Susceptibility to drug-induced apoptosis correlates with differential modulation of Bad, Bcl-2 and Bcl-xL protein levels

To define the responses of apoptotic regulatory proteins to different chemotherapeutic agents, we investigated the expression of Bcl-2 family gene products, the release of cytochrome c, and the activation of pro-caspase-3 during apoptosis induced by Taxol and Thiotepa, in the MCF-7 breast carcinoma and the HL-60 leukemia cell lines. The earliest event induced by drug exposure was increase in Bad protein levels, followed by Bcl-2 down-regulation, cytochrome c release, and Bcl-xL and Bax up-regulation. Bak accumulation was a late event. Activation of pro-caspase-3 and cleavage of Bcl-2 protein occurred in the HL-60 cells only, and followed the cytochrome c release. The overall responses were qualitatively similar in both cell types, but MCF-7 cells treated with Taxol showed a significant delay in apoptosis, correlating with early up-regulation of Bcl-2 and delayed release of cytochrome c. We conclude that Bad up-regulation is an early indicator of a cellular response that will lead to cell death, but may be modulated by survival mechanisms, which cumulatively govern the ultimate susceptibility to apoptosis.     

Effects of Bcl-2 and Bcl-XL protein levels on chemoresistance of hepatoblastoma HepG2 cell line.

The ratio between apoptotic promoters and repressors in the Bcl-2 family determines the chemosensitivity of cells to apoptotic stimuli. This study examines the chemoresistance of a transfected human hepatoblastoma HepG2 cell-line during Taxol and Doxorubicin application. Sense bcl-2, and anti-sense bcl-XL gene fragments were separately inserted into HepG2 cells via stable transfection. The expression profile of the Bcl-2 family proteins was determined by Western blot analysis. Chemosensitivity of the transfected cells was measured by Trypan blue exclusion assay and XTT reduction assay during drug application. In the absence of Bax protein, HepG2 cells with elevated Bcl-2 protein levels did not exhibit any significant increase in chemosensitivity towards the drugs. Transfected cells with reduced Bcl-XL levels became more sensitive to the drugs, and a significant difference in IC50 values was observed. The chemosensitivity of HepG2 cells to Taxol and Doxorubicin was not affected by Bcl-2 levels, while reduction of Bcl-XL levels rendered the cells more sensitive to the drugs. This suggests that the Bcl-2 protein alone could not protect HepG2 cells from drug-induced apoptosis, and that the Bcl-XL protein may be a target for gene therapy in hepatoblastoma treatment.     

Microtubule dysfunction induced by paclitaxel initiates apoptosis through both c-Jun N-terminal kinase (JNK)-dependent and -independent pathways in ovarian cancer cells

The antineoplastic agent paclitaxel (TaxolTM), a microtubule stabilizing agent, is known to arrest cells at the G2/M phase of the cell cycle and induce apoptosis. We and others have recently demonstrated that paclitaxel also activates the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) signal transduction pathway in various human cell types, however, no clear role has been established for JNK/SAPK in paclitaxel-induced apoptosis. To further examine the role of JNK/SAPK signaling cascades in apoptosis resulting from microtubular dysfunction induced by paclitaxel, we have coexpressed dominant negative (dn) mutants of signaling proteins of the JNK/SAPK pathway (Ras, ASK1, Rac, JNKK, and JNK) in human ovarian cancer cells with a selectable marker to analyze the apoptotic characteristics of cells expressing dn vectors following exposure to paclitaxel. Expression of these dn signaling proteins had no effect on Bcl-2 phosphorylation, yet inhibited apoptotic changes induced by paclitaxel up to 16 h after treatment. Coexpression of these dn signaling proteins had no protective effect after 48 h of paclitaxel treatment. Our data indicate that: (i) activated JNK/SAPK acts upstream of membrane changes and caspase-3 activation in paclitaxel-initiated apoptotic pathways, independently of cell cycle stage, (ii) activated JNK/SAPK is not responsible for paclitaxel-induced phosphorylation of Bcl-2, and (iii) apoptosis resulting from microtubule damage may comprise multiple mechanisms, including a JNK/SAPK-dependent early phase and a JNK/SAPK-independent late phase.     

扁蒴藤素对人鼻咽癌HNE2 细胞增殖的抑制作用研究

目的：探讨扁蒴藤素对人鼻咽癌HNE2 细胞增殖的抑制作用,明确HSP70 在肿瘤发展过程中的抑制作用。方法：噻唑蓝法 （MTT）检测扁蒴藤素对HNE2 细胞生长抑制作用,流式细胞术检测扁蒴藤素诱导HNE2 细胞凋亡,免疫印迹法检测Caspase、 PARP、酪氨酸激酶、AKT 及Bcl-2 家族蛋白表达。结果：扁蒴藤素抑制HNE2 细胞的生长,促使Caspase 9,Caspase 3和PARP 蛋 白被切割,上调Bim 等促凋亡蛋白的表达,减少Bcl-xL等抗凋亡蛋白的表达,下调EGFR 等受体酪氨酸激酶, 抑制AKT 磷酸化, 上调热休克蛋白70（HSP70）的表达。用热休克反应抑制剂KNK437 抑制HSP70 的表达可以增强扁蒴藤素促进细胞凋亡的能力。 结论：扁蒴藤素通过下调受体酪氨酸激酶,激活caspase 介导的凋亡通路抑制鼻咽癌HNE2 细胞的增殖,抑制HSP70 的表达可增 强其抗肿瘤作用。     

ELF-MF attenuates quercetin-induced apoptosis in K562 cells through modulating the expression of Bcl-2 family proteins

This study investigated the effects of sinusoidal ELF-MF (1 mT; 50 Hz) on the apoptosis induced by four different compounds, namely vinblastine, etoposide, quercetin, and resveratrol, in human K562 chronic myeloid leukemia cells. The exposure to ELF-MF did not affect growth and viability of untreated K562 cells and did not influence the anti-proliferative effects of resveratrol, vinblastine, and etoposide. On the contrary, in quercetin-treated cells, exposure to ELF-MF significantly reduced the percentage of apoptotic cells and the caspase-3 activity and modified the cell cycle profile especially after 48 h of exposure. In addition, the simultaneous treatments for 24 h with quercetin plus ELF-MF increased Bcl-2 protein expression and prevented quercetin-induced downregulation of Mcl-1 and Bcl-xL. Finally, an increase of HSP70 expression was also observed after prolonged ELF-MF treatment. The ELF-MF-dependent modulation of the expression of anti-apoptotic Bcl-2 family and Hsp70 proteins could act as a pro-survival mechanism in K562 cells.     

Inhibition of JNK2 and JNK3 by JNK inhibitor IX induces prometaphase arrest-dependent apoptotic cell death in human Jurkat T cells

Exposure of human Jurkat T cells to JNK inhibitor IX (JNKi), targeting JNK2 and JNK3, caused apoptotic DNA fragmentation along with G₂/M arrest, phosphorylation of Bcl-2, Mcl-1, and Bim, Δψm loss, and activation of Bak and caspase cascade. These JNKi-induced apoptotic events were abrogated by Bcl-2 overexpression, whereas G₂/M arrest, cyclin B1 up-regulation, Cdk1 activation, and phosphorylation of Bcl-2 family proteins were sustained. In the concomitant presence of the G₁/S blocking agent aphidicolin and JNKi, the cells underwent G₁/S arrest and failed to induce all apoptotic events. The JNKi-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by the Cdk1 inhibitor. Immunofluorescence microscopic analysis revealed that mitotic spindle defect and prometaphase arrest were the underlying factors for the G₂/M arrest. These results demonstrate that JNKi-induced mitochondrial apoptosis was caused by microtubule damage-mediated prometaphase arrest, prolonged Cdk1 activation, and phosphorylation of Bcl-2 family proteins in Jurkat T cells.     

Transfection-enforced Bcl-2 overexpression and an anti-Parkinson drug, rasagiline, prevent nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase induced by an endogenous dopaminergic neurotoxin, N-methyl(R)salsolinol

An endogenous dopaminergic neurotoxin, N-methyl(R)salsolinol, was found to induce apoptosis in human dopaminergic SH-SY5Y cells by step-wise activation of apoptotic cascade; collapse in mitochondrial membrane potential, DeltaPsim, activation of caspases, and fragmentation of DNA. Recently, accumulation of gylceraldehyde-3-phosphate dehydrogenase (GAPDH) in nuclei was proposed to play an important role in apoptosis. In this paper, involvement of GAPDH in apoptosis induced by N-methyl(R)salsolinol was studied. The isoquinoline reduced DeltaPsim within 3 h, as detected by a fluorescence indicator, JC-1, then after 16 h incubation, GAPDH accumulated in nuclei by detection with immunostaining. To clarify the role of GAPDH in apoptotic process, a stable cell line of Bcl-2 overexpressed SH-SY5Y cells was established. Overexpression of Bcl-2 prevented the decline in DeltaPsim and also apoptotic DNA damage induced by N-methyl(R)salsolinol. In Bcl-2 transfected cells, nuclear translocation of GAPDH was also completely suppressed. In addition, a novel antiparkinsonian drug, rasagiline, prevented nuclear accumulation of GAPDH induced by N-methyl(R)salsolinol in control cells. These results suggest that GAPDH may accumulate in nuclei as a consequence of signal transduction, which is antagonized by anti-apoptotic Bcl-2 protein family and rasagiline. The results are discussed in concern to intracellular mechanism underlying anti-apoptotic function of rasagiline analogues.     

Differential regulation of protein expression, growth and apoptosis by natural and synthetic retinoids

All-trans retinoic acid (ATRA) can down regulate the anti-apoptotic protein Bcl-2 and the cell cycle proteins cyclin D1 and cdk2 in estrogen receptor-positive breast cancer cells. We show here that retinoids can also reduce expression of the inhibitor of apoptosis protein, survivin. Here we have compared the regulation of these proteins in MCF-7 and ZR-75 breast cancer cells by natural and synthetic retinoids selective for the RA receptors (RARs) alpha, beta, and gamma then correlated these with growth inhibition, induction of apoptosis and chemosensitization to Taxol. In both cell lines ATRA and 9-cis RA induced the most profound decreases in cyclin D1 and cdk2 expression and also mediated the largest growth inhibition. The RARalpha agonist, Ro 40-6055 also strongly downregulated these proteins although did not produce an equivalent decrease in S-phase cells. Only ATRA induced RARbeta expression. ATRA, 9-cis RA and 4-HPR initiated the highest level of apoptosis as determined by mitochondrial Bax translocation, while only ATRA and 9-cis RA strongly reduced Bcl-2 and survivin protein expression. Enumeration of dead cells over 96 h correlated well with downregulation of both survivin and Bcl-2. Simultaneous retinoid-mediated reduction of both these proteins also predicted optimal Taxol sensitization. 4-HPR was much weaker than the natural retinoids with respect to Taxol sensitization, consistent with the proposed requirement for reduced Bcl-2 in this synergy. Neither the extent of cell cycle protein regulation nor AP-1 inhibition fully predicted the antiproliferative effect of the synthetic retinoids suggesting that growth inhibition requires regulation of a spectrum of RAR-regulated gene products in addition even to pivotal cell cycle proteins.     

Manganese-induced apoptosis in rat myocytes

Manganese can be toxic to the heart, causing dysfunction following long exposure. In our experiments, we examined the cytotoxicity of manganese in neonatal rat ventricular myocytes (NRVM) by MTT assays in vitro. Results showed that after incubation in the different concentrations of manganese for 24 h, apparent cytotoxicity was observed. At 500, 1000, and 1500 2 microM of manganese, the percentage of cell viability dropped to 82% +/- 6.13, 78% +/- 5.28, and 66% +/- 4.22, respectively. When cells were treated for 48 h, all concentrations tested exerted toxic effect; especially from 500 to 1500 microM the cell viability dropped from 67% +/- 4.84 to 37% +/- 3.25. Apoptosis in NRVM was then examined by flow cytometry. Results showed that the percentage of apoptotic cells treated with 500 microM of manganese for 24 h increased from 4% +/- 0.84 to 7% +/- 1.16. After 48 h of incubation, this percentage increased to 11% +/- 0.91. There was no significant difference between control groups (0 microM manganese) after 24 and 48 h incubation. The morphological changes of NRVM nuclei were visualized with the fluorescent DNA-binding dye Hoechst33342 after incubation in 500 microM of manganese for 48 h. Compared with normal nuclei, apoptotic nuclei showed the typical features of fragmentation and condensation. To investigate whether there are any apoptotic gene expression changes during apoptosis, we examined the expression level of Bcl-2, Bax, and P53 mRNAs after treatment with 500 microM of manganese for 48 h. The Bcl-2 mRNA expression decreased while the expression of Bax as well as P53 mRNAs increased. These results suggested that manganese cytotoxicity on NRVM could induce apoptosis in NRVM cells. The apoptosis process might involve, and be promoted by, the changes of the expression levels of P53, Bcl-2, and Bax proteins.     

CDK1 siRNA干扰在Taxol诱导细胞凋亡中的作用

目的研究转染细胞周期依赖性蛋白激酶1（cyclin．dependent kinase1,CDK1）siRNA、以及转染后进行凋亡刺激对细胞周期和凋亡的影响,探讨CDK1在细胞凋亡中的确切作用,揭示细胞周期与细胞凋亡协调的分子机制。方法以人宫颈癌细胞株HeLa细胞为研究对象,脂质体转染CDK1siRNA,转染后48h加紫杉醇（Tax01）（20μg／m1）刺激凋亡,Western印迹检测CDK1和抗凋亡蛋白BCL2表达,AnnexinV／PI法检测细胞的凋亡,流式细胞仪分析DNA含量检测细胞周期。结果转染CDK1 siRNA后,CDK1蛋白的表达下降,细胞周期G2／M期比例增加,细胞凋亡率与对照相比没有明显升高。只加Taxol刺激12h后细胞凋亡率增加并伴有S期和G2／M期比例增加。转染CDKlsiRNA后再用Taxol刺激,其细胞凋亡率没有明显改变,G2／M期阻滞效应也没有叠加。BCL2蛋白只在加Taxol刺激组表达下降,与CDK1表达减少没有相关性。结论siRNA沉默导致的CDK1表达降低只导致细胞周期G2／M期阻滞,没有引起细胞凋亡;CDK1的表达降低对紫杉醇所诱导的细胞周期阻滞和细胞凋亡效应没有明显影响。     

Carnosic acid modulates Akt/IKK/NF-κB signaling by PP2A and induces intrinsic and extrinsic pathway mediated apoptosis in human prostate carcinoma PC-3 cells

This study investigates the efficacy of carnosic acid (CA), a polyphenolic diterpene, isolated from the plant rosemary (Rosemarinus officinalis), on androgen-independent human prostate cancer PC-3 cells. CA induced anti-proliferative effects in PC-3 cells in a concentration- and time-dependent manner, which was due to apoptotic induction as evident from flow-cytometry, DNA laddering and TUNEL assay. Apoptosis was associated with the activation of caspase-8, -9, -3 and -7, increase in Bax:Bcl-2 ratio, release of cytochrome-c and decrease in expression of inhibitor of apoptosis (IAP) family of proteins. Apoptosis was attenuated upon pretreatment with specific inhibitors of caspase-8 (Z-IETD-fmk) and caspase-9 (Z-LEHD-fmk) suggesting the involvement of both intrinsic and extrinsic apoptotic cascades. Further, apoptosis resulted from the inhibition of IKK/NF-κB pathway as evident from decreased DNA binding activity, nuclear translocation of p50 and p65 and IκBα phosphorylation. The down-regulation of IKK/NF-κB was associated with inhibition of Akt phosphorylation and its kinase activity with a concomitant increase in the serine/threonine protein phosphatase 2A (PP2A) activity. Pharmacologic inhibition of PP2A by okadaic acid and calyculin A, significantly reversed CA-mediated apoptotic events in PC-3 cells indicating that CA induced apoptosis by activation of PP2A through modulation of Akt/IKK/NF-κB pathway. In addition, CA induced apoptosis in another androgen refractory prostate cancer DU145 cells via intrinsic pathway as evidenced from the activation of caspase 3, cleavage of PARP, increase in Bax:Bcl-2 ratio and cytochrome-c release. Carnosic acid, therefore, may have the potential for use in the prevention and/or treatment of prostate cancer.     

p38 MAP kinase mediates apoptosis through phosphorylation of BimEL at Ser-65

The stress-activated c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein (MAP) kinase (p38) regulate apoptosis induced by several forms of cellular insults. Potential targets for these kinases include members of the Bcl-2 family proteins, which mediate apoptosis generated through the mitochondria-initiated, intrinsic cell death pathway. Indeed, the activities of several Bcl-2 family proteins, both pro- and anti-apoptotic, are controlled by JNK phosphorylation. For example, the pro-apoptotic activity of Bim(EL), a member of the Bcl-2 family, is stimulated by JNK phosphorylation at Ser-65. In contrast, there is no reported evidence that p38-induced apoptosis is due to direct phosphorylation of Bcl-2 family proteins. Here we report evidence that sodium arsenite-induced apoptosis in PC12 cells may be due to direct phosphorylation of Bim(EL) at Ser-65 by p38. This conclusion is supported by data showing that ectopic expression of a wild type, but not a non-phosphorylatable S65A mutant of Bim(EL), potentiates sodium arsenite-induced apoptosis and by experiments showing direct phosphorylation of Bim(EL) at Ser-65 by p38 in vitro. Furthermore, sodium arsenite induced Bim(EL) phosphorylation at Ser-65, which was blocked by p38 inhibition. This study provides the first example whereby p38 induces apoptosis by phosphorylating a member of the Bcl-2 family and illustrates that phosphorylation of Bim(EL) on Ser-65 may be a common regulatory point for cell death induced by both JNK and p38 pathways.     

Bcl-2 small hairpin RNAs enhance radiation-induced apoptosis in A549 cells

Bcl-2, a prominent member of the family of proteins, is responsible for dys-regulation of apoptosis and resistance to chemotherapy and radiotherapy. This study investigated whether small hairpin RNA (shRNA) targeting Bcl-2 could render A549 cells more susceptible to gamma radiation-induced apoptosis. Recombinant Bcl-2 shRNAs expression vector were transfected into A549 cells with Lipofectamine 2000. Transfected cells were screened in 800 mg/ml G418 screening medium, and after stable transfection, silencing was examined. Expression of the Bcl-2 protein was assayed using Western blot in A549 cells. Inhibition of cell growth was assessed by a MTT assay. Apoptosis was determined by morphological observation and flow cytometry. Expression levels of Bcl-2 protein from A549 cells decreased after stable transfection with Bcl-2 shRNAs. No differences in Bcl-2 protein levels between control shRNA group and untreated cells were noted. After stable transfection with Bcl-2 shRNAs the viability of cells was less than after stable transfection with those with control shRNAs and untransfected A549, respectively (P<0.05). Control shRNA had no significant effect on growth of cells. Radiation significantly inhibited the growth of cells stably transfected with Bcl-2 shRNA (P<0.05). No difference in survival between the cells with control shRNA and untransfected cells was noted. Using Giemsa staining, cells stably transfected with Bcl-2 shRNA combined with radiation at 48 h displayed changes of apoptosis. After treatment with radiation apoptotic rates of the A549 cells stably transfected with Bcl-2 shRNA significantly increased (P<0.05), compared with the cells with control shRNA and untransfected cells. shRNAs against the Bcl-2 mRNA increases radiation-induced apoptosis in A549 cells.     

5-烯丙基-7-二氟亚甲基白杨素（C19H14O4F2）诱导人卵巢癌CoC1细胞凋亡

目的 观察5-烯丙基-7-二氟亚甲基白杨素（ADFMChR）诱导人卵巢癌（CoCl）细胞凋亡的作用。方法 以体外培养的人卵巢癌CoCl为研究对象。采用软琼脂克隆测定ADFMChR对细胞集落的影响;流式细胞术（FCM）检测ADFMChR诱导细胞凋亡率;凝胶电泳观察ADFMChR诱导基因DNA梯形条带。Westernblot分析ADFMChR对CoCl细胞PPARγ,NF-κB,Bcl-2,Bax蛋白表达的影响。结果软琼脂克隆显示ADFMChR呈剂量依赖性抑制细胞集落形成;FCM分析发现ADFMChR呈剂量依赖性诱导细胞凋亡;ADFMChR（30μmol／L）孵育CoCl细胞48h后,DNA琼脂糖凝胶电泳呈现典型梯形条带。Westernblot分析结果表明ADFMChR以剂量依赖方式上调CoCl细胞PPARγ和Bax蛋白表达,下调NF-κB和Bcl-2蛋白表达。结论 ADFMChR诱导人卵巢癌CoCl细胞凋亡与其活化PPARγ,抑制NF-κB表达和提高Bax／Bcl-2比值有关。     

Apoptosis in gastric mucosa with stress-induced gastric ulcers.

The maintenance of gastric mucosal integrity depends upon the interplay between epithelial cell proliferation and apoptosis (programmed cell death). The Bcl-2 family of proteins plays a central role in the regulation of apoptotic cell death by suppressing the apoptosis while some others such as Bax proteins promote this process. Stress-induced gastric ulcerations are accompanied by the fall in gastric mucosal cell proliferation but little is known about the influence of the stress on the apoptosis in gastric mucosa. In the present study, the gastric epithelial apoptosis was determined by means of expression of Bax and Bcl-2 mRNA in the gastric mucosa following acute stress. Wistar rats were exposed to mild water immersion and restraint stress (WRS) for 3.5 h and then sacrificed at 0, 2, 4, 6, 12 and 24 h after the termination of WRS. At each time interval after WRS, the gastric blood flow (GBF) and the proliferating cell nuclear antigen (PCNA) labeling were determined. The apoptosis rate in the gastric mucosa was determined by the terminal deoxynucleotidyl transferase (TDT) mediated 2-deoxyuridine 5-triphosphate (dUTP)-biotin nick end-labeling (TUNEL) staining method and the expression of Bax and Bcl-2 mRNA was analyzed by RT-PCR and southern blot hybridization. WRS produced multiple erosions accompanied by the fall in GBF and PCNA index and by a dramatic enhancement in gastric epithelial apoptosis rate reaching maximum at 4 h after exposure to WRS. Following 6 and 12 h after the end of WRS the apoptotis declined but even 24 h after WRS it failed to reach the value recorded in intact gastric mucosa. The PCNA index was still significantly inhibited at 2 h after WRS but then showed significant rise at 6 and 12 h to reach at 24 h after WRS, the level similar to that measured in intact gastric mucosa. The expression of Bax mRNA was detected in intact gastric mucosa and gradually increased in first 4 h after WRS to decline at 24 h to the level not significantly different from that observed in the intact mucosa. In contrast, the expression of Bcl-2 mRNA was almost undetectable during first 4 h but showed strong signal at 6 and 12 h to decline to the control level 24 h after WRS. We conclude that: 1. Healing of WRS lesions involves an increase in GBF and mucosal cell proliferation and 2. The enhancement in gastric epithelial apoptosis accompanies the mucosal damage induced by stress and this appears to be triggered by the shift from the cell death effector Bax to the cell death repressor Bcl-2 protein.     

Mechanistic aspects of the induction of apoptosis by lauryl gallate in the murine B-cell lymphoma line Wehi 231

The effect of lauryl gallate (antioxidant E-312) has been studied on the mouse B-cell lymphoma line Wehi 231. This compound is able to inhibit protein tyrosine kinases (PTKs) in whole cells and in crude extracts with a better efficiency than other well-known PTK inhibitors such as herbimycin or genistein. Initial events triggered upon the incubation of cells with lauryl gallate in phosphate-buffered saline (up to 1 h) include the inhibition of tyrosine phosphorylation, discharge of the mitochondrial transmembrane potential, and induction of mRNA for Bcl-2. Long-term cultures in complete medium supplemented with fetal calf serum (up to 24 h) in the presence of this compound exhibit clear apoptotic features such as increase in phosphatidylserine in the cell surface, decrease in the functionality of mitochondria, cytochrome c release to the cytosol, activation of caspases, hypodiploidy, and oligonucleosomal breakdown of DNA. Comparison between Wehi cells overexpressing Bcl-2 (Wehi-bcl-2) with Wehi-neo cells shows a delay in the manifestations of the apoptotic signs, indicating that Bcl-2 has a partial protective effect on the apoptosis induced by lauryl gallate. The proapoptotic effect of lauryl gallate is not dependent on DNA or protein synthesis, is not blocked by the chelation of calcium, and is not reverted by N-acetylcysteine.     

Apoptosis in RAW 264.7 cells exposed to 4-hydroxy-2-nonenal: dependence on cytochrome C release but not p53 accumulation

The toxic reactive aldehyde lipid peroxidation byproduct 4-hydroxy-2-nonenal (HNE) is thought to be a major contributor to oxidant stress-mediated cell injury. HNE induced apoptosis in RAW 264.7 murine macrophage cells in a dose-dependent manner within 6-8 h after exposure. Expression of the antiapoptotic protein Bcl-2 in stably transfected RAW 264.7 cells prevented HNE-induced internucleosomal DNA fragmentation and apoptosis, and these cells resume growth after a temporary (24-48 h) growth delay. While parental RAW 264.7 cells released mitochondrial cytochrome c within 3 h after HNE exposure, expression of Bcl-2 prevented cytochrome c release. In control cells, p53 protein levels peaked at 6-9 h after HNE exposure and then declined, while in Bcl-2 expressing cells, p53 levels were maximal at 6-9 h and remained elevated up to 96 h. Expression of SV40 large T-antigen, which forms a stable complex with p53 protein, via stable transfection-blocked transactivation of the p53-regulated gene p21(WAF1/CIP1), but did not affect induction of apoptosis by HNE, suggesting that p53 function is not important in HNE-induced apoptosis. These results suggest that cytochrome c release, but not p53 accumulation, plays an essential role in HNE-induced apoptosis in RAW 264.7 cells.     

Role of mitochondria in aspirin-induced apoptosis in human gastric epithelial cells

This study was undertaken to determine whether the Bcl-2 family proteins and Smac are regulators of aspirin-mediated apoptosis in a gastric mucosal cell line known as AGS cells. Cells were incubated with varying concentrations of acetylsalicylic acid (ASA; 2-40 mM), with or without preincubation of caspase inhibitors. Apoptosis was characterized by Hoechst staining and DNA-histone-associated complex formation. Antiapoptotic Bcl-2, proapoptotic Bax and Bid, Smac, and cytochrome-c oxidase (COX IV) were analyzed by Western blot analyses from cytosol and mitochondrial fractions. ASA downregulated Bcl-2 protein expression and induced Bax translocation into the mitochondria and cleavage of Bid. In contrast, expression of Smac was significantly decreased in mitochondrial fractions of ASA-treated cells. Bax and Bid involvement in apoptosis regulation was dependent on caspase activation, because caspase-8 inhibition suppressed Bax translocation and Bid processing. Caspase-9 inhibition prevented Smac release from mitochondria. Additionally, increased expression of the oxidative phosphorylation enzyme COX IV was observed in mitochondrial fractions exposed to ASA at concentrations >5 mM. Although caspase-8 inhibition had no effect on aspirin-induced apoptosis and DNA-histone complex formation, caspase-9 inhibition significantly decreased both of these events. We conclude that Bcl-2 protein family members and Smac regulate the apoptotic pathway in a caspase-dependent manner. Our results indicate also that mitochondrial integration and oxidative phosphorylation play a critical role in the pathogenesis of apoptosis in human gastric epithelial cells.     

Interconnections between E2-dependent regulation of cell cycle progression and apoptosis in MCF-7 tumors growing on nude mice

Growth of human breast adenocarcinoma MCF-7 cells as a tumor on nude mice is dependent on estrogen. It has been shown that estrogen withdrawal (EW) induces a partial regression of the tumor via an inhibition of cell proliferation and an induction of apoptosis. We investigated in this in vivo model the underlying molecular mechanisms of the hormone-dependent regulation of cell cycle machinery and apoptosis. We found that, 2 days after EW, the tumor protein levels of p21 rose, whereas those of Rb proteins decreased in parallel with the decrease in the proportion of tumor cells in S phase and the increase of the tumor apoptotic index. Between 3 and 7 days after EW, apoptosis was inhibited and tumor proliferation returned to the control value. There was a concomitant decline in p21 and an elevation of Rb tumor protein content. Slight variations of cyclin D protein level were observed in MCF-7 tumors over the time course following EW treatment. Bcl-2 overexpression not only inhibited apoptosis induced by EW but also modulated hormone-dependent cell cycle regulation. First, the analysis of phosphorylation status of Rb protein and the measurement of the proportion of tumor cells in S phase indicated that Bcl-2 overexpression results in a decrease of DNA synthesis induced by estradiol. Furthermore, after EW, Bcl-2-induced inhibition of hormone-dependent apoptosis was associated with an inhibition of Rb protein downregulation, a sustained level of p21 protein, and a prolonged inhibition of cell cycle progression. These results suggest that, in human hormone-dependent breast cancers, cross-talk exists between the signaling pathways which lead to regulation of cell cycle progression and apoptosis.     

Selective involvement of BH3-only proteins and differential targets of Noxa in diverse apoptotic pathways

The BH3-only proteins of the Bcl-2 family are known to mediate mitochondrial dysfunction during apoptosis. However, the identity of the critical BH3-only proteins and the mechanism of their action following treatment by diverse apoptotic stimuli remain to be fully resolved. We therefore used RNAi to screen the entire Bcl-2 family for their involvement in three major apoptotic pathways in HeLa cells. We found that Bcl-xL and Mcl-1 are major inhibitors of apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL), endoplasmic reticulum (ER) stress, and proteasome inhibition. Among the 10 BH3-only proteins, Bid and Noxa were found to be critically involved in TRAIL-induced apoptosis, in which Noxa participates by constitutively binding to Mcl-1. Bim and Noxa were found to be necessary for ER stress-induced apoptosis, in which Noxa assisted Bim function by sequestering Mcl-1 and binding to Bcl-xL. As a critical BH3-only protein, Noxa was strongly upregulated and became associated with both Mcl-1 and Bcl-xL during apoptosis induced by proteasome inhibition. In addition, we found that Noxa became 'Mcl-1 free' following treatment by ER stress and proteasome inhibition, but not after TRAIL treatment. These results defined the critical Bcl-2 network during apoptosis and suggested that Noxa participated in triggering mitochondrial dysfunction in multiple apoptotic pathways through distinct mechanisms.