Zinc Supplementation Enhances the Pro-Death Function of UPR in Lymphoma Cells Exposed to Radiation

We have previously shown that Zinc supplementation triggered ER stress/UPR in cancer cells undergoing treatment by genotoxic agents, reactivated wtp53 in cancer cells harboring mutant p53 (mutp53) and potentiated the activity of wtp53 in those carrying wtp53. In this study, we used Zinc chloride alone or in combination with 2 Gy radiation to treat Primary Effusion Lymphoma (PEL) cells, an aggressive B-cell lymphoma associated with KSHV that harbors wt or partially functioning p53. We found that Zinc triggered a mild ER stress/UPR in these lymphoma cells and activated ERK1/2, molecule known to sustain cell survival in the course of UPR activation. In combination with radiations, Zinc triggered a stronger p53 activation that counteracted its mediated ERK1/2 phosphorylation, further upregulating the UPR molecule CHOP and promoting cell death. These data suggest that Zinc supplementation could be a promising strategy to reduce the doses of radiation and possibly of other DNA-damaging agents to obtain an efficient capacity to induce lymphoma cell death.

Loss of p19Arf Promotes Fibroblast Survival During Leucine Deprivation

Fibroblasts are quiescent and tumor suppressive in nature but become activated in wound healing and cancer. The response of fibroblasts to cellular stress has not been extensively investigated however the p53 tumor suppressor has been shown to be activated in fibroblasts during nutrient deprivation. Since the p19 Alternative reading frame (p19Arf) tumor suppressor is a key regulator of p53 activation during oncogenic stress, we investigated the role of p19Arf in fibroblasts during nutrient deprivation. Here we show that prolonged leucine deprivation resulted in increased expression and nuclear localization of p19Arf, triggering apoptosis in primary murine adult lung fibroblasts (ALFs). In contrast, the absence of p19Arf during long-term leucine deprivation resulted in increased ALF proliferation, migration and survival through upregulation of the Integrated Stress Response pathway and increased autophagic flux. Our data implicates a new role for p19Arf in response to nutrient deprivation.

Potential Effects of Metformin on the Vitality, Invasion, and Migration of Human Vascular Smooth Muscle Cells via Downregulating lncRNA-ATB

Objective. To elucidate the role of metformin in influencing VSMCs via the involvement of lncRNA-ATB. Methods. qRT-PCR was conducted to detect serum levels of lncRNA-ATB and p53 in CHD patients ( n = 50 ) and healthy subjects ( n = 50 ). Correlation in serum levels of lncRNA-ATB and p53 in CHD patients was assessed by Pearson correlation test. ROC curves were depicted for analyzing the predictive potential of lncRNA-ATB in the occurrence of CHD. After metformin induction in VSMCs overexpressing lncRNA-ATB, relative levels of lncRNA-ATB and p53 were detected. Meanwhile, proliferative, migratory, and invasive abilities in VSMCs were, respectively, examined by CCK-8 and transwell assay. The interaction between lncRNA-ATB and p53 was tested by RIP. In addition, the coregulation of lncRNA-ATB and p53 in cell functions of VSMCs was finally determined. Results. Increased serum level of lncRNA-ATB and decreased p53 level were detected in CHD patients than those of healthy subjects. LncRNA-ATB could interact with p53 and negatively regulate its level. In addition, lncRNA-ATB could serve as a potential biomarker for predicting the occurrence of CHD. The overexpression of lncRNA-ATB triggered viability, migratory, and invasive abilities in VSMCs, and the above trends were abolished by metformin induction. The overexpression of p53 partially abolished the promotive effects of lncRNA-ATB on proliferative, migratory, and invasive abilities in VSMCs. Conclusions. Metformin induction inhibits proliferative, migratory, and invasive abilities in VSMCs by downregulating lncRNA-ATB, which may be related to p53 activation.

Fasting Induces a Highly Resilient Deep Quiescent State in Muscle Stem Cells via Ketone Body Signaling

Short-term fasting is beneficial for the regeneration of multiple tissue types. However, the effects of fasting on muscle regeneration are largely unknown. Here we report that fasting slows muscle repair both immediately after the conclusion of fasting as well as after multiple days of refeeding. We show that ketosis, either endogenously produced during fasting or a ketogenic diet, or exogenously administered, promotes a deep quiescent state in muscle stem cells(MuSCs). Although deep quiescent MuSCs are less poised to activate, slowing muscle regeneration, they have markedly improved survival when facing sources of cellular stress. Further, we show that ketone bodies, specifically b hydroxybutyrate, directly promote MuSC deep quiescence via a non-metabolic mechanism. We show that b-hydroxybutyrate functions as an HDAC inhibitor within MuSCs leading to acetylation and activation of an HDAC1 target protein p53. Finally, we demonstrate that p53 activation contributes to the deep quiescence and enhanced resilience observed during fasting.

p57 Suppresses the Pluripotency and Proliferation of Mouse Embryonic Stem Cells by Positively Regulating p53 Activation

Embryonic stem cells (ESCs) are pluripotent stem cells that have indefinite self-renewal capacities under appropriate culture conditions in vitro. The pluripotency maintenance and proliferation of these cells are delicately governed by the concert effect of a complex transcriptional regulatory network. Herein, we discovered that p57Kip2 (p57), a cyclin-dependent kinase inhibitor canonically inhibiting cell proliferation, played a role in suppressing the pluripotency state of mouse ESCs (mESCs). p57 knockdown significantly stimulated the expressions of core pluripotency factors NANOG, OCT4, and SOX2, while p57 overexpression inhibited the expressions of these factors in mESCs. In addition, consistent with its function in somatic cells, p57 suppressed mESC proliferation. Further analysis showed that p57 could interact with and contribute to the activation of p53 in mESCs. In conclusion, the present study showed that p57 could antagonize the pluripotency state and the proliferation process of mESCs. This finding uncovers a novel function of p57 and provides new evidence for elucidating the complex regulatory of network of mESC fate.

circ-Sirt1 Decelerates Senescence by Inhibiting p53 Activation in Vascular Smooth Muscle Cells, Ameliorating Neointima Formation

Vascular smooth muscle cell (VSMC) senescence is a major driver of neointimal formation. We have demonstrated that circ-Sirt1 derived from the SIRT1 gene suppressed VSMC inflammation and neointimal formation. However, the effect of circ-Sirt1 inhibiting inflammation on VSMC senescence during neointimal hyperplasia remains to be elucidated. Here, we showed that circ-Sirt1 was highly expressed in young and healthy arteries, which was decreased in aged arteries and neointima of humans and mice. Overexpression of circ-Sirt1 delayed Ang II-induced VSMC senescence in vitro and ameliorated neointimal hyperplasia in vivo. Mechanically, circ-Sirt1 inhibited p53 activity at the levels of transcription and post-translation modulation. In detail, circ-Sirt1, on the one hand, interacted with and held p53 to block its nuclear translocation, and on the other hand, promoted SIRT1-mediated p53 deacetylation and inactivation. In conclusion, our data suggest that circ-Sirt1 is a novel p53 repressor in response senescence-inducing stimuli, and targeting circ-Sirt1 may be a promising approach to ameliorating aging-related vascular disease.

Synthesis and Biological Evaluation of S-, O- and Se-Containing Dispirooxindoles

A series of novel S-, O- and Se-containing dispirooxindole derivatives has been synthesized using 1,3-dipolar cycloaddition reaction of azomethine ylide generated from isatines and sarcosine at the double C=C bond of 5-indolidene-2-chalcogen-imidazolones (chalcogen was oxygen, sulfur or selenium). The cytotoxicity of these dispiro derivatives was evaluated in vitro using different tumor cell lines. Several molecules have demonstrated a considerable cytotoxicity against the panel and showed good selectivity towards colorectal carcinoma HCT116 p53+/+ over HCT116 p53−/− cells. In particular, good results have been obtained for LNCaP prostate cell line. The performed in silico study has revealed MDM2/p53 interaction as one of the possible targets for the synthesized molecules. However, in contrast to selectivity revealed during the cell-based evaluation and the results obtained in computational study, no significant p53 activation using a reporter construction in p53wt A549 cell line was observed in a relevant concentration range.

Depletion of the miR-34a “sponge” MALAT1 in aging skeletal muscle: Implications for age-related muscle loss

We have recently shown that increased levels of reactive oxygen species (ROS) in aging skeletal muscle are associated with increased expression of the senescence-associated microRNA miR-34a-5p (miR-34a). The histone deacetylase Sirt1 is a validated target of miR-34a, and miR-34a expression is induced by the tumor suppressor p53 which is itself stimulated by ROS. Long noncoding RNAs (lncRNAs) are known to function as “sponges” for microRNAs, but the role of such competing endogenous RNAs (ceRNA) in muscle aging is not well understood. We therefore examined in skeletal muscles of young (4-6 mos) and aged (22-24) male and female mice the expression of several lncRNAs that are predicted to bind miR-34a-5p in silico and whose predicted binding has been validated experimentally. Results indicate a significant decrease in lncRNA MALAT1 expression with aging. MALAT1 is known to be highly expressed during the later stages of myoblast differentiation and myotube maturation. We therefore treated C2C12 cells at 48 hrs with hydrogen peroxide (10 uM) and examined changes in MALAT1 expression. MALAT1 was significantly decreased with H2O2 treatment, whereas miR-34a is increased in C2C12 cells after hydrogen peroxide exposure. Age-related muscle atrophy mediated by ROS may therefore result in part from related mechanisms involving miR-34a activity: an increase in miR-34a targeting Sirt1 resulting from p53 activation and an increase in miR-34a bioavailability resulting from a decline in miR-34a “sponging” due to ceRNA MALAT1 depletion. These findings suggest that therapeutic interventions increasing MALAT1 expression in muscle may potentially enhance the preservation of muscle mass with aging.

Hypoxia-Induced ZWINT Mediates Pancreatic Cancer Proliferation by Interacting With p53/p21

p53/p21 signaling plays a vital role in pancreatic cancer (PC) progression. ZWINT was shown to function as an oncoprotein in the progression of multiple cancers. However, the involvement of ZWINT and p53 activation in the progression of PC remains poorly understood. Bioinformatics and tissue array chip analyses were performed to evaluate ZWINT expression in pancreatic cancer. ZWINT mRNA and protein expression were evaluated in normoxia and hypoxia. CHIP was used to evaluate HIF1α interaction with the ZWINT promoter. CCK8, colony formation, EDU, and cell cycle analysis were used to examine PC cell proliferation. Immunoprecipitation and immunofluorescence were used to examine the interaction of ZWINT, MDM2, and p53. p53 activity was evaluated by q-PCR and luciferase assay. Protein degradation and ubiquitination assays were used to analyze the role of ZWINT in p53 ubiquitination. ZWINT was overexpressed in pancreatic cancer and induced in hypoxia. ZWINT promoted pancreatic cancer growth and cell cycle progression. Bioinformatic analysis revealed that ZWINT may regulate the p53 signal pathway. ZWINT interacts with p53 and promotes its ubiquitination and degradation. ZWINT promoted proliferation via p53/p21. Immunohistochemistry of clinical specimens revealed that that ZWINT expression was significantly negatively correlated with p53/p21. Our data showed that hypoxia regulates the expression of ZWINT, which activated p53/p21 signaling pathway to promote PC growth.

Bcl-xL Reduces Chinese Giant Salamander Iridovirus-Induced Mitochondrial Apoptosis by Interacting with Bak and Inhibiting the p53 Pathway

Chinese giant salamander iridovirus (GSIV) infection could lead to mitochondrial apoptosis in this animal, a process that involves B-cell lymphoma-2 (BCL-2) superfamily molecules. The mRNA expression level of Bcl-xL, a crucial antiapoptotic molecule in the BCL-2 family, was reduced in early infection and increased in late infection. However, the molecular mechanism remains unknown. In this study, the function and regulatory mechanisms of Chinese giant salamander (Andrias davidianus) Bcl-xL (AdBcl-xL) during GSIV infection were investigated. Western blotting assays revealed that the level of Bcl-xL protein was downregulated markedly as the infection progressed. Plasmids expressing AdBcl-xL or AdBcl-xL short interfering RNAs were separately constructed and transfected into Chinese giant salamander muscle cells. Confocal microscopy showed that overexpressed AdBcl-xL was translocated to the mitochondria after infection with GSIV. Additionally, flow cytometry analysis demonstrated that apoptotic progress was reduced in both AdBcl-xL-overexpressing cells compared with those in the control, while apoptotic progress was enhanced in cells silenced for AdBcl-xL. A lower number of copies of virus major capsid protein genes and a reduced protein synthesis were confirmed in AdBcl-xL-overexpressing cells. Moreover, AdBcl-xL could bind directly to the proapoptotic molecule AdBak with or without GSIV infection. In addition, the p53 level was inhibited and the mRNA expression levels of crucial regulatory molecules in the p53 pathway were regulated in AdBcl-xL-overexpressing cells during GSIV infection. These results suggest that AdBcl-xL plays negative roles in GSIV-induced mitochondrial apoptosis and virus replication by binding to AdBak and inhibiting p53 activation.