P53 mRNA Levels Research Articles

Fibronectin protected bovine preantral follicles from the deleterious effects of kisspeptin

Kisspeptin (Kp), a multifunctional neuropeptide critical for initiating puberty and regulating ovulation, was reported to be expressed in mammalian ovaries. Fibronectin (FN), a major secretory product of granulosa cells, provided the extracellular environment for the cumulus cells during maturation. In the current study, we aimed to investigate the potential interplay between FN and Kp in bovine preantral follicles in the context of follicular development and quality. The results showed that Kp significantly reduced the follicular diameters after 14 days in culture, and this was prevented by the addition of FN. Follicles treated with Kp in the presence of FN showed lower levels of apoptotic cells compared to the Kp-treated group. The immunofluorescence analysis showed high levels of cyclooxygenase-2 (COX2), nuclear factor kappa B (NF-κB), and caspase 3, and low levels of sirtuin 1 (Sirt1) and Poly ADP-Ribose Polymerase 1 (PARP1) in the Kp-treated group compared to the control and FN-Kp co-treated groups. The protein expression levels of phosphoinositide 3 kinase (PI3K) increased significantly in the FN and FN-Kp combination treatment groups. Finally, we examined the signal pathway affecting the follicular development after Kp treatment. We detected a significant decrease in the mRNA levels of B-cell lymphoma 2 (BCL2), Sirt1, and PI3K, but the mRNA levels of NF-κB, Caspase3, COX2, P21, and P53 were significantly higher than in the control. Taken together, our results showed the importance of FN for preantral follicle developmental, and, for the first time, we reported that FN could neutralize the deleterious consequences of Kp, suggesting a potential role in the regulation of PI3K/Sirt1 signaling in bovine preantral follicle development.

Inhibition of autophagy aggravates molybdenum-induced mitochondrial dysfunction by aggravating oxidative stress in duck renal tubular epithelial cells

Excessive molybdenum (Mo) has adverse effects on animals. To elucidate the effects of autophagy on Mo-induced nephrotoxicity, the duck renal tubular epithelial cells were cultured in medium in absence and presence of (NH4)6Mo7O24.4H2O (0, 480, 720, 960 μM Mo), 3-Methyladenine (3-MA) (2.5 μM), and the combination of Mo and 3-MA for 12 h. After 12 h exposure, the MDC staining, morphologic observation, LC3 puncta, cell viability, autophagy-related genes mRNA and proteins levels, lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) level, antioxidant indices, mitochondrial membrane potential (MMP), mitochondrial mass, mitochondrial respiratory control ratio (RCR) and oxidative phosphorylation rate (OPR) were determined. The results showed that excessive Mo exposure significantly elevated the number of autophagosome and LC3 puncta, upregulated Beclin-1, Atg5, LC3A and LC3B mRNA levels, and LC3II/LC3I and Beclin-1 protein levels, decreased mTOR, p62 and Dynein mRNA levels and p62 protein level. Besides, co-treatment with Mo and 3-MA dramatically increased LDH release, ROS level, hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents as well as cell dam age, reduced cell viability, the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT), MMP, mitochondrial mass, mitochondrial RCR and OPR compared to treatment with Mo alone. Taken together, these results suggest that excessive Mo exposure can induce autophagy in duck renal tubular epithelial cells, inhibition of autophagy aggravates Mo-induced mitochondrial dysfunction by regulating oxidative stress.

Identification of key genes of hesperidin in inhibition of breast cancer stem cells by functional network analysis

Breast cancer therapy with classical chemotherapy is unable to eradicate breast cancer stem cells (BCSCs). Loss of p53 function causes growth and differentiation in cancer stem cells (CSCs); therefore, p53-targeted compounds can be developed for BCSCs-targeted drugs. Previously, hesperidin (HES), a citrus flavonoid, showed anticancer activities and increased efficacy of chemotherapy in several types of cancer in vitro and in vivo. This study was aimed to explore the key protein and molecular mechanism of hesperidin in the inhibition of BCSCs using bioinformatics and in vitro study. Bioinformatics analysis revealed about 75 potential therapeutic target proteins of HES in BCSCs (TH), in which TP53 was the only direct target protein (DTP) with a high degree score. Furthermore, the results of GO enrichment analysis showed that TH was taken part in the biological process of regulation of apoptosis and cell cycle. The KEGG pathway enrichment analysis also showed that TH is involved in several pathways, including cell cycle, p53 signaling pathway. In vitro experiment results showed that HES inhibited cell proliferation, mammosphere, and a colony formation, and migration in on MCF-7 3D cells (mammospheres). HES induced G0/G1 cell cycle arrest and apoptosis in MCF-7 cells 3D. In addition, HES treatment reduced the mRNA level of p21 but increased the mRNA level of cyclin D1 and p53 in the mammosphere. HES inhibits BCSCs in mammospheres. More importantly, this study highlighted p53 as a key protein in inhibition of BCSCs by HES. Future studies on the molecular mechanism are needed to validate the results of this study.

Histological, ultrastructural, and biochemical study on the possible role of Panax ginseng in ameliorating liver injury induced by Lambda cyhalotherin

BackgroundLambda-cyhalotherin (LCT) is a pyrithroid type 2 pesticide that is broadly utilized in pest control in public health, animal health, and agriculture. Although claiming that LCT has a low mammalian toxicity, several investigations reported its mammalian hepatotoxicity by mediating oxidative stress causes severe hepatotoxicity and liver damage.ResultsLCT significantly decreased catalase (CAT), superoxide dismutase (SOD), and total thiol (T. thiol) and increased lipid peroxidation (LPO). mRNA and protein expression levels of p53 were upregulated, whereas Bcl-2 mRNA and protein expression levels were downregulated in LCT-intoxicated animals. Also, light microscopic and ultrastructure studies for liver tissues of LCT-intoxicated animals showed mononuclear leukocytic infiltration in the parenchyma, congested portal vein with thickened wall, and proliferation of bile duct and hepatocytes with cytoplasmic vacuolations, fatty changes, and collagen fibers. Panax ginseng co-treatment attenuated oxidative stress biomarkers. Both tested doses of Panax ginseng (100 and 200 mg /kg b. wt./day) significantly decreased p53 and elevate Bcl-2 mRNA and protein expression levels and reveals significant amelioration and restoration of normal histology and ultrastructure of liver, but 200 mg/kg b. wt. of Panax ginseng seems to be more potent.ConclusionPanax ginseng exhibited ameliorative effect against hepatic oxidative stress, apoptosis, histopathological, and ultrastructural changes induced by LCT.

Electroacupuncture improves learning and memory functions in a rat cerebral ischemia/reperfusion injury model through PI3K/Akt signaling pathway activation.

Electroacupuncture has been widely used to treat cognitive impairment after cerebral ischemia, but the underlying mechanism has not yet been fully elucidated. Studies have shown that autophagy plays an important role in the formation and development of cognitive impairment, and the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway plays an important role in autophagy regulation. To investigate the role played by the PI3K/Akt signaling pathway in the electroacupuncture treatment of cerebral ischemia/reperfusion rat models, we first established a rat model of cerebral ischemia/reperfusion through the occlusion of the middle cerebral artery using the suture method. Starting at 2 hours after modeling, electroacupuncture was delivered at the Shenting (GV24) and Baihui (GV20) acupoints, with a dilatational wave (1-20 Hz frequency, 2 mA intensity, 6 V peak voltage), for 30 minutes/day over 8 consecutive days. Our results showed that electroacupuncture reduced the infarct volume in a rat model of cerebral ischemia/reperfusion injury, increased the mRNA expression levels of the PI3K/Akt signaling pathway-related factors Beclin-1, mammalian target of rapamycin (mTOR), and PI3K, increased the protein expression levels of phosphorylated Akt, Beclin-1, PI3K, and mTOR in the ischemic cerebral cortex, and simultaneously reduced p53 mRNA and protein expression levels. In the Morris water maze test, the latency to find the hidden platform was significantly shortened among rats subjected to electroacupuncture stimulation compared with rats without electroacupuncture stimulation. In the spatial probe test, the number of times that a rat crossed the target quadrant was increased in rats subjected to electroacupuncture stimulation compared with rats without electroacupuncture stimulation. Electroacupuncture stimulation applied to the Shenting (GV24) and Baihui (GV20) acupoints activated the PI3K/Akt signaling pathway and improved rat learning and memory impairment. This study was approved by the Animal Ethics Committee of the First Affiliated Hospital of Henan University of Traditional Chinese Medicine, China (approval No. 8150150901) on March 10, 2016.

Selenium supplementation protects against oxidative stress-induced cardiomyocyte cell cycle arrest through activation of PI3K/AKT.

Oxidative stress significantly contributes to heart disease, and thus might be a promising target for ameliorating heart failure. Mounting evidence suggests that selenium has chemotherapeutic potential for treating heart disease due to its regulation of selenoproteins, which play antioxidant regulatory roles. Oxidative stress-induced cardiomyocyte cell cycle arrest contributes to the loss of cardiomyocytes during heart failure. The protective effects and mechanism of selenium against oxidative stress-induced cell cycle arrest in cardiomyocytes warrant further study. H9c2 rat cardiomyoblast cells were treated with hydrogen peroxide in the presence or absence of selenium supplementation. Na2SeO3 pretreatment alleviated H2O2-induced oxidative stress, increased thioredoxin reductase (TXNRD) activity and glutathione peroxidase (GPx) activity and counteracted the H2O2-induced cell cycle arrest at the S phase. These effects were accompanied by attenuation of the H2O2-induced strengthening of the G2/M-phase inhibitory system, including increased mRNA and protein levels of cyclin-dependent kinase 1 (CDK1) and decreased p21 mRNA levels. Notably, Na2SeO3 pretreatment activated the PI3K/AKT signaling pathway, and inhibition of PI3K counteracted the protective effects of selenium on H2O2-induced cell cycle arrest. We corroborated our findings in vivo by inducing oxidative stress in pig heart by feeding a selenium deficient diet, which decreased the TXNRD activity, inactivated PI3K/AKT signaling and strengthened the G2/M-phase inhibitory system. We concluded that the cardioprotective effects of selenium supplementation against oxidative stress-induced cell cycle arrest in cardiomyocytes might be mediated by the selenoprotein-associated (GPx and TXNRD) antioxidant capacity, thereby activating redox status-associated PI3K/AKT pathways, which promote cell cycle progression by targeting the G2/M phase inhibitory system. This study provides new insight into the underlying mechanisms of cardioprotection effects of selenium at the cellular level.

Rosiglitazone Prevents Autophagy by Regulating Nrf2-Antioxidant Response Element in a Rat Model of Lithium-pilocarpine-induced Status Epilepticus

Status epilepticus (SE) leads to irreversible neuronal damage and consists of a complex pathogenesis that involves oxidative stress and subsequent autophagy. Rosiglitazone has recently been considered as a potential neuroprotective factor in epilepsy because of its antioxidative function. The aim of this study was to assess the effects of rosiglitazone in SE rat models and investigate whether its mechanisms of action involve autophagy via the antioxidant factor, nuclear factor erythroid 2-related factor 2 (Nrf2). The male Sprague-Dawley rats (200–220 g) were used to establish lithium-pilocarpine-induced SE model. We found that rosiglitazone markedly improved neuronal survival at 24-h post-SE as indicated via Hematoxylin-Eosin and Nissl staining. Furthermore, along with a reduction in reactive oxygen species, rosiglitazone pretreatment enhanced the antioxidative activity of superoxide dismutase and the expression level of Nrf2, as detected via chemical assay kits and Western blotting, respectively. In addition, the microtubule-associated protein light chain 3II (LC3II)/LC3I ratio was increased and peaked at 24 h after SE, whereas p62 mRNA levels were sharply elevated at 72 h after SE, both SE-induced increases of which were reversed via rosiglitazone pretreatment. To further test our hypothesis of the key role of Nrf2 in this process, small-interfering RNA for Nrf2 (siNrf2) was then transfected into SE rats to knockdown Nrf2 expression. We found that siNrf2 partially blocked the above effects of rosiglitazone on autophagy-related proteins in SE rats. Taken together, our findings suggest that rosiglitazone attenuates oxidative-stress-induced autophagy via increasing Nrf2 in SE rats and may be used as a promising therapeutic strategy for SE treatment.

The 40bp Indel Polymorphism rs150550023 in the MDM2 Promoter is Associated with Intriguing Shifts in Gene Expression in the p53-MDM2 Regulatory Hub.

Simple SummaryMany naturally occurring variants in the human DNA sequence have an influence on cancer risk, and most of them are located outside the parts of the genome that code for proteins. Presumably, they have an effect on the amount of messenger RNA and protein that is made from genes in their neighborhood. We analyzed one such DNA sequence variant termed rs150550023, which is near a gene termed MDM2 (human homolog of mouse double minute 2). The main function of MDM2 is to negatively regulate another gene termed p53, which is a very important tumor suppressor, i.e., it counteracts cancer. We studied the DNA sequence variant rs150550023 by comparing it in 407 female patients with breast cancer and 254 females without cancer. We found no evidence that rs150550023 plays an important role in the risk of breast cancer, the average age at which patients get breast cancer, how many patients develop breast cancer metastases, or how many patients die of breast cancer. However, we found evidence that rs150550023 may work together with another DNA sequence variant within the MDM2 gene termed SNP309. We also analyzed the tumor tissue of ≈100 breast cancer patients of this study after it had been surgically removed. We measured the amount of messenger RNA (mRNA) which is made from the genes MDM2, p53, and three other genes (termed p21, BAX, and PERP) for which it is known that p53 has an influence on the amount of their mRNA being made. We found that rs150550023 indeed has an influence on the amount of mRNA made from some of these genes. However, since MDM2 is a negative regulator of p53, it is likely that many of these alterations cancel each other out if both p53 and MDM2 is produced at higher levels.Most low-penetrance genetic risk factors for cancer are located in noncoding regions, presumably altering the regulation of neighboring genes. The poorly characterized Indel polymorphism rs150550023 (rs3730485; del1518) in the promoter of MDM2 (human homolog of mouse double minute 2) is a biologically plausible candidate genetic risk factor, which might influence the expression of MDM2, a key negative regulator of the central tumor suppressor p53. Here, we genotyped rs150550023 in a Central European hospital-based case–control study of 407 breast cancer patients and 254 female controls. mRNA levels of MDM2, p53, and the p53 target genes p21, BAX, and PERP were quantified with qRT-PCR, and p53 protein was assessed with immune histochemistry in ≈100 primary breast tumors with ascertained rs150550023 genotype. We found no evidence for an association of rs150550023 with the risk, age at onset, or prognosis of breast cancer. A possible synergism was observed with SNP309 in promoter P2 of MDM2. Mean mRNA levels of MDM2, p53, p21, and BAX were ≈1.5–3 fold elevated in TP53 wildtype tumors with the minor homozygous Del/Del genotype. However, systematic shifts in p53 protein levels or mutation rates were not observed, suggesting that the elevated p53 mRNA levels are due to regulatory feedback loops that compensate for the effects of rs150550023 on MDM2 expression.

Down-regulation of Survivin and Bcl-2 concomitant with the activation of caspase-3 as a mechanism of apoptotic death in KG1a and K562 cells upon exposure to a derivative from ciprofloxacin family

Ciprofloxacin derivatives belong to a family of antibiotics called fluoroquinolones. Recently, these compounds have been recommended for the treatment of cancer. In the present study, we assessed the cytotoxicity of several new synthetic ciprofloxacin derivatives and the apoptosis-inducing activity of the most efficient derivative in two human myeloid leukemia K562 and KG1-a cell lines. Among the prepared ciprofloxacin derivatives, 1-cyclopropyl-7-(4-(2-((3,7-dimethyloct-6-en-1-yl)oxy)-2-oxoethyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4dihydroquinoline-3-carboxylic acid (4-DMOCP) was more active compound with IC50 of 19.56 and 22.13 μM for K562 and KG1-a, respectively. Apoptotic activity of the 4-DMOCP was examined morphologically through Hoechst 33258 staining, Annexin V/PI double staining, and caspase-3 activity assays. Changes in the expression level of some apoptosis-related genes and protein, including Bcl-2, Bax, Survivin, p53, Caspase-8 and Caspase-9 were evaluated by the real-time quantitative PCR (qRT PCR) and western blotting. The qRT PCR analysis showed that 4-DMOCP induces apoptosis in both cell lines via the down-regulation of Survivin and Bcl2, up-regulation of caspase-8 and -9, as well as a time-dependent increase in the Bax/Bcl2 transcripts. The mRNA level of p53 was also increased in both cell lines. In addition, western blot analysis revealed that treatment with the compound, down-regulated the protein expression levels of Bcl2 and Survivin and up-regulated the protein level of Bax in both cell lines. These findings suggest that these new compounds can be good candidates for the treatment of acute and chronic myeloid leukemia.

FLT3 Inhibitors Induce Instability of p53 By Mir-181 Mediated Downregulation of Deubiquitinase YOD1 in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a hematological malignancy featured by impaired differentiation and uncontrolled proliferation of myeloid blasts. FLT3 internal tandem duplication (ITD) presents in 30-40% patients with AML, which serves as an independent poor prognostic marker and an attractive therapeutic target. Up to date, several tyrosine kinase inhibitors (TKIs) targeting FLT3 have been approved by FDA in the treatment of AML. However, FLT3 TKIs as single agents have limited effects to eliminate AML cells due to multiple drug-resistant mechanisms, including secondary FLT3 mutations, alternatively activated cell survival pathways, upregulation of FLT3 ligand, and downregulation of tumor suppressor genes. In the present study, we found that FLT3 TKIs decreased tumor suppressor p53 protein level by downregulation of YOD1 through miR-181 in FLT3-ITD mutant AML cells. In our previous studies, we generated FLT3-ITD transformed HCD-57 cells. HCD-57 cells are erythroleukemia cells that depend on erythropoietin for survival. When infected with recombinant retroviruses carrying FLT3-ITD, they acquired ability to proliferate in the absence of EPO. By using transcriptome analysis with RNAseq, we identified multiple differentially expressed miRNAs in HCD-57 transformed by FLT3-ITD, compared with parental HCD-57 cells. miR-181a-5p and miR-181b-5p were among these highly differentially expressed miRNAs. These two miRNAs were predicted to bind in 3'UTR of deubiquitinase YOD1 by using TargetScanHuman 7.2, an online tool to predict biological targets of miRNAs. Previous studies have shown that the ubiquitination and protein level of p53 is dysregulated due to overexpressed E3 ligase MDM2/4 in AML cells, but few studies focused on deubiquitinase of p53. We found that deubiquitinase YOD1 interacted with p53 by immunoprecipitation. Overexpression of YOD1 prevented degradation of p53 led by cycloheximide, a protein synthesis inhibitor. We further found that overexpression of YOD1 resulted in decreased ubiquitination of p53, indicating that YOD1 stabilized p53 protein through deubiquitination. Subsequently, we detected the expression of miR-181, YOD1 and p53 in a FLT3-ITD positive AML cell line MV-4-11 treated by FLT3 TKIs sorafenib, sunitinib and quizartinib. Real-time quantitative PCR showed that the treatment of FLT3 TKIs upregulated the expression of miR-181a-5p/miR-181b-5p, and downregulated mRNA level of their predicted target YOD1. The mRNA level of p53 remained unchanged but its protein level decreased with enhanced ubiquitination in MV-4-11 cells treated by FLT3 TKIs in the ubiquitination assay. These data suggested that FLT3 TKIs could reduce the stability of p53 by regulating miRNA-targeted YOD1. In addition, we collected peripheral blood mononuclear cells (PBMCs) from patients with AML and age-matched healthy donors. We found increased expression of miR-181a-5p and miR-181b-5p and decreased expression of YOD1 in PBMCs from AML patients compared with healthy donors, suggesting a pathological role of miR-181 to regulate YOD1/53 pathway in AML. In conclusion, our data showed that FLT3 TKIs induced instability of p53 by miR-181 mediated downregulation of YOD1. YOD1 as a novel deubiquitinase of p53 could play important roles in drug-resistance and progression of AML. Disclosures No relevant conflicts of interest to declare.

Impaired DNA damage response promotes atherosclerosis by enhancing cellular senescence and proinflammatory activation

Abstract Background DNA damage likely contributes to the pathogenesis and progression of atherosclerosis. Chemotherapy, radiotherapy and some DNA damage response (DDR) syndromes are known to increase atherosclerosis. However, causative links between DNA damage and atherosclerosis are yet to be established. Purpose The aim of this study was to investigate the role of impaired DDR in atherosclerosis. Methods and results Ku80, a DNA repair protein, -deficient apolipoprotein E knockout mice (Ku80+/− ApoE−/−) and ApoE−/m- mice were fed on high-fat diet for 4 weeks, and then oil red O staining was performed on their isolated aortas. Plaque area was significantly increased in Ku80+/− ApoE−/− mice. Immunohistochemical analysis of lesions from Ku80+/− ApoE−/− mice revealed enhanced accumulation of DNA double-strand breaks (DSBs). Similar results were obtained from Ku80+/− ApoE−/− mice fed on high-fat diet for 2 weeks which was considered as the early stages of atherosclerosis. mRNA levels of inflammatory cytokines such as IL-6 and MCP-1 in the aorta were significantly elevated in Ku80+/− ApoE−/− mice compared with those of ApoE−/− mice. To investigate whether impaired DDR is associated with the increased expression of inflammatory cytokines, vascular smooth muscle cells were isolated from Ku80 WT and Ku80+/− mice and subjected to quantitative RT-PCR analysis. mRNA levels of IL-6 and MCP-1 were significantly elevated in Ku80+/− cells. In addition, IκBα protein expression was significantly decreased in Ku80+/− cells, which suggested enhanced NF-κB activation in those cells. Immunofluorescent analysis revealed accumulation of DSBs and persistent DDR in Ku80+/− cells. In addition, elevated p16 mRNA level and senescence-associated β-gal activity were observed in Ku80+/− cells. Conclusion These results suggested that impaired DDR promoted atherosclerosis partly through cellular senescence and enhanced proinflammatory activation. Therefore, therapies targeting enhancing DDR may be beneficial in the prevention of DNA damage-induced atherosclerosis. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): KAKENHI

Effect of Photodynamic Therapy on Cyclin D1 and P53 mRNA Levels in Head and Neck Squamous Cell Carcinoma Cell Line

Background: Photodynamic Therapy (PDT) is considered as one of the alternative treatments for Head and Neck Squamous Cell Carcinoma (HNSCC). P53 mRNA is a tumor suppressor gene whose mutation increases the likelihood of uncontrolled cancer-like divisions. The mutation of CCND1 oncogenic increases the production of cyclin D1 as a tumorigenic protein. Objectives: This study aimed to determine the effect of PDT using toluidine blue as a photosensitizer on the CCND1 and P53 mRNA levels in the HNSCC cell line. Methods: A human HNSCC cell line from NCBI.C196 designation HN5 was used. Cells were divided into four groups: Group I (HNSCC cell line under the influence of toluidine blue and laser irradiation), group II (HNSCC cell line under the influence of toluidine blue), group III (HNSCC cell line under laser irradiation), group IV (control group, HNSCC cell line). A 660 nm THOR laser using toluidine blue as a photosensitizer was applied. The RNA extraction was performed in three steps, including cell degradation, purification, and precipitation by alcohol. The cDNA was prepared using Takara Kit. The Amplicon kit and Real-Time PCR analysis were used for the assessment of mRNA relative levels. Results: The P53 mRNA relative levels were 1.50 ± 0.33 in group I (P = 0.65), 1.49 ± 0.23 (P =0.5) in group II, and 1.40 ± 1.05 (P = 0.63) in group III. Compared to the control group, the mean increases in CCND1 mRNA were 18.01 ± 3.37 (p=0.04) in group I, 17.69 ± 3.3 (P = 0.03) in group II, and 9.01 ± 6.17 (P = 0.20) in group III. Conclusions: The comparison of the fold change index for P53 and CCND1 mRNA by the one-way ANOVA test showed that despite the increased expression of P53 and CCND1 mRNA in treatment groups compared to the control group, there was no statistically significant difference between the increases in P53 mRNA (P = 0.99) and CCND1 mRNA (P = 0.22) index between the groups. The results of this study could be a starting point for a better understanding of the mechanism of genes in PDT of the HNSCC cell line.

Inhibition of ROS/NLRP3/Caspase-1 mediated pyroptosis attenuates cadmium-induced apoptosis in duck renal tubular epithelial cells.

Cadmium (Cd) is an occupational and environmental pollutant, which mainly causes nephrotoxicity by damaging renal proximal tubular cells. To evaluate the effects of Cd on pyroptosis and the relationship between pyroptosis and apoptosis in duck renal tubular epithelial cells, the cells were cultured with 3CdSO4·8H2O (0, 2.5, 5.0, or 10.0μM Cd), N-acetyl-L-cysteine (NAC) (100.0μM), Z-YVAD-FMK (10.0μM) or the combination of Cd and NAC or Z-YVAD-FMK for 12h, and then cytotoxicity was assessed. The results evidenced that Cd significantly increased the releases of interleukin-18 (IL-18) and interleukin-1β (IL-1β), lactate dehydrogenase (LDH) and nitric oxide (NO), relative conductivity and cellular reactive oxygen species (ROS) level. Simultaneously, Cd also markedly upregulated NLRP3, Caspase-1, ASC, NEK7, IL-1β and IL-18 mRNA levels and NLRP3, Caspase-1 p20, GSDMD and ASC protein levels. Additionally, NAC notably improved the changes of above indicators induced by Cd. Combined treatment with Cd and Z-YVAD-FMK remarkably elevated Bcl-2 mRNA and protein levels, inhibited p53, Bax, Bak-1, Cyt C, Caspase-9 and Caspase-3 mRNA levels and p53, Bax, Bak-1, Caspase-9/cleaved Caspase-9 and Caspase-3/cleaved Caspase-3 protein levels, increased mitochondrial membrane potential (MMP), decreased apoptosis ratio and cell damage compared to treatment with Cd alone. Taken together, Cd exposure induces duck renal tubular epithelial cell pyroptosis through ROS/NLRP3/Caspase-1 signaling pathway, and inhibiting Caspase-1 dependent pyroptosis attenuates Cd-induced apoptosis.

Role of AMPK/mTOR-independent autophagy in clear cell renal cell carcinoma

We examined the status and role of autophagy, a process of lysosomal recycling of cellular material, in clear cell renal cell carcinoma (ccRCC). Paired samples of tumor and adjacent non-malignant...

Blood functional assay for rapid clinical interpretation of germline TP53 variants

BackgroundThe interpretation of germline TP53 variants is critical to ensure appropriate medical management of patients with cancer and follow-up of variant carriers. This interpretation remains complex and is becoming a...

Association of MicroRNA-21 with p53 at Mutant Sites R175H and R248Q, Clinicopathological Features, and Prognosis of NSCLC

This study aimed to investigate the association of miRNA-21 with mutant p53 expression, prognosis, interaction, and clinicopathological features of non-small cell lung cancer (NSCLC). Tissue specimens from 200 NSCLC patients were collected for qRT-PCR analysis of miR-21 and p53 expression, and p53 mutations were analyzed by Sanger sequencing. NSCLC cell lines were used to determine the effects of miR-21 knockdown on cell viability, cell cycle distribution, and p53 expression. We found that miR-21 expression was upregulated in NSCLC tissues, which was associated with an increase in p53 mRNA levels and with advanced tumor-node-metastasis (TNM) stages and lymph node metastasis. The most common mutant sites of p53 in NSCLC were R175H and R248Q. Moreover, elevated miR-21 and p53 expression levels were associated with shorter overall survival. Knockdown of miR-21 reduced NSCLC cell viability, arrested NSCLC cells at the G0-to-G1 phase of the cell cycle, and downregulated mutant p53 mRNA levels and phosphorylated p53 protein expression in A549 and H1650 cells compared to control cells. miR-21 is associated p53 at mutant sites R175H and R248Q, which seems not to be oncogenic, as it is being reported, since in a normal cell, without a mutated p53, it will probably have a protective role.

Herbal formula Renshenwuweizi decoction induces p53-mediated cell cycle arrest and apoptosis in A549 cells.

To investigate the effect of Renshenwuweizi decoction (RSWWZ decoction) on the growth of non-small cell lung cancer cells in vitro. A549 non-small cell lung cancer cells were divided into two groups: control and RSWWZ decoction treatment groups. Cell Counting Kit-8 was used to measure the inhibitory effect of RSWWZ decoction on the growth of A549 cells. 4', 6-diamidino-2-phenylindole staining and Annexin V-fluorescein isothiocyanate/propidium iodide double staining were used to investigate apoptosis in A549 cells following RSWWZ decoction treatment, and the mitochondrial membrane potential of treated cells was detected with Rhodamine 123. Cell cycle progression was analyzed by flow cytometry. The mRNA levels of p53, Bax, B-cell lymphoma-2 (Bcl-2) and p21 were measured by quantitative real-time reverse transcription polymerase chain reaction. The protein expressions of p53, Bax, Bcl-2, p21, cyclin-dependent kinases 2 (CDK2), and cyclin A were detected by Western blot. RSWWZ decoction reduced the viability of A549 cells in a dose-dependent manner by inducing apoptosis and decreased mitochondrial membrane potential. RSWWZ decoction increased p53 and Bax expression and decreased Bcl-2 expression in a dose-dependent manner. RSWWZ decoction also induced an S-phase cell cycle arrest by increasing p21 and decreasing cyclin A and CDK2 expression. In vitro experiments revealed that the Renshenwuweizi decoction-induced decrease in A549 cell proliferation was achieved by inducing apoptosis and S-phase cell cycle arrest via the p53 pathway. These findings provide the experimental basis for Renshenwuweizi decoction treatment of lung cancer.

Expression of Rab3b in Human Glioma: Influence on Cell Proliferation and Apoptosis.

Glioma is the most common human central nervous system tumour with a high degree of malignancy. Some Rab GTPases have significant effects on glioma. This study aimed to investigate the effect of Rab3b (Rab GTPase3b) on human glioma cell proliferation and apoptosis by silencing Rab3b and to initially verify the value of Rab3b expression for the diagnosis and progression in human glioma. Rab3b was silenced by siRNA transfection. Human glioma tissues and normal brain tissues adjacent to glioma were obtained by surgery. Rab3b, P53, Caspase 7, Bax, and Bim mRNA and protein expression levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. Cell proliferation was detected by the cell counting kit-8 assay, and the cell cycle and apoptosis were analysed using flow cytometry. Rab3b mRNA and protein expression in human glioma U251 and U87 cells were significantly downregulated after Rab3b silencing. Rab3b silencing inhibited glioma cell proliferation by promoting cell cycle arrest and induced apoptosis by upregulating the expression of apoptosis-related proteins. Rab3b expression in human glioma (n = 33) was significantly higher than that in normal brain tissues adjacent to glioma (n = 15). In addition, Rab3b expression levels in high-grade gliomas (WHO III-IV, n = 19) were also significantly higher than those in low-grade gliomas (WHO I-II, n = 14). Rab3b expression levels are significantly related to the progression of gliomas. Moreover, Rab3b silencing not only significantly inhibits cell proliferation in gliomas via cell cycle arrest but also promotes cell apoptosis by upregulating the expression levels of apoptosis-related proteins; however these preliminary in vitro results warrant validation on in vivo studies.

Florfenicol causes excessive lipid peroxidation and apoptosis induced renal injury in broilers

In order to study the effects and mechanism of florfenicol (FFC) on the kidney function of broilers, 180 1-day-old broilers were randomly divided into 6 groups, 30 in each group. Except for the control group, different doses of FFC were added to drinking water in the other 5 groups (0.15 g/L, 0.3 g/L, 0.6 g/L, 1.2 g/L and 1.8 g/L). After continuous administration for 5 days, renal histopathological changes, serum renal function indicators, renal peroxidation products and antioxidant factors, and apoptotic factors were detected in broilers aged 21 and 42 days. The results showed that compared with the control group, the kidney tissue structure was disordered, the glomerulus was atrophic, the cystic cavity was enlarged, and the epithelial cells of renal tubules were seriously vacuolated in broilers of treatment groups. And with the growth of broilers, the kidney injury of broilers in the low-dose FFC group was relieved. FFC significantly increased the contents of uric acid (UA), blood urea nitrogen (BUN), creatinine (CRE) in serum and malondialdehyde (MDA) in kidney of broilers, but significantly reduced the levels of glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) in kidney. FFC significantly inhibited the mRNA relative transcriptional levels of nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase-1 (NQO-1), and increased the mRNA and protein expression levels of p53, Caspase-3 and Caspase-6 in kidney tissue of broilers. It is concluded that FFC has certain nephrotoxicity to broilers, and its effect on kidney is dose-dependent and reversible. FFC causes intense lipid peroxidation in broiler kidney by inhibiting the expression of related factors in the downstream signal pathway of Nrf2. FFC can also up-regulate the expression of pro-apoptotic factors and accelerate the abnormal apoptosis of renal cells, thus seriously affecting the renal function of broilers.

The Effect of Topical Administration of an Ointment Prepared From Trifolium repens Hydroethanolic Extract on the Acceleration of Excisional Cutaneous Wound Healing.

Natural agents with antioxidant and anti-inflammatory properties are safer than synthetic agents and may improve wound healing. The purpose of this study is to evaluate the in vivo wound healing potential of an ointment prepared from Trifolium repens hydroethanolic extract (T repens) concerning excisional wounds in a rat model. Seventy-two adult Wistar rats were divided into 4 groups: a control group and 3 groups of animals treated with 1.5% T repens, 3% T repens, and 6% T repens. A full-thickness wound with an area of 314 mm2 was created in each rat. To investigate the effect of T repens on wound healing, the wound area, histological analyses (eg, angiogenesis, fibroblast, fibrocyte, mast-cell distribution), intracytoplasmic carbohydrate storage, and B-cell lymphoma 2-like protein 4 (BAX), B-cell lymphoma 2 (Bcl-2), and p53 gene expression in the wound tissue were evaluated for 21 days. Antioxidant activity was further measured by 2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 2,2-Di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH) assays. The animals in the treated groups showed higher wound contraction ratios (P ⟨ .05), angiogenesis, fibroblast, fibrocyte, and mast-cell distribution and intracytoplasmic carbohydrate storage compared with the control group (P ⟨ .05). Moreover, the topical administration of T repens increased the messenger ribonucleic acid (mRNA) level of Bcl-2 and reduced the BAX and p53 mRNA levels (P ⟨ .05). These findings further revealed the strong antioxidant activity of T repens. The topical administration of T repens accelerated wound healing by increasing angiogenesis; fibroblast, fibrocyte, and mast-cell distribution; intracytoplasmic carbohydrate storage; and modulation in genes involved in apoptosis in a rat model.