Levels Of Acetylated P53 Research Articles

Rice Germ Attenuates Chronic Unpredictable Mild Stress-Induced Muscle Atrophy.

Chronic stress leads to hypothalamic-pituitary-adrenal axis dysfunction, increasing cortisol levels. Glucocorticoids (GCs) promote muscle degradation and inhibit muscle synthesis, eventually causing muscle atrophy. In this study, we aimed to evaluate whether rice germ supplemented with 30% γ-aminobutyric acid (RG) attenuates muscle atrophy in an animal model of chronic unpredictable mild stress (CUMS). We observed that CUMS raised the adrenal gland weight and serum adrenocorticotropic hormone (ACTH) and cortisol levels, and these effects were reversed by RG. CUMS also enhanced the expression of the GC receptor (GR) and GC-GR binding in the gastrocnemius muscle, which were attenuated by RG. The expression levels of muscle degradation-related signaling pathways, such as the Klf15, Redd-1, FoxO3a, Atrogin-1, and MuRF1 pathways, were enhanced by CUMS and attenuated by RG. Muscle synthesis-related signaling pathways, such as the IGF-1/AKT/mTOR/s6k/4E-BP1 pathway, were reduced by CUMS and enhanced by RG. Moreover, CUMS raised oxidative stress by enhancing the levels of iNOS and acetylated p53, which are involved in cell cycle arrest, whereas RG attenuated both iNOS and acetylated p53 levels. Cell proliferation in the gastrocnemius muscle was reduced by CUMS and enhanced by RG. The muscle weight, muscle fiber cross-sectional area, and grip strength were reduced by CUMS and enhanced by RG. Therefore, RG attenuated ACTH levels and cortisol-related muscle atrophy in CUMS animals.

Resveratrol Relieves Hyperoxia-Induced Brain Injury in Neonatal Rats by Activating Sirt1.

Neonatal rats with hyperoxia-induced brain injury were treated with resveratrol to investigate its protective effects through analyzing changes in reactive oxygen species (ROS), Sirt1, p53, and acetylated p53 levels. Neonatal rats were randomly divided into hyperoxia and resveratrol intervened groups. Rats in both groups were placed in a hyperoxia chamber for 7 days to induce hyperoxia-induced brain injury. The rats in the resveratrol intervened group were administered resveratrol 60 μg/g body weight daily, whereas those in the hyperoxia group were administered a dimethyl sulfoxide-based solvent. Brain tissues were collected, and hematoxylin and eosin (H&E) and TUNEL staining, ROS measurements, real time-polymerase chain reaction, and western blotting were performed. H&E and TUNEL staining revealed increased cell damage and apoptosis in brain tissue from hyperoxia-exposed animals compared with the findings in animals in the resveratrol intervened group. Real time-polymerase chain reaction and western blotting identified increases in Sirt1 expression and decreases in p53 expression in the resveratrol intervened group. In addition, acetylated p53 protein expression was lower in the intervened group than in the hyperoxia group. Resveratrol alleviated brain apoptosis induced by hyperoxia in neonatal rats by upregulating Sirt1-mediated pathways, suggesting its potentially beneficial role in the treatment of brain injury induced by hyperoxia.

Melatonin prevents acute kidney injury in severely burned rats via the activation of SIRT1.

Acute kidney injury (AKI) is a common complication after severe burns. Melatonin has been reported to protect against multiple organ injuries by increasing the expression of SIRT1, a silent information regulator that regulates stress responses, inflammation, cellular senescence and apoptosis. This study aimed to investigate the protective effects of melatonin on renal tissues of burned rats and the role of SIRT1 involving the effects. Rat severely burned model was established, with or without the administration of melatonin and SIRT1 inhibitor. The renal function and histological manifestations were determined to evaluate the severity of kidney injury. The levels of acetylated-p53 (Ac-p53), acetylated-p65 (Ac-p65), NF-κB, acetylated-forkhead box O1 (Ac-FoxO1), Bcl-2 and Bax were analyzed to study the underlying mechanisms. Our results suggested that severe burns could induce acute kidney injury, which could be partially reversed by melatonin. Melatonin attenuated oxidative stress, inflammation and apoptosis accompanied by the increased expression of SIRT1. The protective effects of melatonin were abrogated by the inhibition of SIRT1. In conclusion, we demonstrate that melatonin improves severe burn-induced AKI via the activation of SIRT1 signaling.

Abstract 1645: DNA-damaging compound 0404 effectively inhibits hepatocellular carcinoma by upregulation of miR-34a and miR-200c expression

Abstract Hepatocellular carcinoma (HCC) is the sixth most common type of cancer and the third most common cause of cancer deaths worldwide. DNA-damaging agents have a long history of use in cancer chemotherapy. Unfortunately, chemotherapeutic treatment strategies against HCC are very ineffective. We have screened of a novel DNA-damaging compounds, 0404, by using time-dependent cellular response (TCRP) based on unique TCRP DNA-damage signature. This new compound could induce apoptosis effectively in hepatocellular carcinoma at nM-degree concentration. Since microRNAs (miRNAs) have been show play critical roles in pathogenesis of HCC, the present study was aimed to investigate the role of miRNAs in the anticancer effects of 0404. We found that, HepG2 cells, which retain wild-type p53, were more sensitive to 0404 treatment than Huh7 cells, which with mutant p53. At IC50 and IC99 concentration, 0404 upregulated the expression of miR-34a and miR-200c, two major miRNA regulated by p53, in a dose- and time-dependent manner in HepG2 cells. Western blot analysis further show that up-regulation of miR-34a decreased SIRT1 expression, leading to an increase in acetylated p53 levels and also an increase in p53 targets such as p21 and PUMA. In addition, up-regulation of miR-200c decreased the expression of EMT-inducing transcription factors ZEB1 and ZEB2, which play key role in tumor invasion and metastasis. Furthermore, miR-34a or miR-200c inhibitors impaired the down-regulation of miR-34a or miR-200c target genes, respectively, by 0404 treatment, and partially impaired the tumor suppression effect of 0404. These results suggest that 0404 may be an attractive agent in chemotherapy for HCC, especially in HCC with wt p53, by regulation of miR-34a and miR-200c. Financial support: This study was supported by the National Natural Science Fund (81272679) and the Major national S&T Projects (2013ZX09041003). Citation Format: Caixia Xia, Yanning Liu, Guohua Lou, Xiao Xu, Long Mao, Min Zheng. DNA-damaging compound 0404 effectively inhibits hepatocellular carcinoma by upregulation of miR-34a and miR-200c expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1645. doi:10.1158/1538-7445.AM2015-1645

Differential expression of sirtuins in the aging rat brain.

Although there are seven mammalian sirtuins (SIRT1-7), little is known about their expression in the aging brain. To characterize the change(s) in mRNA and protein expression of SIRT1-7 and their associated proteins in the brain of “physiologically” aged Wistar rats. We tested mRNA and protein expression levels of rat SIRT1-7, and the levels of associated proteins in the brain using RT-PCR and western blotting. Our data shows that SIRT1 expression increases with age, concurrently with increased acetylated p53 levels in all brain regions investigated. SIRT2 and FOXO3a protein levels increased only in the occipital lobe. SIRT3-5 expression declined significantly in the hippocampus and frontal lobe, associated with increases in superoxide and fatty acid oxidation levels, and acetylated CPS-1 protein expression, and a reduction in MnSOD level. While SIRT6 expression declines significantly with age acetylated H3K9 protein expression is increased throughout the brain. SIRT7 and Pol I protein expression increased in the frontal lobe. This study identifies previously unknown roles for sirtuins in regulating cellular homeostasis and healthy aging.

Targeting HPV16 E6-p300 interaction reactivates p53 and inhibits the tumorigenicity of HPV-positive head and neck squamous cell carcinoma

The incidence of human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) has rapidly increased over the past 30 years prompting the suggestion that an epidemic may be on the horizon. Therefore, there is a clinical need to develop alternate therapeutic strategies to manage the growing number of HPV-positive HNSCC patients. High-risk HPV E6 inactivates p53 through two distinct mechanisms; association with E6AP to degrade p53 and association with p300 to block p300-mediated p53 acetylation and activation. In this study, we determined if targeting the E6-p300 interaction is an effective approach to reactivate p53 in HPV-positive HNSCC. Ectopic expression of the CH1 domain of p300 in HPV-positive HNSCC blocks the association between E6 and p300, increases total and acetylated p53 levels, and enhances p53 transcriptional activity. Moreover, expression of p21, miR-34a, and miR-200c are increased demonstrating functional p53 reactivation. CH1 overexpression in HPV-positive HNSCC has a global anti-cancer effect resulting in a decrease in cell proliferation and clonogenic survival and an increase in apoptosis. The in vivo tumor initiating ability of HPV-positive HNSCC is severely compromised with CH1 overexpression, in part through a reduction in the cancer initiating cell population. A novel small molecule CH1 inhibitor, CH1iB, reactivates p53 and potentiates the anti-cancer activity of cis-platinum in HPV-positive HNSCC cells. Our work shows that CH1 domain inhibitors represent a novel class of p53 reactivation therapeutics for managing HPV-positive HNSCC patients.

Sirtuin 1 enzyme activity and autophagy proteins are increased in the kidney during murine sepsis

Sepsis affects over 750,000 patients annually and the development of acute kidney injury (AKI) during sepsis nearly doubles the mortality rate to 75%. We have recently published that the dietary antioxidant, resveratrol (RSV), can improve kidney function and survival in the cecal ligation and puncture (CLP) model of sepsis in the mouse. Since RSV has been reported to lead to the activation of sirtuin1 (SIRT1), which may be protective, we investigated the relationship between SIRT1 and acetylation of p53. SIRT1 enzyme activity was increased at 10 and 18h post CLP as were the autophagy‐related proteins Atg5 and LC3. Moreover, acetylated p53 levels were significantly increased at 6h and 10h, but not 18h. By immunostaining, the number of TUNEL‐positive cells was increased by sepsis and acetylated p53 was localized mainly to the cytoplasm of proximal tubular epithelial cells in the cortex and cortico‐medullary junction. While RSV (10 mg/kg, ip) did not increase SIRT1 activity, RSV did block the increase in acetylated p53. These data suggest that CLP causes both autophagy and apoptosis in the kidney and that RSV may dampen the apoptotic response. Supported by F30 DK085705 (JHH) and R01 DK075991 (PRM).

Pharmacological Inhibition of the Stress-Related Deacetylase SIRT1 Enhances Eradication of CML stem Cells

Abstract 448BCR-ABL tyrosine kinase inhibitors (TKI) are effective in inducing remissions and prolonging survival of CML patients, but fail to eradicate primitive leukemia stem cells (LSC) which remain a potential source of relapse. New strategies to enhance elimination of residual CML LSC in TKI-treated patients are required. We have previously reported that the stress-related deacetylase SIRT1 is expressed at high levels in CML stem/progenitor cells and that inhibition of SIRT1 expression using lentivirus-expressed shRNA induces apoptosis in CML progenitors and increases their sensitivity to imatinib (IM) by activating p53 signaling (Blood 2010, 116: 200A). These results support an important role for SIRT1 in CML LSC maintenance and TKI resistance, and as a potential molecular target for therapy directed against CML LSC. Tenovin-6 (TV) has been identified as a potent small molecule inhibitor of SIRT1 activity (Cancer Cell 2008, 13:454). Here we evaluated whether pharmacological inhibition of SIRT1 activity using TV could selectively inhibit CML stem/progenitor cells. As with shRNA-mediated knock-down of SIRT1, treatment with TV (0.5 μM) significantly increased apoptosis of CML CD34+ cells (TV16±7% vs. Control 3±2%, p=0.04, n=3), but not normal CD34+ cells (TV 6±2% vs. Control 4±2%, p=0.1, n=3). The combination of IM (2.5 μM) and TV induced significantly increased apoptosis in CML progenitors compared to IM alone, and to a significantly greater extent than in normal cells (CML, TV + IM 40±2% vs. IM 19±3%, p=0.009, n=3; CB, TV + IM 15±4% vs. IM 10±2%, p=0.04, n=3). TV (1 μM) increased apoptosis in both CML CD34+CD38− (TV 42±10% vs. Control 4±3%, p=0.04, n=3) and CD34+CD38+ cells (TV 35±7% vs. Control 8±2%, p=0.03, n=3). CFSE labeling indicated that treatment with TV resulted in increased apoptosis of undivided CML CD34+CD38− cells identified on the basis of high CFSE fluorescence (TV 20±7% vs. Control 2±1%, p=0.04, n=3). The combination of TV with IM resulted in a significant increase in apoptosis in CML CD34+CD38− CFSEhigh cells compared to IM alone (TV plus IM 35±5% vs. IM 10±4%, p=0.03, n=3). Treatment with TV (0.5 μM) reduced CML CFC frequency (70±9% inhibition with TV compared to untreated controls, p=0.009, n=3) without affecting normal CFC frequency. Combination of TV (0.5 μM) with IM resulted in enhanced inhibition of CML CFC compared to IM alone, but did not enhance inhibition of normal CFC (CML: TV plus IM 82±6% inhibition vs. IM 57±10%, p=0.02, n=3; CB: TV plus IM 38±7% inhibition vs. IM 36±9%, p=0.1, n=3). TV treatment effectively inhibited the growth of Baf3 cells expressing T315I-mutated BCR-ABL, and significantly enhanced apoptosis of IM-resistant CML blast crisis CD34+ cells [TV (1 μm) 30±1% vs. Control 19±5%, p=0.04, n=3], suggesting SIRT1 inhibition can also target TKI-resistant CML cells. Ex vivo treatment with TV (1 μM) significantly reduced longer-term (12 weeks) engraftment of CML CD34+ cells in NSG mice following TV treatment (TV treated 0.2*105±0.1*105 human CD45+ cells in murine BM vs. Control 1.8*105±0.6*105, p=0.009, n=5). Significant reduction in engraftment of CD33+ (p=0.008) and CD14+ myeloid cells (p=0.009) was seen. Q-PCR and FISH analysis confirmed that engrafted human cells were leukemic in origin. Interestingly, engraftment of CB CD34+ cells was not reduced after treatment with TV (TV 2.7*106±0.7*106 human CD45+ cells in murine BM, vs. Control 2.4*106±0.8*106, p=0.2, n=6). These results show that SIRT1 inhibition by TV effectively targets primitive human CML cells with in vivo multi-lineage engraftment capacity. Treatment with TV significantly enhanced acetylated p53 levels in CML CD34+ cells, indicating effective inhibition of SIRT1 activity. TV treatment also increased total p53 levels, possibly related to reduced p53 degradation. TV treatment did not increase acetylated p53 or total p53 levels in normal CD34+ cells. Importantly shRNA-mediated knock-down of p53 resulted in significant reduction of TV-induced apoptosis in CML CD34+ cells (13±6% apoptosis with p53 shRNA; 33±7% apoptosis with control shRNA, p=0.04, n=3), indicating that the effects of TV on CML CD34+ cells are related to p53 acetylation and activation. In conclusion, our studies indicate that pharmacological inhibition of SIRT1 can activate p53 and enhance eradication of CML LSC in combination with TKI treatment, and support further evaluation of targeted inhibition of SIRT1 as a therapeutic strategy in CML. Disclosures:No relevant conflicts of interest to declare.