Failure of stress-induced downregulation of Bcl-2 contributes to apoptosis resistance in senescent human diploid fibroblasts

We previously reported that senescent human diploid fibroblasts (HDFs) are resistant to apoptosis induced by H(2)O(2) and staurosporine. We report here that senescent HDFs are resistant to thapsigargin-induced apoptosis as well. These agonists caused the reductions in mitochondrial membrane potential (MMP) and in the apoptosis inhibitory protein (B-cell lymphoma) only in young HDFs but not in senescent HDFs. In addition, downregulation of Bcl-2 increased the sensitivity of senescent HDFs to apoptosis induction, suggesting the significant role of Bcl-2 in apoptosis resistance of the senescent HDFs. We further found that P-cAMP response element-binding protein (CREB), a positive regulator of Bcl-2, decreased in stress-induced apoptosis of young HDFs but not in senescent HDFs, and that Bcl-2 was markedly reduced in CREB small interfering RNA (siRNA), transfected senescent HDFs. In addition, activity of protein phosphatase 2A (PP2A), which dephosphorylates p-CREB, significantly increased in young HDFs but not in senescent HDFs treated with H(2)O(2), staurosporine or thapsigargin. Taken together, these results suggest that failure of stress-induced downregulation of Bcl-2 underlies resistance of senescent HDFs to apoptosis.     

Aging-associated increase of gelsolin for apoptosis resistance

Gelsolin, a Ca(2+)-dependent actin regulatory protein, was recently suggested to participate in apoptosis regulation. In this study, we found that the level of gelsolin is elevated in senescent human diploid fibroblasts (HDFs) and also in the tissues of old rats, i.e., in the liver, kidney, heart, spleen, stomach, and brain, etc. The ubiquitous increase of gelsolin in the aged organs and cells led us to assume that it might be related with one of the cardinal senescent phenotypes, aging-associated apoptosis resistency. Thus, we tested the sensitivity of senescent cells to apoptosis by menadione, an apoptosis-inducing agent, before and after the down-regulation of gelsolin. The down-regulation of gelsolin in senescent HDFs, independently of Bcl-2 family expression, resulted in an increased sensitivity to menadione-induced apoptotic cell death. The observed ubiquitous increase of gelsolin in the senescent states of cells and tissues, and the increased sensitivity to apoptosis-induction by gelsolin down-regulation, suggests that gelsolin would be partly responsible for age-related apoptosis resistance.     

Defective nuclear translocation of stress-activated signaling in senescent diploid human fibroblasts: a possible explanation for aging-associated apoptosis resistance

In order to study the nature of aging-dependent apoptosis resistance, we compared the activation pattern of mitogen-activated protein kinases (MAPK) in response to three different stress modalities: hydrogen peroxide (H₂O₂), staurosporine, and thapsigargin. We observed the agonist-specific activation pattern of MAP kinases in human diploid fibroblasts (HDFs). When young HDFs were treated with PD98059, a specific inhibitor of extracellular signal-regulated kinase (ERK), H₂O₂-induced apoptosis was blocked, whereas staurosporine-induced apoptosis was inhibited by treatment with SB203580, a specific inhibitor of p38. In addition, the levels of anti-apoptotic protein Bcl-2 (B-cell lymphoma protein-2) were restored by PD98059 or SB239063 in cells treated with H₂O₂ or staurosporine, respectively. We also found that inhibition of the nuclear import of p-Erk and p-p38 using wheat germ agglutinin induced apoptosis resistance in young HDF cells in response to H₂O₂ or staurosporine. These data indicate a potential role of the nuclear translocation of apoptotic signals in the induction of apoptosis. Moreover, the nuclear translocation of activated ERK1/2 and p38 in response to H₂O₂ or staurosporine was significantly compromised in senescent HDFs, compared with young cells. Taken together, we propose that the apoptosis resistance of senescent HDFs might be related to the defective nuclear translocation of stress-activated signals in an agonist-specific manner, which would imply the operation of an aging-dependent functional nucleo-cytoplasmic trafficking barrier.     

Vinblastine-induced phosphorylation of Bcl-2 and Bcl-XL is mediated by JNK and occurs in parallel with inactivation of the Raf-1/MEK/ERK cascade

Microtubule-damaging agents arrest cells at G(2)/M and induce apoptosis in association with phosphorylation of the anti-apoptotic proteins Bcl-2 and Bcl-X(L). Because microtubule inhibitors activate JNK, we sought to determine whether JNK was responsible for Bcl-2/Bcl-X(L) phosphorylation in KB-3 cells treated with vinblastine. Two major endogenous forms of JNK, p46(JNK1) and p54(JNK2), were present in KB-3 cells, and both isoforms were activated by vinblastine as determined by Mono Q chromatography. We used antisense oligonucleotides (AS) to specifically inhibit their expression. A combination of AS-JNK1 with AS-JNK2 inhibited by 80% vinblastine-induced phosphorylation of two known JNK substrates, c-Jun and ATF-2. In addition, AS-JNK1/2 inhibited vinblastine-induced phosphorylation of Bcl-2 by 85% and that of Bcl-X(L) by 65%. Stable expression of the JNK scaffold protein JIP-1 blocked vinblastine-induced phosphorylation of c-Jun and ATF-2, but did not affect Bcl-2/Bcl-X(L) phosphorylation, confirming a bifurcation in JNK signaling involving both nuclear and non-nuclear substrates. Vinblastine-induced phosphorylation of Raf-1 was unaffected by AS-JNK1/2 and was associated with loss of activity for MEK substrate in vitro and inactivation of ERK in vivo. These results provide evidence for a direct role of the JNK pathway in apoptotic regulation through Bcl-2/Bcl-X(L) phosphorylation.     

MAPKs and NF-κB-mediated acrylamide-induced neuropathy in rat striatum and human neuroblastoma cells SY5Y

Acrylamide (ACR) is a potent neurotoxin that can be produced during high-temperature food processing, but the underlying toxicological mechanism remains unclear. In this study, the detrimental effects of ACR on the striatal dopaminergic neurons and the roles of mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NF-κB) in ACR-induced neuronal apoptosis were investigated. Acute ACR exposure caused dopaminergic neurons loss and apoptosis as revealed by decreased tyrosine hydroxylase (TH)-positive cells and TH protein level and increased terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells in the striatum. ACR-decreased glutathione content, increased levels of malondialdehyde, proinflammatory cytokines tumor necrosis factor α, and interleukin 6. In addition, nuclear NF-κB and MAPKs signaling pathway with c-Jun N-terminal kinase (JNK) and p38 were activated by ACR. Specific inhibitors were used to explore the roles of MAPKs and NF-κB pathways in ACR-induced apoptosis in SH-SY5Y cells. Pretreatment with JNK-specific inhibitors SP600125 markedly upregulated the reduced B-cell lymphoma 2 (Bcl-2) content and downregulated the increased Bcl-2-associated X protein (Bax) level and thereby eventually reduced the proportions of early and late apoptotic cells induced by ACR, while p38 suppression by SB202190 only reversed the decrease in Bcl-2 expression. Inhibition of NF-κB by BAY 11-7082 markedly upregulated Bax level and decreased Bcl-2 expression, and eventually increasing the proportions of neuronal apoptosis compared with that in ACR alone. These results suggested that JNK contributed to ACR-induced apoptosis, while NF-κB acted as a protective regulator in response to ACR-induced neuropathy. This study helps to offer a deeper insight into the mechanism of ACR-induced neuropathy.     

高危型人乳头瘤病毒感染与宫颈癌中miR-218表达的关系

目的观察高危型人乳头瘤病毒(HPV)感染与宫颈癌中miR-218表达的关系。方法收集2015年6月至2018年12月我院手术切除的宫颈癌组织并检测高危型HPV感染情况和miR-218表达量。培养HPV16阳性的SiHa细胞株并进行分组,阴性对照(NC)组转染NC模拟物、miR-218组转染miR-218模拟物,检测两组细胞凋亡率、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关x蛋白(Bax)、Bcl-2相互作用细胞死亡介导因子(Bim)、含半胱氨酸的天冬氨酸蛋白水解酶(Caspase)-9、Caspase-3的mRNA表达量及凋亡通路分子c-Jun氨基末端激酶(JNK)、c-Jun基因(c-Jun)、磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(AKT)、哺乳动物雷帕霉素靶蛋白(mTOR)的蛋白表达量。结果高危型HPV阳性的宫颈癌组织中miR-218表达量减少。转染24 h后,miR-218组细胞凋亡率、细胞中Bax、Bim、Caspase-9、Caspase-3的mRNA表达量及JNK、c-Jun的蛋白表达量均明显高于NC组,而细胞中Bcl-2的mRNA表达量及PI3K、AKT、mTOR的蛋白表达量均低于NC组,差异均有统计学意义(均P<0.05)。结论miR-218在高危型HPV感染的宫颈癌组织中表达减少。增加miR-218的表达能够促进HPV感染宫颈癌细胞的凋亡。该调控作用与JNK/c-Jun通路的激活及PI3K/AKT/mTOR通路的抑制有关。     

The expression of oncogenes in tumour tissues of breast carcinoma patients

The balance between Bcl-2 and c-Myc and c-Jun seems to be an important determinant of cellular sensitivity to the induction of apoptosis. High expression of Bcl-2 was noticed to be strongly related to low rates of apoptotic cell death. The mean value of the apoptotic index was 45.0% in Bcl-2-negative tumours and 7.5% in Bcl-2-positive tumours. C-Myc and c-Jun accumulation were associated with the absence of Bcl-2 expression and with increased apoptotic activity. The loss of Bd-2 expression was strongly correlated with increased apoptotic cell death. The inverse correlation is between apoptotic and mitotic index. A high mitotic index exists in most patients with a low apoptotic index. Bcl-2, c-Myc and c-Jun does not only take part in cell death, but also in cell division in breast carcinoma cells in which the regulation of cell division and cell death are strictly connected.     

IL-2 deprivation triggers apoptosis which is mediated by c-Jun N-terminal kinase 1 activation and prevented by Bcl-2

A variety of environmental stresses, as well as inflammatory cytokines, induce activation of c-Jun N-terminal kinases. We describe here that IL-2 deprivation-induced apoptosis in TS1alphabeta cells does not modify c-Jun protein levels and correlates Bcl-2 downregulation and an increase in JNK1, but not JNK2, activity directly related to the induction of apoptosis. Indeed, downregulation of JNK1 expression using antisense oligonucleotides inhibits apoptosis induced by IL-2 withdrawal. Overexpression of Bcl-2 promotes cell survival and blocks JNK1 activation as well as apoptosis caused by IL-2 deprivation. This suggests that inhibition of the JNK1 signaling pathway may be a mechanism through which Bcl-2 promotes cell survival and prevents apoptosis triggered by growth factor withdrawal.     

胸苷激酶基因在人二倍体成纤维细胞衰老过程中的表达调控

为研究人胸苷激酶 (humanthymidinekinase ,hTK)基因在复制衰老细胞及早衰细胞中表达下调的分子机制 ,构建了含hTK启动子的荧光素酶报告基因载体 .转染结果显示 ,复制衰老细胞与早衰细胞中hTK启动子的转录活性比年轻细胞中下降了近 3倍 ,表明转录水平的调控是hTK在衰老细胞中表达下降的主要调控机制 .定点突变的结果显示 ,转录因子Sp1、NF Y结合位点的突变可使hTK启动子活性降低近 5 0 % ,而E2F结合位点的突变可使其活性升高 2倍多 ,提示Sp1和NF Y是hTK基因的转录活化因子 ,而E2F为转录抑制因子 .电泳迁移率变更实验发现 ,与年轻细胞相比 ,Sp1、NF Y与hTK启动子的DNA结合活性在复制衰老细胞和早衰细胞中无明显改变 ,提示转录活化因子Sp1、NF Y并非hTK在衰老细胞中下调的主要因素 .染色质免疫共沉淀结果显示 ,在细胞内Rb结合在hTK启动子上 ,且同年轻细胞相比 ,复制衰老细胞及早衰细胞中的hTK启动子结合着更多的Rb ,这提示细胞衰老过程中Rb的去磷酸化可能与hTK基因在衰老过程中的下调有关 .     

The role of mitogen-activated protein kinase in cadmium-induced primary rat cerebral cortical neurons apoptosis via a mitochondrial apoptotic pathway

Cadmium (Cd) is an extremely toxic metal capable of severely damaging several organs, including the brain. Studies have shown that Cd induces neuronal apoptosis partially by activating the mitogen-activated protein kinase (MAPK) pathways. However, the underlying mechanism of MAPK involving the mitochondrial apoptotic pathway in neurons remains unclear. In this study, primary rat cerebral cortical neurons were exposed to Cd, which significantly decreased cell viability and the B-cell lymphoma 2/Bcl-2 associate X protein (Bcl-2/Bax) ratio and increased the percentage of apoptotic cells, release of cytochrome c, cleavages of caspase-3 and poly (ADP-ribose) polymerase (PARP), and nuclear translocation of apoptosis-inducing factor (AIF). In addition, Cd induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK. Inhibition of ERK and JNK, but not p38 MAPK, partially protected the cells from Cd-induced apoptosis. ERK and JNK inhibition also blocked alteration of the Bcl-2/Bax ratio, release of cytochrome c, cleavages of caspase-3 and PARP, and nuclear translocation of AIF. Taken together, these data suggest that the ERK- and JNK-mediated mitochondrial apoptotic pathways play important roles in Cd-induced neuronal apoptosis.     

Induction of apoptosis by casticin in cervical cancer cells: reactive oxygen species-dependent sustained activation of Jun N-terminal kinase

Casticin, a polymethoxyflavone from Fructus viticis used as an anti-inflammatory agent in Chinese traditional medicine, has been reported to have anti-cancer activity. The purpose of this study was to examine the apoptotic activity of casticin on human cervical cancer cells and its molecular mechanism. We revealed a novel mechanism by which casticin-induced apoptosis occurs and showed for the first time that the apoptosis induced by casticin is mediated through generation of reactive oxygen species (ROS) and sustained activation of c-Jun N-terminal kinase (JNK) in HeLa cells. Casticin markedly increased the levels of intracellular ROS and induced the expression of phosphorylated JNK and c-Jun protein. Pre-treatment with N-acetylcysteine and SP600125 effectively attenuated induction of apoptosis by casticin in HeLa cells. Moreover, casticin induced ROS production and apoptotic cell death in other cervical cancer cell lines, such as CasKi and SiHa. Importantly, casticin did not cause generation of ROS or induction of apoptosis in normal human peripheral blood mononuclear cells and embryonic kidney epithelium 293 cells. These results suggest that ROS generation and sustained JNK activation by casticin play a role in casticin-induced apoptosis and raise the possibility that treatment with casticin might be promising as a new therapy against human cervical cancer.     

Inhibition of cell proliferation and cell cycle progression by specific inhibition of basal JNK activity: evidence that mitotic Bcl-2 phosphorylation is JNK-independent

The c-Jun NH(2)-terminal kinase (JNK) subgroup of mitogen-activated protein kinases has been implicated largely in stress responses, but an increasing body of evidence has suggested that JNK also plays a role in cell proliferation and survival. We examined the effect of JNK inhibition, using either SP600125 or specific antisense oligonucleotides, on cell proliferation and cell cycle progression. SP600125 was selective for JNK in vitro and in vivo versus other kinases tested including ERK, p38, cyclin-dependent protein kinase 1 (CDK1), and CDK2. SP600125 inhibited JNK activity and KB-3 cell proliferation with the same dose dependence, suggesting that inhibition of proliferation was a direct consequence of JNK inhibition. Inhibition of proliferation by SP600125 was associated with an increase in the G(2)-M and apoptotic fractions of cells but was not associated with p53 or p21 induction. Antisense oligonucleotides to JNK2 but not JNK1 caused highly significant inhibition of cell proliferation. Wild-type mouse fibroblasts responded similarly with proliferation inhibition and apoptosis induction, whereas c-jun(-/-) fibroblasts were refractory to the effects of SP600125, suggesting that JNK signaling to c-Jun is required for cell proliferation. Studies in synchronized KB-3 cells indicated that SP600125 delayed transit time through S and G(2)-M phases. Correspondingly, JNK activity increased in late S phase and peaked in late G(2) phase. During synchronous mitotic progression, cyclin B levels increased concomitant with phosphorylation of c-Jun, H1 histone, and Bcl-2. In the presence of SP600125, mitotic progression was prolonged, and c-Jun phosphorylation was inhibited, but neither H1 nor Bcl-2 phosphorylation was inhibited. However, the CDK inhibitor roscovitine inhibited mitotic Bcl-2 phosphorylation. These results indicate that JNK, and more specifically the JNK2 isoform, plays a key role in cell proliferation and cell cycle progression. In addition, conclusive evidence is presented that a kinase other than JNK, most likely CDK1 or a CDK1-regulated kinase, is responsible for mitotic Bcl-2 phosphorylation.     

Coordination of JNK1 and JNK2 is critical for GADD45alpha induction and its mediated cell apoptosis in arsenite responses

Arsenite is a well documented environmental pathogen, whereas it has also been applied as medication to treat various neoplasmas. The pathogenic and therapeutic effects of arsenite are associated with cellular apoptotic responses. However, the molecular mechanisms of arsenite-induced apoptosis are not very well understood. Our previous study has shown that arsenite exposure is able to activate JNKs, which subsequently mediate the apoptotic outcome. The present study further revealed that the coordination of JNK1 and JNK2 was critical for the arsenite-induced expression of GADD45alpha (growth arrest and DNA damage 45alpha), which in turn mediated the cellular apoptosis. The arsenite-induced apoptosis and GADD45alpha expression were significantly impaired in mouse embryonic fibroblasts deficient in either jnk1 (JNK1-/-) or jnk2 (JNK2-/-). Knockdown of GADD45alpha by its specific small interfering RNA also dramatically reduced the apoptotic responses, and overexpression of GADD45alpha in either JNK1-/- or JNK2-/- mouse embryonic fibroblasts partially resensitized the cell death. Furthermore, it was found that the regulation of GADD45alpha by JNK1 and JNK2 was achieved through mediating the activation of c-Jun, since in the JNK1-/- and JNK2-/- cells the c-Jun activation was impaired, and overexpression of the dominant negative mutant of c-Jun (TAM67) in wild type cells could also block GADD45alpha induction as well as cellular apoptosis. Our results demonstrate that the coordination of JNK1 and JNK2 is critical for c-Jun/GADD45alpha-mediated cellular apoptosis induced by arsenite.     

E2F1-directed activation of Bcl-2 is correlated with lactoferrin-induced apoptosis in Jurkat leukemia T lymphocytes

Lactoferrin (Lf) has been shown to control the proliferation of a variety of mammalian cells. Recently, we reported that human Lf induces apoptosis via a c-Jun N-terminal kinases (JNK)-associated Bcl-2 pathway that stimulates programmed cell death. In order to gain insight into the mechanism underlying Lf-triggered apoptotic features, we attempted to determine the mechanisms whereby the Lf-induced Bcl-2 family proteins exert their pro- or anti-apoptotic effects in Jurkat leukemia T lymphocytes. Treatment of the cells with high concentrations of Lf resulted in a significant reduction in in vitro growth and cell viability. As the levels of Lf increased, greater quantities of CDK6 and hyper-phosphorylated retinoblastoma protein were produced, resulting in the induction of E2F1-dependent apoptosis. Simultaneously, PARP and caspases were efficiently cleaved during Lf-induced apoptosis. The E2F1-induced apoptotic process occurred preferentially in p53-deficient Jurkat leukemia cells. Therefore, we attempted to determine whether E2F1-regulated Bcl-2 family proteins involved in the apoptotic process were relevant to Lf-induced apoptosis. We found that Lf increased the interaction of Bcl-2 with the pro-apoptotic protein Bad, whereas the total protein levels did not change significantly. Our results, collectively, suggest that Lf exploits the control mechanism of E2F1-regulated target genes or Bcl-2 family gene networks involved in the apoptotic process in Jurkat human leukemia T lymphocytes.