Hyperglycemia-induced GLP-1R downregulation causes RPE cell apoptosis

Glucagon-like peptide-1 receptor (GLP-1R) is closely associated with the onset of diabetes and its complications. However, its roles in diabetic retinopathy are unknown. Retinal pigment epithelial (RPE) cells are a crucial component of the outer blood–retina barrier and their death is related to the progression of diabetic retinopathy. Thus, we examined the pathophysiological role of GLP-1R in RPE cell apoptosis. We found that GLP-1R expression was lower in the isolated neuroretina and RPE cells of streptozotocin-treated rats than in vehicle-treated rats. High-glucose treatment also decreased GLP-1R expression in a human RPE cell line (ARPE-19 cells). GLP-1R was silenced in ARPE-19 cells, in order to elucidate the pathophysiological roles of GLP-1R. This increased intracellular reactive oxygen species (ROS) generation and activated p53-mediated Bax promoter and endoplasmic reticulum (ER) stress signaling. We also found that GLP-1R knockdown-mediated p53 expression was regulated by ER stress. Interestingly, antioxidant treatment and peroxiredoxin 1 (Prx1) overexpression attenuated GLP-1R knockdown-induced ER stress signaling and p53 expression. Finally, to confirm that GLP-1R activation has protective effects, ARPE-19 cells were treated with exendin-4, a synthetic GLP-1R agonist. This attenuated high-glucose-induced ROS generation, ER stress signaling, and p53 expression. Collectively, these results indicated that hyperglycemia decreases GLP-1R expression in RPE cells. Such a decrease generates intracellular ROS, which increases ER stress-mediated p53 expression, and subsequently causes apoptosis by increasing Bax promoter activity. Our data suggested that regulation of GLP-1R expression is a promising approach for the treatment of diabetic retinopathy.     

Concurrent expression of heme oxygenase-1 and p53 in human retinal pigment epithelial cell line

Heme oxygenase-1 (HO-1) is a stress-responsive protein that is known to regulate cellular functions such as cell proliferation, inflammation, and apoptosis. Here, we investigated the effects of HO activity on the expression of p53 in the human retinal pigment epithelium (RPE) cell line ARPE-19. Cobalt protoporphyrin (CoPP) induced the expression of both HO-1 and p53 without significant toxicity to the cells. In addition, the blockage of HO activity with the iron chelator DFO or with HO-1 siRNA inhibited the CoPP-induced expression of p53. Similarly, zinc protoporphyrin (ZnPP), an inhibitor of HO, suppressed p53 expression in ARPE-19 cells, although ZnPP increased the level of HO-1 protein while inhibiting HO activity. Also, CoPP-induced p53 expression was not affected by the formation of reactive oxygen species (ROS). Based on these results, we conclude that HO activity is involved in the regulation of p53 expression in a ROS-independent mechanism, and also suggest that the expression of p53 in ARPE-19 cells is associated with heme metabolites such as biliverdin/bilirubin, carbon monoxide, and iron produced by the activity of HO.     

Cell death induced by Pteris semipinnata L. is associated with p53 and oxidant stress in gastric cancer cells

In this study, we demonstrated that Ent-11alpha-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F) had stronger cytotoxicity against MKN-45, a gastric cancer cell line bearing wild-type p53 than MKN-28, another gastric cancer cell line containing missense mutation in p53. The rapid increase of ROS level was involved in the mechanism of cytotoxicity. Classical features of apoptosis induced by 5F were observed in MKN-45 cells only or more significant in MKN-45 cells than MKN-28 cells. Translocation of Bax from cytosol to mitochondria, reduction of delta psi m and DNA fragmentation were induced by 5F in the p53-dependent manner. We conclude that the expression of Bax and its downstream molecules requires the presentation of a wild-type p53 in the cells treated by 5F.     

SIRT1 regulates apoptosis and Nanog expression in mouse embryonic stem cells by controlling p53 subcellular localization

Nuclear tumor suppressor p53 transactivates proapoptotic genes or antioxidant genes depending on stress severity, while cytoplasmic p53 induces mitochondrial-dependent apoptosis without gene transactivation. Although SIRT1, a p53 deacetylase, inhibits p53-mediated transactivation, how SIRT1 regulates these p53 multifunctions is unclear. Here we show that SIRT1 blocks nuclear translocation of cytoplasmic p53 in response to endogenous reactive oxygen species (ROS) and triggers mitochondrial-dependent apoptosis in mouse embryonic stem (mES) cells. ROS generated by antioxidant-free culture caused p53 translocation into mitochondria in wild-type mES cells but induced p53 translocation into the nucleus in SIRT1(-/-) mES cells. Endogenous ROS triggered apoptosis of wild-type mES through mitochondrial translocation of p53 and BAX but inhibited Nanog expression of SIRT1(-/-) mES, indicating that SIRT1 makes mES cells sensitive to ROS and inhibits p53-mediated suppression of Nanog expression. Our results suggest that endogenous ROS control is important for mES cell maintenance in culture.     

Pharmacological activation of a novel p53-dependent S-phase checkpoint involving CHK-1

We have recently shown that induction of the p53 tumour suppressor protein by the small-molecule RITA (reactivation of p53 and induction of tumour cell apoptosis; 2,5-bis(5-hydroxymethyl-2-thienyl)furan) inhibits hypoxia-inducible factor-1α and vascular endothelial growth factor expression in vivo and induces p53-dependent tumour cell apoptosis in normoxia and hypoxia. Here, we demonstrate that RITA activates the canonical ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related DNA damage response pathway. Interestingly, phosphorylation of checkpoint kinase (CHK)-1 induced in response to RITA was influenced by p53 status. We found that induction of p53, phosphorylated CHK-1 and γH2AX proteins was significantly increased in S-phase. Furthermore, we found that RITA stalled replication fork elongation, prolonged S-phase progression and induced DNA damage in p53 positive cells. Although CHK-1 knockdown did not significantly affect p53-dependent DNA damage or apoptosis induced by RITA, it did block the ability for DNA integrity to be maintained during the immediate response to RITA. These data reveal the existence of a novel p53-dependent S-phase DNA maintenance checkpoint involving CHK-1.     

Inhibition of p53 by Lentiviral Mediated sh RNA Abrogates G1 Arrest and Apoptosis in Retinal Pigmented Epithelial Cell Line

We silenced p53 gene expression in ARPE-19, a human retinal pigmented epithelial cell line using RNA interference. The effect of silencing the p53 gene in proliferating ARPE-19 cells was studied. Four short hairpin RNAs (shRNAs) targeting different regions of human p53 mRNA were delivered individually into ARPE-19 cells using lentiviral vector to produce stable cell lines. p53 mRNA and protein levels were reduced to varying extents in the four shRNA-transduced ARPE-19 cell lines. The cell line that showed greatest reduction (85-90%) of p53 expression showed decreased p21 promoter activation after DNA damage with camptothecin, etoposide and MMS. Whereas treatment of wild type ARPE-19 cells with camptothecin resulted in apoptosis, silencing p53 expression increased their survival. Cell cycle analyses indicated that irradiation resulted in a G1 arrest in ARPE-19 cells, and that the arrest was significantly reduced in p53-silenced cells. Thus, p53 plays a central role in the response of ARPE-19 cells to DNA damaging agents that act via different mechanisms. Additionally, ARPE-19 cells with reduced p53 expression behave similar to tumor cell lines with mutated or non-functional p53. The present data demonstrate the utility of lentiviral vectors to create stable isogenic cell lines with reduced expression of a specific gene, thereby permitting the study of the function of a gene, the pathways controlled by it, and the effect of therapeutics on a cell with altered genetic makeup in a pair-wise fashion.     

Trichomonas vaginalis: reactive oxygen species mediates caspase-3 dependent apoptosis of human neutrophils

There are many neutrophils in the vaginal discharge from women infected with Trichomonas vaginalis. The aim of our study was to determine whether human neutrophil apoptosis may be regulated by reactive oxygen species (ROS) in response to trichomonads infection. Incubation of human neutrophils with live trichomonads caused marked receptor shedding of CD16, decrease of mitochondrial membrane potential (MMP) and caspase-3 activation in human neutrophils. These proapoptotic effects of T. vaginalis on neutrophils were inhibited by pretreatment of neutrophils with an inhibitor of NADPH oxidase, diphenyleneiodonium chloride (DPI), suggesting an important role of intracellular ROS accumulation in T. vaginalis-triggered apoptosis. Indeed, large amounts of ROS levels were detected in neutrophils incubated with live trichomonads, and were also effectively inhibited by DPI. However, pan-caspase inhibitor z-VAD-fmk or caspase-3 inhibitor z-DEVD-fmk did not affect T. vaginalis-induced ROS generation in neutrophils. These results suggest that ROS-dependent caspase-3 activation plays an important role in apoptosis of human neutrophils induced by T. vaginalis.     

Phosphatidylcholine-specific phospholipase C, p53 and ROS in the association of apoptosis and senescence in vascular endothelial cells

Previously, we found that phosphatidylcholine-specific phospholipase C (PC-PLC) participated in apoptosis signaling of vascular endothelial cells (VECs). Here, to explore whether PC-PLC is involved in the association of apoptosis and senescence in VECs, we analyzed p53 expression and intracellular reactive oxygen species (ROS) levels in young and senescent VECs before and after inhibiting PC-PLC activity. The results showed that suppressing PC-PLC inhibited apoptosis and the elevation of p53 expression induced by apoptosis in young cells, but not in senescent cells, and that inhibiting PC-PLC depressed intracellular ROS levels both in young and senescent cells. The data suggested that PC-PLC was involved in the association of apoptosis and senescence. Its function might be closely related to the level of p53 in VECs.     

2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone,from buds of Cleistocalyx operculatus,induces apoptosis in human hepatoma SMMC-7721 cells through a reactive oxygen species-dependent mechanism

Nowadays, much effort is being devoted to detect new substances that not only significantly induce the death of tumor cells, but also have little side effect on normal cells. Our previous study showed that 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC) exhibited significant cytotoxic potential with an IC₅₀ value of 32.3 ± 1.13 μM against SMMC-7721 cells and could induce SMMC-7721 cells apoptosis. In the present study, we found that DMC was almost nontoxic to human normal liver L-02 and human normal fetal lung fibroblast HFL-1 cells as their IC₅₀ values (111.0 ± 4.57 and 152.0 ± 4.83 µM for L-02 and HFL-1 cells, respectively) were much higher. To further explore the apoptotic mechanism of DMC, we investigated the role of the reactive oxygen species (ROS) in the apoptosis induced by DMC in SMMC-7721 cells. Our results suggested that the cytotoxicity and the generation of intracellular ROS were inhibited by N-acetylcysteine (NAC). Reversal of apoptosis in NAC pretreated cells indicated the involvement of ROS in DMC-induced apoptosis. The loss of mitochondrial membrane potential (ΔΨm) induced by DMC was significantly blocked by NAC. NAC also prevented the decrease of Caspase-3 and -9 activities, the increase of Bcl-2 protein expression and the decrease of p53 and PUMA protein expressions. Together, these results indicated that ROS played a key role in the apoptosis induced by DMC in human hepatoma SMMC-7721 cells.     

TNF-alpha/cycloheximide-induced apoptosis in intestinal epithelial cells requires Rac1-regulated reactive oxygen species

Previously we have shown that both Rac1 and c-Jun NH(2)-terminal kinase (JNK1/2) are key proapoptotic molecules in tumor necrosis factor (TNF)-alpha/cycloheximide (CHX)-induced apoptosis in intestinal epithelial cells, whereas the role of reactive oxygen species (ROS) in apoptosis is unclear. The present studies tested the hypothesis that Rac1-mediated ROS production is involved in TNF-alpha-induced apoptosis. In this study, we showed that TNF-alpha/CHX-induced ROS production and hydrogen peroxide (H(2)O(2))-induced oxidative stress increased apoptosis. Inhibition of Rac1 by a specific inhibitor NSC23766 prevented TNF-alpha-induced ROS production. The antioxidant, N-acetylcysteine (NAC), or rotenone (Rot), the mitochondrial electron transport chain inhibitor, attenuated mitochondrial ROS production and apoptosis. Rot also prevented JNK1/2 activation during apoptosis. Inhibition of Rac1 by expression of dominant negative Rac1 decreased TNF-alpha-induced mitochondrial ROS production. Moreover, TNF-alpha-induced cytosolic ROS production was inhibited by Rac1 inhibition, diphenyleneiodonium (DPI, an inhibitor of NADPH oxidase), and NAC. In addition, DPI inhibited TNF-alpha-induced apoptosis as judged by morphological changes, DNA fragmentation, and JNK1/2 activation. Mitochondrial membrane potential change is Rac1 or cytosolic ROS dependent. Lastly, all ROS inhibitors inhibited caspase-3 activity. Thus these results indicate that TNF-alpha-induced apoptosis requires Rac1-dependent ROS production in intestinal epithelial cells.     

Restoration of p53/miR-34a regulatory axis decreases survival advantage and ensures Bax-dependent apoptosis of non-small cell lung carcinoma cells

Tumor-suppressive miR-34a, a direct target of p53, has been shown to target several molecules of cell survival pathways. Here, we show that capsaicin-induced oxidative DNA damage culminates in p53 activation to up-regulate expression of miR-34a in non-small cell lung carcinoma (NSCLC) cells. Functional analyses further indicate that restoration of miR-34a inhibits B cell lymphoma-2 (Bcl-2) protein expression to withdraw the survival advantage of these resistant NSCLC cells. In such a proapoptotic cellular milieu, where drug resistance proteins are also down-regulated, p53-transactivated Bcl-2 associated X protein (Bax) induces apoptosis via the mitochondrial death cascade. Our results suggest that p53/miR-34a regulatory axis might be critical in sensitizing drug-resistant NSCLC cells.     

The Ganglioside GM3 Is Associated with Cisplatin-Induced Apoptosis in Human Colon Cancer Cells

Cisplatin (cis-diamminedichloroplatinum, CDDP) is a well-known chemotherapeutic agent for the treatment of several cancers. However, the precise mechanism underlying apoptosis of cancer cells induced by CDDP remains unclear. In this study, we show mechanistically that CDDP induces GM3-mediated apoptosis of HCT116 cells by inhibiting cell proliferation, and increasing DNA fragmentation and mitochondria-dependent apoptosis signals. CDDP induced apoptosis within cells through the generation of reactive oxygen species (ROS), regulated the ROS-mediated expression of Bax, Bcl-2, and p53, and induced the degradation of the poly (ADP-ribosyl) polymerase (PARP). We also checked expression levels of different gangliosides in HCT116 cells in the presence or absence of CDDP. Interestingly, among the gangliosides, CDDP augmented the expression of only GM3 synthase and its product GM3. Reduction of the GM3 synthase level through ectopic expression of GM3 small interfering RNA (siRNA) rescued HCT116 cells from CDDP-induced apoptosis. This was evidenced by inhibition of apoptotic signals by reducing ROS production through the regulation of 12-lipoxigenase activity. Furthermore, the apoptotic sensitivity to CDDP was remarkably increased in GM3 synthase-transfected HCT116 cells compared to that in controls. In addition, GM3 synthase-transfected cells treated with CDDP exhibited an increased accumulation of intracellular ROS. These results suggest the CDDP-induced oxidative apoptosis of HCT116 cells is mediated by GM3.     

mtCLIC/CLIC4, an organellular chloride channel protein, is increased by DNA damage and participates in the apoptotic response to p53

mtCLIC/CLIC4 (referred to here as mtCLIC) is a p53- and tumor necrosis factor alpha-regulated cytoplasmic and mitochondrial protein that belongs to the CLIC family of intracellular chloride channels. mtCLIC associates with the inner mitochondrial membrane. Dual regulation of mtCLIC by two stress response pathways suggested that this chloride channel protein might contribute to the cellular response to cytotoxic stimuli. DNA damage or overexpression of p53 upregulates mtCLIC and induces apoptosis. Overexpression of mtCLIC by transient transfection reduces mitochondrial membrane potential, releases cytochrome c into the cytoplasm, activates caspases, and induces apoptosis. mtCLIC is additive with Bax in inducing apoptosis without a physical association of the two proteins. Antisense mtCLIC prevents the increase in mtCLIC levels and reduces apoptosis induced by p53 but not apoptosis induced by Bax, suggesting that the two proapoptotic proteins function through independent pathways. Our studies indicate that mtCLIC, like Bax, Noxa, p53AIP1, and PUMA, participates in a stress-induced death pathway converging on mitochondria and should be considered a target for cancer therapy through genetic or pharmacologic approaches.