Nuclear P53 Expression Research Articles

HIF-1α Dependent Upregulation of ZIP8, ZIP14, and TRPA1 Modify Intracellular Zn2+ Accumulation in Inflammatory Synoviocytes.

Intracellular free zinc ([Zn2+]i) is mobilized in neuronal and non-neuronal cells under physiological and/or pathophysiological conditions; therefore, [Zn2+]i is a component of cellular signal transduction in biological systems. Although several transporters and ion channels that carry Zn2+ have been identified, proteins that are involved in Zn2+ supply into cells and their expression are poorly understood, particularly under inflammatory conditions. Here, we show that the expression of Zn2+ transporters ZIP8 and ZIP14 is increased via the activation of hypoxia-induced factor 1α (HIF-1α) in inflammation, leading to [Zn2+]i accumulation, which intrinsically activates transient receptor potential ankyrin 1 (TRPA1) channel and elevates basal [Zn2+]i. In human fibroblast-like synoviocytes (FLSs), treatment with inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-1α (IL-1α), evoked TRPA1-dependent intrinsic Ca2+ oscillations. Assays with fluorescent Zn2+ indicators revealed that the basal [Zn2+]i concentration was significantly higher in TRPA1-expressing HEK cells and inflammatory FLSs. Moreover, TRPA1 activation induced an elevation of [Zn2+]i level in the presence of 1 μM Zn2+ in inflammatory FLSs. Among the 17 out of 24 known Zn2+ transporters, FLSs that were treated with TNF-α and IL-1α exhibited a higher expression of ZIP8 and ZIP14. Their expression levels were augmented by transfection with an active component of nuclear factor-κB P65 and HIF-1α expression vectors, and they could be abolished by pretreatment with the HIF-1α inhibitor echinomycin (Echi). The functional expression of ZIP8 and ZIP14 in HEK cells significantly increased the basal [Zn2+]i level. Taken together, Zn2+ carrier proteins, TRPA1, ZIP8, and ZIP14, induced under HIF-1α mediated inflammation can synergistically change [Zn2+]i in inflammatory FLSs.

A novel supplement with yeast β-glucan, prebiotic, minerals and Silybum marianum synergistically modulates metabolic and inflammatory pathways and improves steatosis in obese mice

ObjectiveTo evaluate the synergic effects of a novel oral supplement formulation, containing prebiotics, yeast β-glucans, minerals and silymarin (Silybum marianum), on lipid and glycidic metabolism, inflammatory and mitochondrial proteins of the liver, in control and high-fat diet-induced obese mice. MethodsAfter an acclimation period, 32 male C57BL/6 mice were divided into the following groups: nonfat diet (NFD) vehicle, NFD supplemented, high-fat diet (HFD) vehicle and HFD supplemented. The vehicle and experimental formulation were administered orally by gavage once a day during the last four weeks of the diet (28 consecutive days). We then evaluated energy homeostasis, inflammation, and mitochondrial protein expression in these groups of mice. ResultsAfter four weeks of supplementation, study groups experienced reduced glycemia, dyslipidemia, fat, and hepatic fibrosis levels. Additionally, proliferator-activated receptor-α, AMP-activated protein kinase-1α, peroxisome proliferator-activated receptor γ co-activator-1α, and mitochondrial transcription factor A expression levels were augmented; however, levels of inhibitor of nuclear factor-κB kinase subunit α and p65 nuclear factor-κB expression, and oxidative markers were reduced. Notably, the cortisol/C-reactive protein ratio, a well-characterized marker of the hypothalamic–pituitary–adrenal axis immune interface status, was found to be modulated by the supplement. ConclusionWe discovered that the novel supplement was able to modify different antioxidant, metabolic and inflammatory pathways, improving the energy homeostasis and inflammatory status, and consequently alleviated hepatic steatosis.

The association between the expression of nuclear Yes-associated protein 1 (YAP1) and p53 protein expression profile in breast cancer patients.

BackgroundYes-associated protein 1 (YAP1) is a key effector molecule regulated by the Hippo pathway and described as a poor prognostic factor in breast cancer. Tumor protein 53 (TP53) mutation is well known as a biomarker related to poor survival outcomes. So far clinical characteristics and survival outcome according to YAP1 and TP53 mutation have been poorly identified in breast cancer.Patients and methodsRetrospectively, 533 breast tumor tissues were collected at the Seoul St Mary’s hospital and Gangnam Severance Hospital from 1992 to 2017. Immunohistochemistry with YAP1 and p53 specific antibodies were performed, and the clinical data were analyzed.ResultsMutant p53 pattern was associated with aggressive tumor features and advanced anatomical stage. Inferior overall survival (OS) and recurrence free survival (RFS) were related with mutant p53 pattern cases with low nuclear YAP1 expression (P = 0.0009 and P = 0.0011, respectively). Multivariate analysis showed that mutant p53 pattern was an independent prognostic marker for OS [hazard ratios (HR): 2.938, 95% confidence intervals (CIs): 1.028–8.395, P = 0.044] and RFS (HR: 1.842, 95% CIs: 1.026–3.304). However, in cases with high nuclear YAP1 expression, there were no significantly difference in OS and RFS according to p53 staining pattern.ConclusionWe found that mutant p53 pattern is a poor prognostic biomarker in breast tumor with low nuclear YAP1 expression. Our findings suggest that interaction between nuclear YAP1 and p53 expression pattern impact survival outcomes.

ROC1 promotes the malignant progression of bladder cancer by regulating p-I\u03baB\u03b1/NF-\u03baB signaling

BackgroundRegulator of cullins 1 (ROC1) is an important catalytic subunit of cullin–RING E3 ligase. Nuclear factor-kappa B (NF-κB) signaling is closely related to tumor invasion and metastasis. Earlier, we reported that ROC1 was associated with a poor prognosis in patients with bladder cancer (BCa). However, it is unclear whether ROC1 is involved in the NF-κB signaling associated with malignant BCa progression.MethodsThe expression of ROC1 and p65 in bladder cancer and paracancerous tissues were detected by immunohistochemistry (IHC). Pearson correlation was used to assess correlation between ROC1 and p65 protein expressions. The wound-healing and transwell assays were used to monitor cell invasion and migration. The effect of ROC1 on the expression of key proteins in the NF-κB signaling was determined by immunofluorescence and western blot (WB). Cycloheximide (CHX), MG132 and immunoprecipitation assays were used to evaluate the effect of ROC1 on the ubiquitination of phosphorylated inhibitor of kappa B alpha (p-IκBα). A lung metastasis mouse model was generated to detect the role of ROC1 in tumor metastasis.ResultsWe found that ROC1 was up-regulated in BCa tissues and cell lines, and high ROC1 levels were positively correlated with higher tumour grade, lymph node metastasis, distant metastasis and poor prognosis. Linear-regression analysis showed significant a Pearson correlation between ROC1 and nuclear p65 expression in BCa tissue microarray (TMA) samples. Functional studies demonstrated that ROC1 promoted BCa cell invasion and migration. In vitro and in vivo experiments showed that ROC1 activated NF-κB signaling by enhancing the ubiquitination of p-IκBα, which caused p65 nuclear translocation and promoted the transcription of some metastasis-related target genes, such as urokinase-type plasminogen activator receptor (uPAR), intracellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), and matrix metalloproteinase 9 (MMP9), resulting in promoting BCa metastasis.ConclusionROC1 plays an important role in the progression of BCa and serves as a potential diagnostic and therapeutic target for patients with BCa.

The Role of Immunohistochemical Markers for the Diagnosis and Prognosis of Adrenocortical Neoplasms.

Adrenal cortical carcinoma (ACC) is a rare cancer with poor prognosis that needs to be distinguished from adrenocortical adenomas (ACAs). Although, the recently developed transcriptome analysis seems to be a reliable tool for the differential diagnosis of adrenocortical neoplasms, it is not widely available in clinical practice. We aim to evaluate histological and immunohistochemical markers for the distinction of ACCs from ACAs along with assessing their prognostic role. Clinical data were retrospectively analyzed from 37 patients; 24 archived, formalin-fixed, and paraffin-embedded ACC samples underwent histochemical analysis of reticulin and immunohistochemical analysis of p27, p53, Ki-67 markers and were compared with 13 ACA samples. Weiss and Helsinki scores were also considered. Kaplan−Meier and univariate Cox regression methods were implemented to identify prognostic effects. Altered reticulin pattern, Ki-67% labelling index and overexpression of p53 protein were found to be useful histopathological markers for distinguishing ACAs from ACCs. Among the studied markers, only pathological p53 nuclear protein expression was found to reach statistically significant association with poor survival and development of metastases, although in a small series of patients. In conclusion, altered reticulin pattern and p53/Ki-67 expression are useful markers for distinguishing ACCs from ACAs. Immunohistopathology alone cannot discriminate ACCs with different prognosis and it should be combined with morphological criteria and transcriptome analysis.

Valproic acid mitigates spinal nerve ligation-induced neuropathic pain in rats by modulating microglial function and inhibiting neuroinflammatory response

Spinal inflammation is a pathophysiological state of neuropathic pain (NP). The subsequent microglial activation and neuroinflammatory response are contributing factors for long-lasting behavioral hypersensitivity. Valproic acid (VPA), a histone deacetylase inhibitor, has promising anti-inflammatory and neuroprotective properties for clinical use in the treatment of neurological disorders. However, the underlying mechanisms of its effects on NP have not been determined. This study aimed to clarify the possible mechanisms by which VPA alleviates NP in rat models induced by spinal nerve ligation (SNL).Intraperitoneal injection of VPA (300 mg/kg) efficiently attenuated mechanical allodynia in rats with NP. VPA exerted anti-inflammatory effects by downregulating proinflammatory cytokines (tumor necrosis factor-α, cytokines interleukin-1β, cytokines interleukin-6; TNF-α, IL-1β, and IL-6) and upregulating anti-inflammatory cytokines (transforming growth factor-β, cytokines interleukin-10, cytokines interleukin-4; TGF-β, IL-10 and IL-4). Additionally, VPA suppressed spinal microgliosis and promoted the polarization of microglia towards the M2 phenotype to further ameliorate spinal neuroinflammation. VPA also exerted neuroprotective effects by decreasing spinal cell apoptosis. The anti-inflammatory and neuroprotective effects may have depended on changes in nuclear histone deacetylase 3 (HDAC3) expression following VPA treatment. Moreover, VPA treatment inhibited nuclear factor-κB (NF-κB) p65 nuclear expression and upregulated acetylated the signal transducer and activator of transcription 1 (STAT1). In addition, VPA suppressed SNL-induced phosphorylation of Janus Kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). Taken together, our results demonstrate that VPA is a promising anti-inflammatory agent suitable for NP therapy that regulates microglial function and suppresses spinal neuroinflammation via the STAT1/NF-κB and JAK2/STAT3 signaling pathways.

Physcion 8-O-β-glucopyranoside ameliorates liver fibrosis through inflammation inhibition by regulating SIRT3-mediated NF-κB P65 nuclear expression

Physcion 8-O-β-glucopyranoside (PSG), an anthraquinone extracted from Rumex japonicus Houtt, has various pharmacological effects, however, the effect of PSG on liver fibrosis and its related mechanism remain to be determined. We here showed that PSG ameliorated liver injury and liver fibrosis, decreased collagen deposition and inhibited inflammation in carbon tetrachloride (CCl4)-induced rats. Consistent with the in vivo results, PSG suppressed the transforming growth factor-β1 (TGF-β1)-induced cell viability, liver fibrosis and secretion of inflammatory factors in hepatic stellate cells (HSCs). Interestingly, PSG increased the enzyme activity and promoter activity of sirtuin 3 (SIRT3) in fibrotic liver and activated HSCs. In addition, PSG notably increased the mRNA and protein expression of SIRT3 both in vivo and in vitro. Depletion of SIRT3 either by using 3-TYP (SIRT3 selective inhibitor) or SIRT3 siRNA attenuated the anti-inflammatory effect of PSG in activated HSCs. Further study found that TGF-β1 increased the nuclear expression of NF-κB p65, but showed no obvious effect on the total NF-κB p65 expression. Compared to the control adenovirus (Ad.mk), overexpression of SIRT3 by infecting adenovirus encoding SIRT3 (Ad.SIRT3) notably decreased the nuclear expression of NF-κB p65 in activated HSCs. Our results demonstrated that PSG attenuated inflammation by regulating SIRT3-mediated NF-κB P65 nuclear expression in liver fibrosis, providing novel molecular insights into the anti-fibrotic effect of PSG.

Autophagy modulates mesenchymal-to-endothelial transition via p53.

Mesenchymal-to-endothelial transition (MEndT) is one of the mechanisms that influences cardiac fibrosis, which is a key process in cardiac remodeling. It has been reported that autophagy inhibits endothelial cell transition. However, whether autophagy could modulate MEndT in cardiac fibrosis has not yet been investigated. Here, we discussed the association between autophagy and MEndT and its possible mechanism. In this study, we induced endothelial-to-mesenchymal transition using transforming growth factor-β to generate mesenchymal cells and fibroblasts in wild-type human umbilical vein endothelial cells and cells with p53 knockout or overexpression. Then, autophagy was induced by Earle's balanced salt solution (EBSS) and was inhibited by bafilomycin A1 or lentivirus-ATG5-shRNA. The expression levels of MEndT and the autophagy markers CD31, VE-Cadherin, Vimentin, α-SMA, LC3, p62 and p53 were examined. We found that activation of autophagy could promote MEndT and increase cytoplasmic and total expression of p53, that but nuclear p53 expression was decreased, and that inhibition of autophagy activation could reverse the effect of EBSS. Moreover, after knockout of nuclear p53, autophagy promoted MEndT, while autophagy inhibited MEndT in p53 overexpressing cells. Our results demonstrate that autophagy modulate MEndT by nuclear p53 provide a new strategy for the treatment of fibrosis diseases.

KPNA4 regulated by miR-548b-3p promotes the malignant phenotypes of papillary thyroid cancer

AimKaryopherin α4 (KPNA4, importin α3) has been verified to be an oncogene in many cancers. However, its role in papillary thyroid cancer (PTC), the most frequent endocrine malignancy, is still unclear. Materials and methodsKPNA4 expression was analyzed in PTC tissues and cells. The effects of KPNA4 on the proliferation, invasion, and apoptosis of PTC cells were evaluated after overexpression or downregulation of KPNA4. The influence of KPNA4 on NF-κB activation was evaluated by nuclear NF-κB p65 expression and NF-κB-luciferase reporter assays. Moreover, we also explored whether KPNA4 was regulated by miR-548b-3p. Additionally, the roles of miR-548b-3p and KPNA4 were explored in a xenograft mouse model. Key findingsKPNA4 expression was increased in PTC tissues and cells, and its expression was significantly related to patients' clinicopathologic features and overall survival. Overexpression of KPNA4 significantly promoted PTC cell proliferation and invasion, enhanced nuclear p65 expression and augmented NF-κB luciferase activity. However, KPNA4 silencing showed opposite effects on the above indexes, and induced apoptosis of PTC cells. KPNA4 was a target of miR-548b-3p, which was downregulated in PTC and inhibited proliferation and invasion, but promoted apoptosis of PTC cells. KPNA4 overexpression abrogated the suppression of miR-548b-3p on the malignant phenotypes of PTC cells. Both miR-548b-3p overexpression and KPNA4 downregulation inhibited tumor growth and Ki-67 expression, elevated numbers of Tunel-positive cells, and deceased nuclear p65 expression in mouse tumor tissues. SignificanceKPNA4 was negatively regulated by miR-548b-3p and promoted the development of PTC via activating the NF-κB pathway.

Activation of toll like receptor 4 (TLR4) promotes cardiomyocyte apoptosis through SIRT2 dependent p53 deacetylation

Cardiomyocyte inflammation followed by apoptosis and fibrosis is an important mediator for development and progression of heart failure. Activation of toll-like receptor 4 (TLR4), an important regulator of inflammation, causes the progression of cardiac hypertrophy and injury. However, the precise mechanism of TLR4-mediated adverse cardiac outcomes is still elusive. The present study was designed to find the role of TLR4 in cardiac fibrosis and apoptosis, and molecular mechanism thereof. Rats were treated with TLR4 agonist (LPS 12.5 μg/kg/day) through osmotic pump for 14 days. To simulate the condition in vitro, H9c2 cells were treated with LPS (1 μg/ml). Similarly, H9c2 cells were transfected with TLR4 and SIRT2 c-DNA clone for overexpression. Myocardial oxidative stress, inflammation, fibrosis and mitochondrial parameters were evaluated both in vitro and in vivo. Cardiac inflammation after LPS treatment was confirmed by increased TNF-α and IL-6 expression in rat heart. There was a marked increase in oxidative stress as observed by increased TBARS and decreased endogenous antioxidants (GSH and catalase), along with mitochondrial dysfunction as measured by mitochondrial complex activity in LPS-treated rat hearts. Histopathological examination showed the presence of cardiac fibrosis after LPS treatment. Protein expression of nuclear p53 and cleaved caspase-7/caspase-9 was significantly increased in LPS treated heart. Similar to in vivo study, nuclear translocation of p53, mitochondrial dysfunction and cellular apoptosis were observed in H9c2 cells treated with LPS. Our data also indicate that decreased expression of SIRT2 was associated with increased acetylation of p53 after LPS treatment. In conclusion, TLR4 activation in rats promotes cardiac inflammation, mitochondrial dysfunction, apoptosis and fibrosis. p53 and caspase 7/caspase 9 were found to play an important role in TLR4-mediated apoptosis. Our data suggest that, reducing TLR4 mediated fibrosis and apoptosis could be a novel approach in the treatment of heart failure, keeping in the view the major role played by TLR4 in cardiac inflammation.

Mdm2 Loss of Heterozygosity Cooperates with Mutant p53 in Acute Myeloid Leukemia

TP53 mutations are observed in 5 to10% of de novo AML cases with dismal disease prognosis and response to therapy. We found that MDM2 loss of heterozygosity (MDM2 LOH) in AML was always concomitant with TP53 missense mutations (log2 odds ratio>3, p<.001), and not TP53 deletions or truncations whereas in lymphomas, MDM2 LOH and TP53 mutations were mutually exclusive. To understand the functional association of MDM2 LOH and TP53 mutation in AML, we generated a conditional somatic Trp53 mutant AML mouse by combining a Trp53R172H mutation with Mdm2 haploinsufficiency in hematopoietic cells using Vav-cre transgenic mice combined with RosamTmG (mTmG) allele as tracing reporter. The conditional mutant Trp53 allele used in this study harbored a R172H mutation downstream of a wildtype Trp53 cDNA flanked by loxP sites (Trp53wm-R172H) so that the expression of the wildtype Trp53 was preserved before the recombination. Mouse cohorts were generated to harbor one Trp53wm-R172H allele and a Trp53fl or were homozygous for the floxed Trp53 allele (Trp53fl/fl). The expression of Cre in hematopoietic stem cells resulted in heterozygous deletion of Mdm2 and conversion of WT Trp53 to Trp53R172, which corresponds to TP53R175H, one of the most frequent mutations in AML. Mice with somatic Trp53 mutation died due to lymphoma, however, Mdm2 haploinsufficient mice combined with Trp53 mutation succumbed to AML. The disease was immunophenotypically and histologically characterized as AML based on the bone marrow histopathology and expression of Cd11b and Ly6A on the surface of leukemic cells. Immunohistopathology of bone marrow and spleen of MdmLOH p53Mut showed a highly proliferative AML characterized by strong nuclear expression of Ki67 and mutant p53 in the bone marrow of mice. Together, our data identifies Mdm2 LOH to be essential for malignant transformation of p53 mutant myeloid lineage leading to AML development. Disclosures Andreeff: Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Amgen: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees.

Elevated P21 (CDKN1a) Mediates Apoptosis of Beta-Thalassemic Erythroid Cells in Mice but Its Ablation Doesn't Improve Erythroid Maturation

Beta-thalassemias are caused by mutations in the β-globin gene leading to anemia. In β-thalassemia, excessive accumulation of unpaired α globin chains in erythroblasts, triggers redox-mediated reactions, which is associated with increased production of immature erythroid precursors that fail to mature. This impaired maturation is in part due to increased apoptosis of late maturing erythroblasts in β-thalassemic patients that aggravates anemia despite enhanced erythropoiesis leading to what is called ineffective erythropoiesis and ultimately resulting in extramedullary expansion of hematopoiesis. The mechanism of apoptosis in beta-thalassemia remains poorly understood. To investigate this, we examined the status of mediators of stress response during erythroid cell maturation ofHbbth3/+ (th3/+) mice, a model that mimics the beta-thalassemia intermedia phenotype in humans. We found that both Foxo3 and p53 were prematurely activated in th3/+ beta-thalassemic erythroblasts as compared to wild type controls. We crossed Hbbth3/+ (th3/+) and Foxo3-/- mice and found that red blood cell (RBC) count and hemoglobin content were improved (by 1g/L, n=10), and erythroblast apoptosis was decreased to similar levels as in the WT during erythroblast maturation of double mutant mice. However, loss of Foxo3 did not ameliorate the splenomegaly of th3/+mice. We also found that p53 direct target, p21 the cyclin-dependent kinase inhibitor was greatly upregulated in th3/+erythroblasts as well as in beta-thalassemic patients' erythroblasts. To address the contribution of p21, we crossed p21-/- and Th3/+. It showed a significant decrease of apoptosis in CD45- TER119+ erythroblasts both in the bone marrow and spleen of double mutant mice (30% and 23% reduction respectively, n=6 mice each genotype). Although, as in beta-thalassemic patients, serum erythropoietin (Epo) was elevated in the peripheral blood of th3/+mice, the double mutant mice had significantly lower level of Epo than th3/+ (45% reduction, n=3 mice per genotype).In p21-/-th3/+, CD45- TER119+ cells also showed lesser ROS accumulation(12% less, n=3 per genotypes). However, to our surprise, the deletion of p21 on beta-thalassemic background did not have any effect on splenomegaly (n=6 mice each genotype), complete blood count, hemoglobin, RBC production or bone marrow erythroid cell maturation (n=12 mice each genotype). To further examine the underlying mechanism, we analyzed cell cycle in double mutant p21-/-th3/+ erythroblast at distinct stages of maturation identified by CD45, TER119, CD44 and cell size (n=3 mice per genotype) using ki67 staining at distinct stages of maturation. We found p21-/-th3/+erythroblasts proliferate much less than their th3/+ counterparts (basophilic erythroblasts G2 14% less, polychromatic erythroblasts 20% less, p<0.05 n=3 mice per genotype). This may partially explain lack of improvement of RBC production and anemia despite enhanced erythroblast survival. ROS levels were also reduced in double mutant p21-/-th3/+ erythroblasts as compared to controls. Next we investigated the status of p53 and Foxo3 in double mutant p21-/-th3/+ erythroblasts as compared to controls. We confirmed as we had observed earlier that nuclear p53 and Foxo3 expression were greater in th3/+ primitive erythroid (TER119-/low, c-KIT+, CD71Hi) cells than in wild type (n=3 mice per genotype) controls. Strikingly, the double mutant p21-/-th3/+ erythroblasts exhibited the greatest nuclear Foxo3 in all four groups, while nuclear p53 was dramatically reduced by over 80% (n=2 mice each genotype. Each mouse taking >=30 cells to calculate nuclear MFI) even as compared to wild type. These combined studies suggest that ameliorating apoptosis may not improve anemia in beta-thalassemia. Disclosures Liang: Hemogenyx Pharmaceuticals LLC: Current Employment.

Arctiin antagonizes triptolide-induced renal toxicity in rats via anti-inflammatory pathway

To investigate the protective effect of arctiin with anti-inflammatory bioactivity against triptolide-induced nephrotoxicity in rats and explore the underlying mechanism. Forty SD rats were divided into 4 groups for gastric lavage of normal saline, arctiin (500 mg/kg), triptolide (500 μg/kg), or both arctiin (500 mg/kg) and triptolide (500 μg/kg). Blood samples were collected for analysis of biochemical renal parameters, and the renal tissues were harvested for determining the kidney index and for pathological evaluation with HE staining. In the in vitro experiment, HK-2 cells were treated with arctiin and triptolide either alone or in combination, and the cell viability was determined with MTT assay; the cell morphological changes was observed using laser confocal microscopy, cell apoptosis was detected using flow cytometry, and the expressions of inflammation-related protein expression were detected by Western blotting. In SD rats, arctiin significantly antagonized triptolide-induced elevation of BUN, Scr and kidney index (P < 0.05) and obviously improved renal tissue damages induced by triptolide including cell swelling, vacuolization and spotty necrosis. Arctiin significantly inhibited triptolide-induced cytotoxicity in HK-2 cells and increased the cell viability at 24 h (P < 0.05). Arctiin also attenuated triptolide-induced cell morphological changes, decreased cell apoptosis rate (P < 0.05) and reversed the expressions of IκBα and nuclear p65 (P < 0.05). Arctiin can protect the kidney from triptolide-induced damages in rats possibly through the anti-inflammatory pathway.

P16 Immunostaining Can Eliminate Approximately 20% Inaccurate Diagnosis of Low-Grade Squamous Intraepithelial Lesions (SIL) in Cervical Biopsies

Abstract Introduction/Objective Differentiating low-grade SIL from reactive changes on light microscopy has remained controversial. P16 immunostaining is widely accepted in high-grade SIL unlike in low-grade SIL. We focused on the use of p16 immunostaining in eliminating inaccurate diagnosis of low-grade SIL on light microscopy. Methods A retrospective study on 60 cervical biopsies results over a 2 year-period was conducted using 35 suspicious cases for low-grade SIL (bi-nucleation and some nuclear atypia) and 25 low suspicion cases (enlarged nuclei but nuclear atypia cannot differentiate from reactive changes). All cases were stained for p16, significant nuclear expression of p16 was determined as low- grade SIL. Results After p16 immunostaining, 30/35 (85%) cervical biopsies from the suspicious group were found to be truly positive for low-grade SIL while 5/35 (15%) biopsies negative for p16 immunostaining were regarded as reactive changes. In the low suspicious group, 7/25 (28%) were found to be positive for low-grade SIL and 18/25 (72%) biopsies were negative, favoring reactive changes. The overall percentage of all 60 biopsies that showed a changed diagnosis based on p16 immunostaining was 20% (5 were truly negative in the suspicious group and 7 were truly positive in the low suspicious group). Using a 2 x 2 table with p16 as a gold standard for all biopsies, sensitivity and specificity of light microscopy for low-grade SIL were only 81% and 78% respectively. Conclusion Our study showed that p16 immunostaining can also be utilized to eliminate inaccurate diagnosis of low- grade SIL up to 20% in cervical biopsies.

SMARCA4/SMARCA2-deficient Carcinoma of the Esophagus and Gastroesophageal Junction.

Undifferentiated carcinoma of the esophagus and gastroesophageal junction is a recently recognized entity in the fifth edition of the World Health Organization Classification of Digestive Tumors and is diagnostically challenging, particularly on small biopsies. SMARCA4 and SMARCA2 are chromatin remodeling genes with key roles in oncogenesis. We retrieved 14 cases of SMARCA4/SMARCA2-deficient undifferentiated carcinoma of the gastroesophageal junction and esophagus from the authors' institutions. The tumors showed similar histologic findings: the sheet-like proliferation of tumor cells characterized by discohesion, large nuclei, and prominent macronucleoli with many tumor cells exhibiting a rhabdoid appearance. In 8 cases, adjacent specialized intestinal metaplasia was noted and 3 cases exhibited adjacent high-grade dysplasia. Immunohistochemically, tumors variably expressed keratins and disclosed loss of expression of SMARCA4 in 12 and SMARCA2 in 7 cases. In 2 cases SMARCA2 alone was lost without SMARCA4 loss. A mutant p53 immunohistochemical pattern was seen in 4 of 4 cases, 3 of which showed diffuse, strong nuclear expression, and 1 case displayed a complete loss of nuclear expression of p53, including invasive carcinoma and associated dysplasia, when present. Limited clinical follow-up was available, but 3 patients died of disease within 0.6, 2, and 7 months of diagnosis. We present the first series of undifferentiated carcinoma of the esophagus and gastroesophageal junction with this characteristic morphology associated with loss of SMARCA4 and/or SMARCA2 expression. This tumor type likely arises from dedifferentiation of a lower grade carcinoma in some cases, and Barrett esophagus and appears to be associated with an aggressive clinical course.

Anti-inflammatory effect of Ganluyin, a Chinese classic prescription, in chronic pharyngitis rat model

BackgroundGanluyin (GLY) is a famous classical prescription with a long history of use as a treatment for inflammatory conditions such as chronic pharyngitis (CP) in many parts of China. However, it has not been developed as a modern pharmaceutic and its anti-inflammatory mechanisms remain unclear. The aim of this study was to assess the anti-inflammatory efficacy of GLY and potential mechanisms in a rat model of CP.MethodsThe chemical profile of GLY was analyzed by HPLC-UV. We used a mouse model of ear edema and a rat model of paw edema. Specifically, xylene was used to induce edema on the surface of one ear in mice, and carrageenan was injected subcutaneously into the right hind paws of rats to induce paw edema. The paw thickness, ear weight, and ear perfusion were measured and recorded. The CP model in rats was induced by irritating the throat with 5% ammonia and was used to evaluate the therapeutic efficacy of GLY. Levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor (TNF-α), and prostaglandin E2 (PGE2) were measured by ELISA in serum, and protein expression of cyclooxygenase-2 (COX-2) and nuclear factor kappa-B p65 (NF-κB p65) in the throat were detected by immunohistochemistry and Western blot to evaluate the anti-inflammatory mechanism of GLY. Hematological assays were also conducted.ResultsThere were four flavonoids identified in GLY: naringin, neohesperidin, baicalin, and wogonoside. The oral administration of GLY showed a significant inhibitory effect on xylene-induced ear swelling and ear blood flow in mice and significantly ameliorated rat right hind paw edema at doses of 6.2 and 12.4 g/kg. Mechanistic studies found that the anti-inflammatory activity of GLY was related to the inhibition of pro-inflammatory cytokines such as IL-1β, IL-6, TNF-α, and PGE2 and that GLY reduced the expression of COX-2 and NF-κB p65 proteins in the throat, attenuated throat injury, and reduced inflammatory exudates. Hematological analysis showed that treatment with GLY prevented increases in white blood cell (WBC), neutrophil (NEUT), lymphocyte (LYMPH) and monocyte (MONO) levels.ConclusionsThese studies indicated that GLY has beneficial anti-inflammatory effects on CP and that it acts through reducing pro-inflammatory factors such as IL-1β, IL-6, TNF-α, and PGE2, as well as decreasing WBC, NEUT, LYMPH and MONO levels and decreasing the expression of COX-2 and NF-κB p65 proteins. These findings may lay the groundwork for further studies of GLY as a suitable candidate for the treatment of inflammatory diseases such as CP.

ІМУНОГІСТОХІМІЧНІ КРІТЕРІАЛЬНО-ВАЖЛИВІ ПОКАЗНИКИ ЛІМФАНГІОГЕНЕЗУ РАКУ ЯЄЧНИКІВ

An in-depth study of the biology of tumor growth will help to identify factors that allow us to understand the pathogenetic mechanisms of the development of ovarian cancer metastasis and progression, as well as to become a theoretical basis for developing new approaches to the treatment of this disease. The aim of this study was to determine immunohistochemical and endothelial criteria for ovarian cancer. The postoperative samples of ovarian tumor tissues were divided into 3 groups: comparison group - ovarian cancer; main group - borderline ovarian tumor; benign ovarian tumors. The study was conducted according to the FIGO 2009 classification. The International Histological Classification of WHO 2013 Female Genital Tumors was used for morphological characteristics. Level of growth factors - sVEGF-A was performed by ELISA using standard test systems (BenderMedSystem, Austria). IGC material studies were performed on serial paraffin sections using a standard method with murine monoclonal antibodies to p53 (clone D0-7. Dilution 1: 100. "Dako"). Ventana Medical Systems, Inc. was used as the detection system. Positive and negative control reactions were performed. The label index (MI) was used to evaluate p53 nuclear expression. WCIF ImageJ and Aperio Image Scope were used to estimate the number and degree of cell staining. Statistical analysis of the obtained data was performed using Statistica 6.0. The results of morphological studies of ovarian cancer showed that in our study patients with serous cancer predominated - 78.5%. The second most frequently diagnosed cancer was undifferentiated. In the second stage of our study, we conducted a comparative analysis of the concentration of p53 in the serum and tissue of patients in the study groups, which showed the existence of significant differences. In patients of the POY and DOY groups, both total and local p53 protein activity were significantly higher than in the comparison group, p &lt;0.05. There was a positive correlation between p53 protein activity in serum and ovarian tissue. Serum VEGF A scores were statistically significantly correlated with the disease stage: Spearman rank correlation coefficient rho = 0.30; 95% CI = 0.02 - 0.536, p &lt;0.05. There were no correlations with patients' age, histological subtype, and degree of tumor differentiation. Considering the results of our study, we can conclude that the criterion-important indicators of QA are serum levels of p53 and the index of serum VEGF A, which is confirmed by the results of ROC analysis p = 0.0026, and indicates a good informativeness of the method.

Impact of Sacubitril and RAAS inhibitors on p53 expression in rat-induced heart failure. A new approach for ischemic heart disease therapy

Background: p53 is a well-known protein that prevents cancer formation, which is recognized as the main protein in the adaptation to many harmful stimuli, like oxidative stress. Among actions of p53, studies have shown that it has an important role in the development of heart failure (HF), and arteriosclerosis. Several clinical studies were done to investigate the role of p53 in the progression of HF and with the intention to improve management of heart failure. Objective: The purpose of this work was to investigate the mechanisms of myocardial injury that precipitates heart failure that is mediated by both β-adrenergic signaling and p53, then compare the results with administered sacubitril and angiotensin system blockers. Methods: Thirty female albino rats were allocated into five groups; group I: served as a control group; group II: were injected with isoproterenol for HF induction; and groups III, IV, and V (HF treated groups ) whereas rats received sacubitril alone, combination of sacubitril with ramipril and combination of sacubitril with aliskiren respectively, orally on daily basis. Results: Results revealed that rats of group II (HF induced) were significantly (P = 0.002) showed more myocardial injury and higher nuclear p53 expression compared to rats of the control group. Furthermore, rats of group III, IV, V (HF treated groups) showed significantly (P = 0.037) less myocardial injury and significantly (P = 0.015) less nuclear p53 expression compared to rats of the group II. Conclusions: It was concluded that rats received either sacubitril alone or with combination of ramipril or aliskiren for HF treatment were alleviated myocardial injury and lower nuclear p53 expression. It was concluded that anti-p53 approach may provide a novel therapeutic strategy for human ischemic heart diseases and myocardial infarction.

Association between NF-κB Activation in Peripheral Blood Mononuclear Cells and Late Skin and Subcutaneous Fibrosis following Radiotherapy.

Background This study aimed at evaluating the association between the speed of nuclear factor-kappa B (NF-κB) activation in peripheral blood mononuclear cells (PBMCs) and late skin and subcutaneous fibrosis in patients with head and neck squamous cell carcinoma (HNSCC) after radiotherapy. Methods The speed of NF-κB activation was represented by the nuclear p65 expression ratio before and after irradiation. The optimal time point to measure the ratio was determined by Western blot in the PBMCs from healthy outpatients ranging from 0 to 12 hours after ex vivo irradiation. We recruited patients with HNSCC who had received ratiotherapy and who were under regular follow-up care. We assessed the association between the risk of developing ≥grade 2 late fibrosis and the nuclear p65 expression ratio in the PBMCs after ex vivo irradiation in these patients. Results The maximum nuclear p65 ratio was observed at 1 hour after ex vivo irradiation in the PBMCs from the healthy outpatients. The speed of NF-κB activation was then represented by the nuclear p65 ratio in the PBMCs before and 1 hour after ex vivo irradiation. A total of 200 patients with HNSCC were recruited, 32.50% (n = 65) of which presented with ≥grade 2 late fibrosis. There was a significant association between the speed of NF-κB activation in the PBMCs and an increased risk of developing ≥grade 2 late fibrosis in these patients (P = 0.004). Subgroup analysis suggested that this finding was independent of the known clinical characteristics. Conclusions The speed of NF-κB activation might be a potential predictor of late toxicity in cancer patients after radiotherapy. Prospective studies are needed for validation.

Metastatic HPV-Associated Oropharyngeal Versus Primary Pulmonary Squamous Cell Carcinoma: is p16 Immunostain Useful?

The lungs are a common site of metastasis of head and neck (H&N) squamous cell carcinomas (SCC). This study attempts to define p16 immunoexpression and presence of HPV in primary SCC of the lung and determine their usefulness in discriminating between primary lung SCC and metastasis from HPV-associated oropharyngeal primary. Pathology archives were searched for patients with SCC of the lung without SCC elsewhere. Tissue microarray was constructed and immunohistochemistry performed using anti-p40 and anti-p16 antibodies. All cases were tested for HPV viral proteins E6/E7 by RNA in situ hybridization (ISH) and available positive cases for HPV DNA by polymerase chain reaction (PCR). Eight of 25 (32%) showed cytoplasmic and nuclear expression of p16: 2 (8%) strong and 2 (8%) moderate in > 70% of tumor cells; 1 (4%) strong, 1 (4%) moderate, and 1 (4%) weak in 50-70% of tumor cells; 1 (4%) weak in < 50% of tumor cells. E6/E7 mRNA ISH was negative in all cases. Seven of 8 (87.5%) p16-expressing cases were available for testing by HPV PCR; all were negative for HPV DNA. A retrospective control group of 12 patients with possible SCC metastatic to lung was also identified; high-risk HPV DNA was present in 3, confirming metastasis. p16 expression in lung SCC is not uncommon and may not discriminate between primary pulmonary SCC and metastasis from HPV-associated oropharyngeal primary. Confirmatory HPV testing (high risk HPV DNA or E6/E7 mRNA) is recommended to differentiate metastasis from oropharyngeal primary from two separate primaries.