prevent the induction of apoptosis by sulforaphane,a promising anti-cancer drug

Summary We investigated apoptosis induction by sulforaphane on three cell lines characterized by a different p53 status. In particular, we used p53-knock-out fibroblasts from newborn mice transfected with the p53-Ser220 mutation, observed in Li-Fraumeni Syndrome patients, as a model of mutated p53 status. Moreover, immortalized fibroblasts from newborn mice expressing or lacking p53 (p53 +/+ andp53-/-, respectively) have been used to verify whether mutated p53 status could prevent sulforaphane-induced apoptotic events. Sulforaphane was able to induce apoptosis on all three cell lines. Indeed, the caspase-3 assays and poly(ADP-ribose)polymerase (PARP) cleavage data indicated that sulforaphane stimulated caspase-3-like activity and degradation of PARP. However, cells with a wild-type or mutated p53 appeared to be more sensitive to the effects of sulforaphane than cells lacking p53. Taken together, our results suggest that sulforaphane could act by a p53-independent pathway. For this reason, sulforaphane can be viewed as a novel agent useful not only in the treatment of Li-Fraumeni-associated tumors but also drug resistant tumors where p53 dysregulation is a feature.     

Induction of PUMA-α and down-regulation of PUMA-β expression is associated with benzo(a)pyrene-induced apoptosis in MCF-7 cells

Benzo(a)pyrene (BP) forms benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE)-DNA adducts in human breast adenocarcinoma MCF-7 cells, leading to p53 protein induction and phosphorylation. Although BP-induced apoptosis in rodent cells is known, it is still unclear in human cells. Here we have analyzed the effects of BP on p53 related apoptotic proteins, cell cycle and cell death in MCF-7 cells. PUMA-protein (p53 up-regulated modulator of apoptosis) levels were changed after BP exposure so that PUMA-α protein was statistically significantly increased whereas PUMA-β protein was statistically significantly decreased. PUMA-protein levels were also investigated in ZR-75-1 cells, where PUMA-α protein was statistically significantly increased. Cytochrome c, which is released from mitochondria during apoptosis to form the apoptosome, was increased in cytoplasmic fraction after BP exposure in MCF-7 cells. Increased apoptosis was also seen after 48 and 72 h BP exposure (2.5 and 5 μM). In addition, BP decreased dose dependently cell viability (2.5 and 5 μM) and increased ROS formation (1 and 10 μM). Our results suggest that PUMA-α protein is involved in BP-induced cell death most likely through a p53 dependent apoptotic pathway.