Increased P53 Gene Expression Research Articles

Abstract P426: Inactivating Grk5 Impairs Basal Cardiac Function And Survival Via P53 Modulation

Introduction: G protein-coupled receptor (GPCR) kinase 5 (GRK5) is a multifunctional protein and depending on its localization within the cell, it has been shown to elicit either protective or deleterious effects. For instance in the heart, when anchored to the plasma membrane, this kinase can regulate specific GPCRs via canonical phosphorylation that can confer cardioprotection. However, when it accumulates in the nucleus its non-canonical activity can drive pathological hypertrophic gene transcription. Interestingly, the latter effects may not be kinase-dependent. Hypothesis: The role played by GRK5’s catalytic activity in the heart has not been fully elucidated and for that reason we sought to assess the in vivo consequences of inactivating the catalytic site of GRK5 with an initial focus at examining the basal cardiac phenotype and response to stress. Methods: We used CRISPR/Cas9 technology to generate a novel knock-in mouse model, with the ATP binding lysine (K) 215 in the catalytic cleft replaced by arginine (R) (GRK5-K215R) resulting in mice devoid of any GRK5 catalytic activity. We studies baseline cardiac function in these mutant mice compared to wild-type (WT) littermates and then stressed them via transverse aortic constriction (TAC). In vitro, we used H9c2 cardiomyocytes and various GRK5 mutants for mechanistic studies. Results: Compared to age-matched WT littermates, GRK5-K215R mice revealed marked and early (9 weeks) deterioration of cardiac function, with augmented apoptosis and fibrosis basally. Importantly, mutant knock-in mice displayed increased p53 gene expression (both at mRNA and protein levels). Moreover, TAC induced increased dysfunction and fibrosis in GRK5-K215R mice compared to WT. Mechanistically, we transduced H9c2 cells with adenoviruses (Ad), encoding for WT GRK5 (Ad-GRK5) or a mutant GRK5 lacking its nuclear localization signal (Ad-NLS) and when GRK5 was localized only outside the nucleus, there was a significant protection against apoptosis, with reduced p53 protein and mRNA levels. Conversely, when we overexpressed a mutant GRK5 without nuclear export signal (GRK5-ΔNES) to trap GRK5 within the nucleus, we found a significant increase in apoptosis, with high p53 protein expression levels. Conclusions: Inactivating GRK5’s catalytic activity impairs its nuclear regulation of p53. This can result in higher levels of p53 mRNA and protein resulting in higher rates of apoptosis in the heart leading to significant cardiac dysfunction and an intolerance to stress.

Dasatinib plus quercetin prevents uterine age-related dysfunction and fibrosis in mice.

The uterine fibrosis contributes to gestational outcomes. Collagen deposition in the uterus is related to uterine aging. Senolytic therapies are an option for reducing health complications related to aging. We investigated effects of aging and the senolytic drug combination of dasatinib plus quercetin (D+Q) on uterine fibrosis. Forty mice, 20 young females (03-months) and 20 old females (18-months), were analyzed. Young (Y) and old (O) animals were divided into groups of 10 mice, with one treatment (T) group (YT and OT) and another control © group (YC and OC). Comparative analysis of Pi3k/Akt1/mTor and p53 gene expression and related microRNAs (miR34a, miR34b, miR34c, miR146a, miR449a, miR21a, miR126a, and miR181b) among groups was performed to test effects of age and treatment on collagen deposition pathways. Aging promoted downregulation of the Pi3k/Akt1/mTor signaling pathway (P = 0.005, P = 0.031, and P = 0.028, respectively) as well as a reduction in expression of miR34c (P = 0.029), miR126a (P = 0.009), and miR181b (P = 0.007). D+Q treatment increased p53 gene expression (P = 0.041) and decreased miR34a (P = 0.016). Our results demonstrate a role for the Pi3k/Akt1/mTor signaling pathway in uterine aging and suggest for the first time a possible anti-fibrotic effect in the uterus of D+Q senolytic therapy.

Amelioration of Combination of Paclitaxel and Di Allyl Sulfide on the Alterations of Bcl2, P53 and Apoptosis Changes Against 7,12 Di Methyl Benz (A) Anthracene Induced Skin Cancer in Experimental Animals.

The purpose of this study was to investigate the Bcl2, P53 and apoptosis changes against skin cancer in experimental animals. Skin cancer is the most common form of human cancer. It is estimated that over 1 million new cases occur annually. The annual rates of all forms of skin cancer are increasing each year, representing a growing public concern. It has also been estimated that nearly half of all Americans who live to age 65 are likely to develop skin cancer at least once. Skin cancer was induced in rats by Di Methyl Benz (a) Anthracene at the dosage of DMBA (5µg) per animal, three times a week for 28weeks after conformation of skin cancer treated with Paclitaxel and Di allyl sulfide for 30days. The levels of Bcl2 gene expression were significantly decreased and P53gene expression were markedly increased in Paclitaxel and Di allyl sulfide treated animals when compared with cancer bearing animals. The treatment with combination of Paclitaxel and Di allyl sulfide effectively reduced Bcl2 protein expression and also increased P53gene expression. Moreover, the levels of Bcl2 and P53 a good indicators of restoring the skin architecture, were also reversed in skin damage subjects after treatment with the herbalcompounds preparation. So, from the obtained results it is concluded that a combination of Paclitaxel and Di allyl sulfide is capable of restoring the skin architecture and can also increase the apoptosis activities in skin cancer rats.

Impact of lithiated cobalt oxide and phosphate nanoparticles on rainbow trout gill epithelial cells

Metal oxide and phosphate nanoparticles (NPs) are ubiquitous in emerging applications, ranging from energy storage to catalysis. Cobalt-containing NPs are particularly important, where their widespread use raises questions about the relationship between composition, structure, and potential for environmental impacts. To address this gap, we investigated the effects of lithiated metal oxide and phosphate NPs on rainbow trout gill epithelial cells, a model for environmental exposure. Lithium cobalt oxide (LCO) NPs significantly reduced cell viability at10 µg/mL, while a 10-fold higher concentration of lithiated cobalt hydroxyphosphate (LCP) NPs was required to significantly reduce viability. Exposure to Li+ and Co2+ alone, at concentrations relevant to ion released from the NPs, did not reduce cell viability and minimally impacted reactive oxygen species (ROS) levels. Both LCO- and LCP-NPs were found within membrane-bound organelles. However, only LCP-NPs underwent rapid and complete dissolution in artificial lysosomal fluid. Unlike LCP-NPs, LCO-NPs significantly increased intracellular ROS, could be found within abnormal multilamellar bodies, and induced formation of intracellular vacuoles. Increased p53 gene expression, measured in individual cells, was observed at sub-toxic concentrations of both LCO- and LCP-NPs, implicating both in inductions of cellular damage and stress at concentrations approaching predicted environmental levels. Our results implicate the intact NP, not the dissolved ions, in the observed adverse effects and show that LCO-NPs significantly impact cell viability accompanied by increase in intracellular ROS and formation of organelles indicative of cell stress, while LCP-NPs have minimal adverse effects, possibly due to their rapid dissolution in acidic organelles.

Antioxidant, cytotoxic and antineoplastic effects of Carissa carandas Linn. leaves

For scientific clarification of some traditional uses, this study was designed to explore the antioxidant, cytotoxic and antineoplastic properties of leaf extract of Carissa carandas Linn., a traditional medicinal plant of Bangladesh. The methanol extract of Carissa carandas leaves (MELC) was applied on DPPH and ABTS experiments to determine its antioxidant activity. In vitro the cytotoxic effect of MELC was evaluated against colonic adenocarcinoma cell lines (SW-480 and SW-48) whereas in vivo its antineoplastic property was tested against Ehrlich ascites carcinoma (EAC). The DPPH and ABTS assays revealed the antioxidant activity of MELC with IC50 10.5±1.2 and 1.75±0.3μg/ml that was comparable to L-ascorbic acid. In vitro cytotoxic study, MELC reduced the viability of adenocarcinoma cells in dose dependent manner and in vivo, administration of MELC (25mg/kg) resulted in a significant (p<0.05) decrease in viable EAC cell count thereby increasing the life span of the EAC cell bearing mice. Restoration of hematological parameters such as red blood cells (RBC), hemoglobin and white blood cells (WBC) to normal levels in MELC-treated mice was also observed. Moreover, treatment with MELC induced apoptosis of EAC cells as observed in fluorescence microscopic view of DAPI (4,6-diamidino-2-phenylindole) stained cells and also increased p53 gene expression MELC-treated cells in respect to untreated EAC control. In addition, the MELC was rich in polyphenol content and its GC–MS chromatogram confirmed the presence of some compounds all of which showed anticancer and cytotoxic activities in previous studies. In a word, this study supports the use of Carissa carandas in traditional medicine as well as highlights the need to further explore the potentials of MELC as an antineoplastic agent.

Influence of triptolide on P53/P73 gene methylation and inhibition effect against proliferation of breast carcinoma MCF-7 cells

Objective To study the effect of triptolide(TP) on the proliferation of breast carcinoma cell line MCF-7 and its association with P53/P73 gene expression and methylation.Methods MCF-7 cells were treated with different concentrations of TP(10 ng/ml,20 ng/ml,and 40 ng/ml),and the proliferation of MCF-7 cells was measured by MTT method.The expressions of methyltransferase DNMT1,DNMT3a and DNMT3b mRNA were measured by RT-PCR in MCF-7 cells,P53/P73 gene methylation was analyzed by methylation specific PCR,and the protein expression of p53/P73 in MCF-7 cells was examined by Western blotting assay.Results TP inhibited the proliferation of MCF-7 cells in a dose-dependent manner(P0.05,P0.01),with the inhibitory rate being(70.1±3.52)% at 40 ng/ml TP,and the IC50 of TP was 20 ng/ml.TP significantly inhibited DNMT1,DNMT3a,and DNMT3b mRNA expression in MCF-7 cells(P0.05,P0.01),and it also significantly inhibited methylation of P53 promoter region.TP increased P53 gene expression at 20 ng/ml and the increase was significant at 40 ng/ml(P0.01).TP reversed the hypermethylation of P73 gene in MCF-7 cells;it also significantly increased P73 mRNA expression at 10 ng/ml(P0.05),and the increase was in a dose-dependent manner.Western blotting analysis showed that TP(20 ng/ml) increased the protein expression of P53 and P73 in MCF-7 cells.Conclusion TP can promote the expression of P53 and P73 genes through inhibiting methyltransferase-dependent gene methylation,and further inhibit the proliferation of MCF-7 cells.

Effects of propranolol on the proliferation and apoptosis of hemangioma-derived endothelial cells

Background/PurposePropranolol, a non-selective beta-blocker, has recently been introduced as a novel treatment modality for proliferating hemangiomas. However, the mechanism of action of this therapy is unknown. In this study, we investigated propranolol in the etiology of hemangiomas that are associated with the proliferation and apoptosis of hemangioma-derived endothelial cells (HemECs). MethodsHemECs were isolated from freshly resected hemangioma specimens. We studied propranolol-treated HemECs in vitro. We measured the effect of propranolol on HemEC viability using the Cell Counting Kit-8 (CCK-8) assay and proliferation and apoptosis using a BrdU labeling assay, annexin-V-fluorescein isothiocyanate/propidium iodide flow cytometry, and Hoechst 33342 fluorescent staining. We explored the potential mechanisms of propranolol-induced HemEC dysfunction using western blot analysis, a caspase assay kit, and real-time quantitative PCR. ResultsWe observed that propranolol had inhibitory effects on the viability and proliferation of HemECs. HemEC apoptosis significantly increased with 100μM propranolol treatment. Vascular endothelial growth factor (VEGF) expression was down-regulated by propranolol in a dose-dependent manner. We also demonstrated activation of the caspase cascade, including caspase-9 and caspase-3 of the intrinsic pathway, and an increased p53 gene expression and Bax/Bcl-xL ratio in HemECs treated with 100μM propranolol. ConclusionsWe obtained novel data that suggests propranolol could inhibit HemEC proliferation and induce apoptosis. The effects were likely mediated through the suppression of VEGF expression, activation of caspase-9 and caspase-3, up-regulation of the pro-apoptotic genes p53 and Bax and down-regulation of the anti-apoptotic gene Bcl-xL.

Investigation on Mechanism of Growth Arrest Induced by Iron Oxide Nanoparticles in PC12 Cells

Iron oxide nanoparticles (Fe3O4 NPs) have recently received increasing interest in the biotechnology and life science. However, little is known about the nanoneurotoxicity of Fe3O4 NPs following exposure to neurons. This study was to elucidate the cytotoxicity and DNA damage of Fe3O4 NPs on PC12 cells line which derived from Rattus norvegicus pheochromocytoma. The cell viability was observed by MTS assay and cell cycle status was analyzed using flow cytometry. DNA damage related gene (P53) and its downstream targets (P21 and GADD45) were determined by semiquantitative RT-PCR. The results showed that exposure to Fe3O4 NPs at dosage levels between 25 and 200 microg/ml decreased cell viability respectively. The nanoparticles treatment caused cell cycle arrest in G2/M phase and the mRNA levels of P53 increased when PC12 cells were incubated with different concentrations Fe3O4 NPs. However, P21 and GADD45, the downstream targets of P53, were not affected. In summary, exposure to Fe3O4 NPs resulted in a dose-dependent cytotoxicity in cultured PC12 cells that was associated with increased P53 gene expression and much attention should be paid to the potential impact of Fe3O4 NPs on the central nervous system.

Developmental toxicity of cypermethrin in embryo-larval stages of zebrafish

Cypermethrin, a type II pyrethroid insecticide, is widely used throughout the world in agriculture, forestry, horticulture and homes. Though the neurotoxicity of cypermethrin has been thoroughly studied in adult rodents, little is so far available regarding the developmental toxicity of cypermethrin to fish in early life stages. To explore the potential developmental toxicity of cypermethrin, 4-h post-fertilization (hpf) zebrafish embryos were exposed to various concentrations of cypermethrin (0, 25, 50, 100, 200 and 400 μg L −1) until 96 h. Among a suite of morphological abnormalities, the unique phenotype curvature was observed at concentrations as low as 25 μg L −1. Studies revealed that 400 μg L −1 cypermethrin significantly increased malondialdehyde production. In addition, activity of antioxidative enzymes including superoxide dismutase and catalase were significantly induced in zebrafish larvae in a concentration-dependent manner. To further investigate the toxic effects of cypermethrin on fish, acridine orange (AO) staining was performed at 400 μg L −1 cypermethrin and the result showed notable signs of apoptosis mainly in the nervous system. Cypermethrin also down-regulated ogg1 and increased p53 gene expression as well as the caspase-3 activity. Our results demonstrate that cypermethrin was able to induce oxidative stress and produce apoptosis through the involvement of caspases in zebrafish embryos. In this study, we investigated the developmental toxicity of cypermethrin using zebrafish embryos, which could be helpful in fully understanding the potential mechanisms of cypermethrin exposure during embryogenesis and also suggested that zebrafish could serve as an ideal model for studying developmental toxicity of environmental contaminants.

Differential effects of naturally occurring and synthetic organoselenium compounds on biomarkers in androgen responsive and androgen independent human prostate carcinoma cells

Epidemiological studies and clinical trials show that selenium supplementation results in reduction of prostate cancer incidence; however, the form of selenium and mechanisms underlying protection remain largely unknown. Toward this end, we compared the effects of naturally occurring selenomethionine (SM) and Se-methylselenocysteine (MSC) and synthetic 1,4-phenylenebis(methylene)selenocyanate (p-XSC) and p-xylylbis(methylselenide) p-XMS) organoselenium compounds in androgen responsive (AR) LNCaP and its androgen independent clone (AI) LNCaP C4-2 human prostate carcinoma cells on cell growth, secretion of prostate specific antigen (PSA), intracellular redox status and genomic profiles with emphasis on identifying redox sensitive genes. Both p-XSC and p-XMS reduced cell number and total protein concentration compared to control-treated AR and AI cells, while SM and MSC exhibited no effect on growth of AR and AI cells. SM, p-XSC and p-XMS but not MSC inhibited levels of secreted PSA in AR cells. SM, MSC and p-XMS increased glutathione (GSH) levels in AI LNCaP cells. By contrast, in both cell types, only p-XSC significantly decreased GSH concentrations to <50% of control suggesting either an increase in intracellular oxidative stress or a change in GSH/GSSG ratio. On the basis of RT-PCR analysis, SM and p-XSC increased p53 gene expression by 2-fold in AR cells but not in AI cells and only SM enhanced epidermal growth factor receptor in AR cells. Depending on the structure, organoselenium compounds exhibit differential effects on growth, PSA secretion, oxidative stress and selective gene responses in human prostate cancer cells and suggest the potential of developing novel organoselenium compounds as chemopreventive agents in models of human prostate cancer.

P53 Expression after Treatment with Zebularine Is Not Due to Demethylation

To the Editor: In their recent article, Hodge et al. suggested that the expression of the p53 gene in the two myeloma cell lines KAS6/1 and IM-9 is regulated by methylation of CpG and CpA dinucleotides in the promoter region ( [1][1]). This hypothesis is based on the observation that the

Polyamine depletion stabilizes p53 resulting in inhibition of normal intestinal epithelial cell proliferation.

The p53 nuclear phosphoprotein plays a critical role in transcriptional regulation of target genes involved in growth arrest and apoptosis. The natural polyamines, including spermidine, spermine, and their precursor putrescine, are required for cell proliferation, and decreasing cellular polyamines inhibits growth of the small intestinal mucosa. In the current study, we investigated the mechanisms of regulation of p53 gene expression by cellular polyamines and further determined the role of the gene product in the process of growth inhibition after polyamine depletion. Studies were conducted both in vivo and in vitro using rats and the IEC-6 cell line, derived from rat small intestinal crypt cells. Levels for p53 mRNA and protein, transcription and posttranscription of the p53 gene, and cell growth were examined. Depletion of cellular polyamines by treatment with alpha-difluoromethylornithine (DFMO) increased p53 gene expression and caused growth inhibition in the intact small intestinal mucosa and the cultured cells. Polyamine depletion dramatically increased the stability of p53 mRNA as measured by the mRNA half-life but had no effect on p53 gene transcription in IEC-6 cells. Induction of p53 mRNA levels in DFMO-treated cells was paralleled by an increase in the rate of newly synthesized p53 protein. The stability of p53 protein was also increased after polyamine depletion, which was associated with a decrease in Mdm2 expression. When polyamine-deficient cells were exposed to exogenous spermidine, a decrease in p53 gene expression preceded an increase in cellular DNA synthesis. Inhibition of the p53 gene expression by using p53 antisense oligodeoxyribonucleotides significantly promoted cell growth in the presence of DFMO. These findings indicate that polyamines downregulate p53 gene expression posttranscriptionally and that growth inhibition of small intestinal mucosa after polyamine depletion is mediated, at least partially, through the activation of p53 gene.

Effects of Selaginella tamariscina on in vitro tumor cell growth, p53 expression, G 1 arrest and in vivo gastric cell proliferation

Selaginella tamariscina, an oriental medicinal plant, was extracted using water and several organic solvents, and each fraction was assayed for its tumoricidal effects with 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT). Influences on expression of p53 tumor suppressor gene and induction of G 1 arrest in the cell cycle were analyzed by Northern blotting and flow cytometry, respectively. The modifying effects of pulverized Selaginella tamariscina on cell turnover in the stomach were also investigated in rats given 150 mg/kg of N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) by gavage and then fed a diet containing 5, 1 or 0% Selaginella tamariscina. Fractions I–V showed significant tumoricidal effects against cultured human leukemia cells whereas these fractions did not affect normal human lymphocytes. Among the effective fractions, the water-extracted fraction (V) efficiently increased p53 gene expression and induced G 1 arrest. The 1% Selaginella tamariscina feeding caused a significant reduction ( P<0.05) in the proliferating cell nuclear antigen-(PCNA) labeling index of the glandular stomach epithelium as compared with the MNNG-alone group value although 5% Selaginella tamariscina feeding was only associated with a tendency for decrease. These results suggest that Selaginella tamariscina could be a candidate chemopreventive agent against gastric cancer.