Cellular carbonyl stress enhances the expression of plasminogen activator inhibitor-1 in rat white adipocytes via reactive oxygen species-dependent pathway

Carbonyl stress is one of the important mechanisms of tissue damage in vascular complications of diabetes. In the present study, we observed that the plasminogen activator inhibitor-1 (PAI-1) levels in serum and its gene expression in adipose tissue were up-regulated in aged OLETF rats, model animals of obese type 2 diabetes. To study the mechanism of PAI-1 up-regulation, we examined the effect of advanced glycation end products (AGEs) and the product of lipid peroxidation (4-hydroxy-2-nonenal (HNE)), both of which are endogenously generated under carbonyl stress. Stimulation of primary white adipocytes by either AGE or HNE resulted in the elevation of PAI-1 in culture medium and at mRNA levels. The up-regulation of PAI-1 was also observed by incubating the cells in high glucose medium (30 mm, 48 h). The stimulatory effects by AGE or high glucose were inhibited by antioxidant, pyrrolidine dithiocarbamate, and reactive oxygen scavenger, probucol, suggesting a pivotal role of oxidative stress in white adipocytes. We also found that the effect by HNE was inhibited by antioxidant, N-acetylcysteine and that a specific inhibitor of glutathione biosynthesis, l-buthionine-S,R-sulfoximine, augmented the effect of subthreshold effect of HNE. Bioimaging of reactive oxygen species (ROS) by a fluorescent indicator, 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, revealed ROS production in white adipocytes treated with AGE or HNE. These results suggest that cellular carbonyl stress induced by AGEs or HNE may stimulate PAI-1 synthesis in and release from adipose tissues through ROS formation.     

Mequindox induced cellular DNA damage via generation of reactive oxygen species

Mequindox, a quinoxaline-N-dioxide derivative that possesses antibacterial properties, has been widely used as a feed additive in the stockbreeding industry in China. While recent pharmacological studies have uncovered potential hazardous effects of mequindox, exactly how mequindox induces pathological changes and the cellular responses associated with its consumption remain largely unexplored. In this study, we investigated the cellular responses associated with mequindox treatment. We report here that mequindox inhibits cell proliferation by arresting cells at the G2/M phase of the cell cycle. Interestingly, this mequindox-associated deleterious effect on cell proliferation was observed in human, pig as well as chicken cells, suggesting that mequindox acts on evolutionarily conserved target(s). To further understand the mequindox-host interaction and the mechanism underlying mequindox-induced cell cycle arrest, we measured the cellular content of DNA damage, which is known to perturb cell proliferation and compromise cell survival. Accordingly, using γ-H2AX as a surrogate marker for DNA damage, we found that mequindox treatment induced cellular DNA damage, which paralleled the chemical-induced elevation of reactive oxygen species (ROS) levels. Importantly, expression of the antioxidant enzyme catalase partially alleviated these mequindox-associated effects. Taken together, our results suggest that mequindox cytotoxicity is attributable, in part, to its role as a potent inducer of DNA damage via ROS.     

Simvastatin rises reactive oxygen species levels and induces senescence in human melanoma cells by activation of p53/p21 pathway

Recent studies demonstrated that simvastatin has antitumor properties in several types of cancer cells, mainly by inducing apoptosis and inhibiting growth. The arrest of proliferation is a feature of cellular senescence; however, the occurrence of senescence in melanoma cells upon simvastatin treatment has not been investigated until now. Our results demonstrated that exposure of human metastatic melanoma cells (WM9) to simvastatin induces a senescent phenotype, characterized by G1 arrest, positive staining for senescence-associated β-galactosidase assay, and morphological changes. Also, the main pathways leading to cell senescence were examined in simvastatin-treated human melanoma cells, and the expression levels of phospho-p53 and p21 were upregulated by simvastatin, suggesting that cell cycle regulators and DNA damage pathways are involved in the onset of senescence. Since simvastatin can act as a pro-oxidant agent, and oxidative stress may be related to senescence, we measured the intracellular ROS levels in WM9 cells upon simvastatin treatment. Interestingly, we found an increased amount of intracellular ROS in these cells, which was accompanied by elevated expression of catalase and peroxiredoxin-1. Collectively, our results demonstrated that simvastatin can induce senescence in human melanoma cells by activation of p53/p21 pathway, and that oxidative stress may be related to this process.     

Regulation of cellular senescence by p53.

Many normal cells respond to potentially oncogenic stimuli by undergoing cellular senescence, a state of irreversibly arrested proliferation and altered differentiated function. Cellular senescence very likely evolved to suppress tumorigenesis. In support of this idea, it is regulated by several tumor suppressor genes. At the heart of this regulation is p53. p53 is essential for the senescence response to short telomeres, DNA damage, oncogenes and supraphysiological mitogenic signals, and overexpression of certain tumor suppressor genes. Despite the well-documented central role for p53 in the senescence response, many questions remain regarding how p53 senses senescence-inducing stimuli and how it elicits the senescent phenotype.     

Sarsasapogenin induces apoptosis via the reactive oxygen species-mediated mitochondrial pathway and ER stress pathway in HeLa cells

Sarsasapogenin is a sapogenin from the Chinese medical herb Anemarrhena asphodeloides Bunge. In the present study, we revealed that sarsasapogenin exhibited antitumor activity by inducing apoptosis in vitro as determined by Hoechst staining analysis and double staining of Annexin V-FITC/PI. In addition, cell cycle arrest in G2/M phase was observed in sarsasapogenin-treated HeLa cells. Moreover, the results revealed that perturbations in the mitochondrial membrane were associated with the deregulation of the Bax/Bcl-2 ratio which led to the upregulation of cytochrome c, followed by activation of caspases. Meanwhile, treatment of sarsasapogenin also activated Unfolded Protein Response (UPR) signaling pathways and these changes were accompanied by increased expression of CHOP. Salubrinal (Sal), a selective inhibitor of endoplasmic reticulum (ER) stress, partially abrogated the sarsasapogenin-related cell death. Furthermore, sarsasapogenin provoked the generation of reactive oxygen species, while the antioxidant N-acetyl cysteine (NAC) effectively blocked the activation of ER stress and apoptosis, suggesting that sarsasapogenin-induced reactive oxygen species is an early event that triggers ER stress mitochondrial apoptotic pathways. Taken together, the results demonstrate that sarsasapogenin exerts its antitumor activity through both reactive oxygen species (ROS)-mediate mitochondrial dysfunction and ER stress cell death.     

Reactive oxygen species as mediators of cellular senescence

Aging has often been viewed as a random process arising from the accumulation of both genetic and epigenetic changes. Increasingly, the notion that aging is a stochastic process is being supplanted by the concept that maximum lifespan of an organism is tightly regulated. This knowledge has led to a growing overlap between classical signal transduction paradigms and the biology of aging. We review certain specific examples where these seemingly disparate disciplines intersect. In particular, we review the concept that intracellular reactive oxygen species function as signalling molecules and that oxidants play a central role as mediators of cellular senescence.     

CHIP-dependent p53 regulation occurs specifically during cellular senescence

p53 regulates several biological processes, including senescence. Its protein stability is regulated by ubiquitination and proteasomal degradation, mainly mediated by Mdm2. However, other E3 ligases have been identified, such as the chaperone-associated ligase CHIP, although their precise function regarding p53 degradation remains elusive. Interestingly, CHIP deficiency has been recently shown to result in accelerated aging in mice, although the molecular basis of this phenotype was not completely understood. In this study, we explore the role of CHIP in regulating p53 in senescence. We demonstrate that in senescent human fibroblasts, CHIP is up-regulated concomitant with a significant down-regulation of p53. Moreover, CHIP partially translocates to the nucleus and acquires higher ubiquitination levels in senescent cells. Notably, CHIP overexpression in young cells, to levels similar to those recorded during senescence, leads to p53 degradation to below its basal levels. In addition, whereas CHIP silencing has no effect on p53 stability in young cells, a considerable p53 accumulation occurs in their senescent counterparts. Finally, we have observed an attenuation of the CHIP-associated molecular folding-refolding machinery during senescence, and supportively, inhibition of Hsp90 activity leads to rapid p53 degradation only in senescent cells. Taking these results together, we conclude that CHIP-dependent p53 regulation occurs specifically during senescence.     

Synthesis of carbazoloquinone derivatives and their antileukemic activity via modulating cellular reactive oxygen species

Carbazoloquinone alkaloids are of great interest as privileged structures for anticancer drug molecules. The purpose of this study was to investigate the structure-activity relationships of carbazoloquinone derivatives as anticancer agents. A series of carbazoloquinones including murrayaquinone A, koeniginequinones A and B, and related analogues were therefore prepared. Palladium-catalyzed intramolecular cyclization reaction mechanism was well elucidated by DFT calculations. Treatment of the synthesized derivatives showed cytotoxicity on human leukemia HL-60 cells in a dose-dependent fashion. In addition, murrayaquinone A and β-brazanquinone elevated cellular levels of reactive oxygen species (ROS), thereby triggering apoptosis. Our findings emphasize the excellent potential of carbazoloquinone derivatives as ROS-inducing anticancer agents.     

The p53/miRNAs/Ccna2 pathway serves as a novel regulator of cellular senescence: Complement of the canonical p53/p21 pathway

Aging is a multifactorial process characterized by the progressive deterioration of physiological functions. Among the multiple molecular mechanisms, microRNAs (miRNAs) have increasingly been implicated in the regulation of Aging process. However, the contribution of miRNAs to physiological Aging and the underlying mechanisms remain elusive. We herein performed high‐throughput analysis using miRNA and mRNA microarray in the physiological Aging mouse, attempted to deepen into the understanding of the effects of miRNAs on Aging process at the “network” level. The data showed that various p53 responsive miRNAs, including miR‐124, miR‐34a and miR‐29a/b/c, were up‐regulated in Aging mouse compared with that in Young mouse. Further investigation unraveled that similar as miR‐34a and miR‐29, miR‐124 significantly promoted cellular senescence. As expected, mRNA microarray and gene co‐expression network analysis unveiled that the most down‐regulated mRNAs were enriched in the regulatory pathways of cell proliferation. Fascinatingly, among these down‐regulated mRNAs, Ccna2 stood out as a common target of several p53 responsive miRNAs (miR‐124 and miR‐29), which functioned as the antagonist of p21 in cell cycle regulation. Silencing of Ccna2 remarkably triggered the cellular senescence, while Ccna2 overexpression delayed cellular senescence and significantly reversed the senescence‐induction effect of miR‐124 and miR‐29. Moreover, these p53 responsive miRNAs were significantly up‐regulated during the senescence process of p21‐deficient cells; overexpression of p53 responsive miRNAs or knockdown of Ccna2 evidently accelerated the cellular senescence in the absence of p21. Taken together, our data suggested that the p53/miRNAs/Ccna2 pathway might serve as a novel senescence modulator independent of p53/p21 pathway.     

Rhein induces apoptosis in HL-60 cells via reactive oxygen species-independent mitochondrial death pathway

Rhein is an anthraquinone compound enriched in the rhizome of rhubarb, a traditional Chinese medicine herb showing anti-tumor promotion function. In this study, we first reported that rhein could induce apoptosis in human promyelocytic leukemia cells (HL-60), characterized by caspase activation, poly(ADP)ribose polymerase (PARP) cleavage, and DNA fragmentation. The efficacious induction of apoptosis was observed at 100 microM for 6h. Mechanistic analysis demonstrated that rhein induced the loss of mitochondrial membrane potential (DeltaPsi(m)), cytochrome c release from mitochondrion to cytosol, and cleavage of Bid protein. Rhein also induced generation of reactive oxygen species (ROS) and the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 kinase. However, these actions seem not to be associated with the apoptosis induction because antioxidants including N-acetyl cysteine (NAC), Tiron, and catalase did not block rhein-induced apoptosis, although they could block the generation of ROS and the phosphorylation of JNK and p38 kinase. Our data demonstrate that rhein induces apoptosis in HL-60 cells via a ROS-independent mitochondrial death pathway.     

CUL4B impedes stress-induced cellular senescence by dampening a p53-reactive oxygen species positive feedback loop

Tumor suppressor p53 is known to regulate the level of intracellular reactive oxygen species (ROS). It can either alleviate oxidative stress under physiological and mildly stressed conditions or exacerbate oxidative stress under highly stressed conditions. We here report that a p53–ROS positive feedback loop drives a senescence program in normal human fibroblasts (NHFs) and this senescence-driving loop is negatively regulated by CUL4B. CUL4B, which can assemble various ubiquitin E3 ligases, was found to be downregulated in stress-induced senescent cells, but not in replicative senescent cells. We observed that p53-dependent ROS production was significantly augmented and stress-induced senescence was greatly enhanced when CUL4B was absent or depleted. Ectopic expression of CUL4B, on the other hand, blunted p53 activation, reduced ROS production, and attenuated cellular senescence in cells treated with H₂O₂. CUL4B was shown to promote p53 ubiquitination and proteosomal degradation in NHFs exposed to oxidative stress, thus dampening the p53-dependent cellular senescence. Together, our results established a critical role of CUL4B in negatively regulating the p53–ROS positive feedback loop that drives cellular senescence.     

Oncogenic ras and p53 cooperate to induce cellular senescence

Oncogenic activation of the mitogen-activated protein (MAP) kinase cascade in murine fibroblasts initiates a senescence-like cell cycle arrest that depends on the ARF/p53 tumor suppressor pathway. To investigate whether p53 is sufficient to induce senescence, we introduced a conditional murine p53 allele (p53(val135)) into p53-null mouse embryonic fibroblasts and examined cell proliferation and senescence in cells expressing p53, oncogenic Ras, or both gene products. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras or activated mek1 with p53 enhanced both p53 levels and activity relative to that observed for p53 alone and produced an irreversible cell cycle arrest that displayed features of cellular senescence. p19(ARF) was required for this effect, since p53(-/-) ARF(-/-) double-null cells were unable to undergo senescence following coexpression of oncogenic Ras and p53. Although the levels of exogenous p53 achieved in ARF-null cells were relatively low, the stabilizing effects of p19(ARF) on p53 could not explain the cooperation between oncogenic Ras and p53 in promoting senescence. Hence, enforced p53 expression without oncogenic ras in p53(-/-) mdm2(-/-) double-null cells produced extremely high p53 levels but did not induce senescence. Taken together, our results indicate that oncogenic activation of the MAP kinase pathway in murine fibroblasts converts p53 into a senescence inducer through both quantitative and qualitative mechanisms.     

Replicative senescence induced by Romo1-derived reactive oxygen species

Persistent accumulation of DNA damage induced by reactive oxygen species (ROS) is proposed to be a major contributor toward the aging process. Furthermore, an increase in age-associated ROS is strongly correlated with aging in various species, including humans. Here we showed that the enforced expression of the ROS modulator 1 (Romo1) triggered premature senescence by ROS production, and this also contributed toward induction of DNA damage. Romo1-derived ROS was found to originate in the mitochondrial electron transport chain. Romo1 expression gradually increased in proportion to population doublings of IMR-90 human fibroblasts. An increase in ROS production in these cells with high population doubling was blocked by the Romo1 knockdown using Romo1 small interfering RNA. Romo1 knockdown also inhibited the progression of replicative senescence. Based on these results, we suggest that age-related ROS levels increase, and this contributes to replicative senescence, which is directly associated with Romo1 expression.