Liproxstatin-1 alleviates cartilage degradation by inhibiting chondrocyte ferroptosis in the temporomandibular joint.

BGROUND INFORMATION: Ferroptosis contributes to temporomandibular joint osteoarthritis (TMJOA) lesion development and is still poorly understood. RESULTS: In this study, we used different TMJOA animal models to examine whether ferroptosis was related to disease onset in TMJOA induced by monosodium iodoacetate (MIA), IL-1ß, occlusion disorder (OD), and unilateral anterior crossbite (UAC). Immunohistochemical staining and Western blot analysis were used to detect ferroptosis- and cartilage degradation-related protein expression. Our results revealed reduced levels of the ferroptosis-related protein GPX4 in the cartilage layer, but the levels of ACSL4 and P53 were increased in the condyle. Injection of the ferroptosis inhibitor liproxstatin-1 (Lip-1) effectively decreased ACSL4, P53 and TRF expression. In vitro, IL-1ß reduced cartilage extracellular matrix expression in mandibular condylar chondrocytes (MCCs). Lip-1 maintained the morphology and function of mitochondria and ameliorated the exacerbation of lipid peroxidation and reactive oxygen species (ROS) production induced by IL-1ß. CONCLUSION: These results suggest that chondrocyte ferroptosis plays an important role in the development and progression of TMJOA. SIGNIFICANCE: Inhibiting condylar chondrocyte ferroptosis could be a promising therapeutic strategy for TMJOA.

Bifidobacterium longum SX-1326 ameliorates gastrointestinal toxicity after irinotecan chemotherapy via modulating the P53 signaling pathway and brain-gut axis.

BACKGROUND: Colorectal cancer (CRC) is a prevalent malignant malignancy affecting the gastrointestinal tract that is usually treated clinically with chemotherapeutic agents, whereas chemotherapeutic agents can cause severe gastrointestinal toxicity, which brings great pain to patients. Therefore, finding effective adjuvant agents for chemotherapy is crucial. METHODS: In this study, a CRC mouse model was successfully constructed using AOM/DSS, and the treatment was carried out by probiotic Bifidobacterium longum SX-1326 (B. longum SX-1326) in combination with irinotecan. Combining with various techniques of modern biomedical research, such as Hematoxylin and Eosin (H&E), Immunohistochemistry (IHC), Western blotting and 16S rDNA sequencing, we intend to elucidate the effect and mechanism of B. longum SX-1326 in improving the anticancer efficacy and reducing the side effects on the different levels of molecules, animals, and bacteria. RESULTS: Our results showed that B. longum SX-1326 enhanced the expression of Cleaved Caspase-3 (M vs. U = p < 0.01) and down-regulated the expression level of B-cell lymphoma-2 (Bcl-2) through up-regulation of the p53 signaling pathway in CRC mice, which resulted in an adjuvant effect on the treatment of CRC with irinotecan. Moreover, B. longum SX-1326 was also able to regulate the gut-brain-axis (GBA) by restoring damaged enterochromaffin cells, reducing the release of 5-hydroxytryptamine (5-HT) in brain tissue (I vs. U = 89.26 vs. 75.03, p < 0.05), and further alleviating the adverse effects of nausea and vomiting. In addition, B. longum SX-1326 reversed dysbiosis in CRC model mice by increasing the levels of Dehalobacterium, Ruminnococcus, and Mucispirillum. And further alleviated colorectal inflammation by downregulating the TLR4/MyD88/NF-κB signaling pathway. CONCLUSIONS: In conclusion, our work reveals that B. longum SX-1326 has a favorable effect in adjuvant irinotecan for CRC and amelioration of post-chemotherapy side effects, and also provides the theoretical basis and data for finding a safe and efficient chemotherapeutic adjuvant.

Hypoxic stress caused apoptosis of MDBK cells by p53/BCL6-mitochondrial apoptosis pathway.

Hypoxia is an important characteristic of Tibetan plateau environment. It can lead to apoptosis, but the mechanism of apoptosis caused by hypoxic stress needs further clarification. Here, cattle kidney cell MDBK were used as cell model. The effect of hypoxic stress on apoptosis and its molecular mechanism were explored. MDBK cells were treated with hypoxic stress, apoptosis and mitochondrial apoptotic pathway were significantly increased, and the expression of B-cell lymphoma 6 (BCL6) was significantly decreased. Overexpressing or inhibiting BCL6 demonstrated that BCL6 inhibited the apoptosis. And the increase of apoptosis controlled by hypoxic stress was blocked by BCL6 overexpressing. MDBK cells were treated with hypoxic stress, the expression and the nuclear localization of p53 were significantly increased. Overexpressing or inhibiting p53 demonstrated that hypoxic stress suppressed the expression of BCL6 through p53. Together, these results indicated that hypoxic stress induced the apoptosis of MDBK cells, and BCL6 was an important negative factor for this regulation process. In MDBK cells, hypoxic stress suppressed the expression of BCL6 through p53/BCL6-mitochondrial apoptotic pathway. This study enhanced current understanding of the molecular mechanisms underlying the regulation of apoptosis by hypoxic stress in MDBK cells.

Naringenin protects myocardial ischemia/reperfusion injury by regulating miR-24-3p to inhibit cell death-inducing p53 target 1 expression.

Myocardial ischemia/reperfusion (I/R) causes serious threats to human life. Naringenin, a polyphenolic compound naturally occurring in citrus fruit, has cardioprotective effects against myocardial I/R injury. Besides, miR-24-3p is also reported to have cardioprotective effects. We intended to explore whether the cardioprotective effects of naringenin relate to miR-24-3p and its underlying mechanism. In this study, we used an in vivo rat myocardial I/R model and an in vitro cardiomyocyte H9c2 hypoxia/reoxygenation (H/R) model. Myocardial injury was detected by hematoxylin-eosin staining and ELISA for creatine kinase (CK), malondialdehyde (MDA), and lactate dehydrogenase (LDH). miR-24-3p and cell death inducing p53 target 1 (Cdip1) mRNA expressions were examined by RT-PCR. We find that naringenin pretreatment significantly relieves myocardial I/R injury, reduces LDH, CD, and MDA levels, and increases miR-24-3p expression. Furthermore, miR-24-3p alleviates myocardial I/R injury partially through regulating Cdip1. Moreover, naringenin protects myocardial I/R injury partially by regulating miR-24-3p to inhibit Cdip1 expression. In conclusion, our data suggest naringenin protects myocardial I/R injury partially through miR-24-3p/Cdip1 axis.

TuBG1 promotes hepatocellular carcinoma via ATR/P53-apoptosis and cycling pathways.

BACKGROUND: As reported, γ-tubulin (TuBG1) is related to the occurrence and development of various types of malignant tumors. However, its role in hepatocellular cancer (HCC) is not clear. The present study was to investigate the relationship between TuBG1 and clinical parameters and survival in HCC patients. METHODS: The correlation between TuBG1 and clinical parameters and survival in HCC patients was explored by bioinformatics analysis. Immunohistochemistry was used for the verification. The molecular function of TuBG1 was measured using colony formation, scratch assay, trans-well assay and flow cytometry. Gene set enrichment analysis (GSEA) was used to pick up the enriched pathways, followed by investigating the target pathways using Western blotting. The tumor-immune system interactions and drug bank database (TISIDB) was used to evaluate TuBG1 and immunity. Based on the TuBG1-related immune genes, a prognostic model was constructed and was further validated internally and externally. RESULTS: The bioinformatic analysis found high expressed TuBG1 in HCC tissue, which was confirmed using immunohistochemistry and Western blotting. After silencing the TuBG1 in HCC cell lines, more G1 arrested cells were found, cell proliferation and invasion were inhibited, and apoptosis was promoted. Furthermore, the silence of TuBG1 increased the expressions of Ataxia-Telangiectasia and Rad-3 (ATR), phospho-P38 mitogen-activated protein kinase (P-P38MAPK), phospho-P53 (P-P53), B-cell lymphoma-2 associated X protein (Bax), cleaved caspase 3 and P21; decreased the expressions of B-cell lymphoma-2 (Bcl-2), cyclin D1, cyclin E2, cyclin-dependent kinase 2 (CDK2) and CDK4. The correlation analysis of immunohistochemistry and clinical parameters and survival data revealed that TuBG1 was negatively correlated with the overall survival. The constructed immune prognosis model could effectively evaluate the prognosis. CONCLUSIONS: The increased expression of TuBG1 in HCC is associated with poor prognosis, which might be involved in the occurrence and development of HCC.

Noise and silver nanoparticles induce hepatotoxicity via CYP450/NF-Kappa B 2 and p53 signaling pathways in a rat model.

Co-exposure to noise and nanomaterials, such as silver nanoparticles (Silver-NPs), is a common occurrence in today's industries. This study aimed to investigate the effects of exposure to noise and the administration of silver-NPs on the liver tissue of rats. Thirty-six adult male albino Wistar rats were randomly divided into six groups: a control group (administered saline intraperitoneally), two groups administered different doses of Silver-NPs (50 mg/kg and 100 mg/kg, 5 days a week for 28 days), two groups exposed to noise in addition to Silver-NPs (at the same doses as mentioned before), and a group exposed only to noise (104 dB, 6 hours a day, 5 days a week for 4 weeks). Blood samples were taken to assess hepatic-functional alterations, such as serum ALP, ALT, and AST levels. Additionally, biochemical parameters (MDA, GPX, and CAT) and the silver concentration in the liver were measured. Histopathological analysis, mRNA expression (P53 and NF-κB), protein expression (CYP450), and liver weight changes in rats were also documented. The study found that the administration of Silver-NPs and exposure to noise resulted in elevated levels of ALP, ALT, AST, and MDA (p < .01). Conversely, GPX and CAT levels decreased in all groups compared with the control group (p < .0001). There was a significant increase (p < .05) in liver weight and silver concentration in the liver tissues of groups administered Silver-NPs (50 mg/kg) plus noise exposure, Silver-NPs (100 mg/kg), and Silver-NPs (100 mg/kg) plus noise exposure, respectively. The expression rate of P53, NF-κB, and cytochromes P450 (CYPs-450) was increased in the experimental groups (p < .05). These findings were further confirmed by histopathological changes. In conclusion, this study demonstrated that exposure to noise and the administration of Silver-NPs exacerbated liver damage by increasing protein and gene expression, causing hepatic necrosis, altering biochemical parameters, and affecting liver weight.

Topical metformin accelerates wound healing by promoting collagen synthesis and inhibiting apoptosis in a diabetic wound model.

The wound healing process, which is a pathophysiological process that includes various phases, is interrupted in diabetes due to hyperglycemia, and since deterioration occurs in these phases, a normal healing process is not observed. The aim of the current study is to investigate the proliferative and antiapoptotic effects of metformin on wound healing after topical application on diabetic and non-diabetic wounds. For this purpose, we applied metformin topically on the full-thickness excisional wound model we created in diabetic and nondiabetic groups. We investigated the effects of metformin on the apoptotic index by the Terminal deoxynucleotidyl transferase mediated dUTP Nick-End Labeling method and on collagen-I, collagen-III, p53, and c-jun expression levels by quantitative reverse transcription polymerase chain reaction technique in wound biopsy tissues. Our results showed that c-jun and p53 mRNA levels and apoptotic index increased with the effect of diabetes, while collagen synthesis was disrupted. As a result of the study, we showed that metformin increases cellular proliferation and has anti-apoptotic effects by increasing collagen-I/III expression and decreasing p53/c-jun level, especially in diabetic wounds and also in normal wounds. In conclusion, the topical effect of metformin on diabetic wounds reversed the adverse effects caused by diabetes, increasing the wound healing rate and improving the wound repair process.

Effects of Imatinib Combined With Everolimus on Mouse Pituitary Tumor Cell AtT-20.

Context: Pituitary adenoma is a clinical syndrome in which excessive production of pituitary corticotropin (ACTH). For ACTH tumor cells, researchers know little about the influence of the cell-cycle process on ACTH production and cell proliferation. Some research has shown that imatinib can induce apoptosis of tumor cells. Objective: The study intended to explore the effects and molecular mechanisms of imatinib combined with everolimus on AtT-20 cells in AtT-20 mouse pituitary tumors. Design: The research team performed a laboratory study using murine corticotropin tumor AtT-20 cells. Setting: The study took place at the Department of Neurosurgery at Renmin Hospital of the Hubei University of Medicine in Shiyan, Hubei, China. Intervention: The research team cultured the cells in AtT-20-cell-specific medium containing 100 µg/mL of streptomycin, 100 U/mL of penicillin, and 10% fetal bovine serum at 37°C and 5% CO2. The team divided the cells into a control group, a normal culture without the drug, and an intervention group, incubated for 24 hours with 1 µM of imatinib and 3 µM of everolimus when the cells grew to 40% confluence. Outcome Measures: The research team: (1) determined the effects of the combined drugs on cell viability using a methyl thiazolyl tetrazolium (MTT) assay; (2) detected the cell's mitochondrial membrane potential and LDH leakage using "sytox blue, 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide," CBIC2(3) or JC-1, and lactate dehydrogenase (LDH) assay kits, respectively; (3) detected AtT-20 cell apoptosis using a "terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling" (TUNEL) kit; (4) analyzed the expression of protein kinase B (p-Akt), cAMP-response element binding protein (p-CREB), p27, p53, and cyclin E using a Western blot test; (5) detected the mRNA expression of opioid melanin procorticotropin (POMC)), caspase-3, and pituitary tumor transforming gene 1 (PTTG1) using reverse transcription-polymerase chain reaction (RT-PCR); (6) measure the concentration of adreno-cortico-tropic-hormone (ACTH) in the supernatant using an enzyme-linked immunoassay (ELISA) kit; and (7) assessed the cell cycle distribution using flow cytometry. Results: No differences existed in cell viability between the groups at the baseline (0 h) of the culture period (P > .05). Compared to the control group, the intervention group's: (1) cell viability was significantly lower at 4, 8, and 12 hours and postintervention at 16 hours (P < .001); (2) LDH concentration was significantly higher (P < .001); (3) mitochondrial membrane potential was significantly lower (P < .001); (4) apoptosis rate of TUNEL was significantly higher (P < .001 ); (5) expression of p-Akt, p-CREB phosphorylation, and cyclin E was significantly lower (P < .001), (6) expression of p27 and p53 protein was significantly higher (P < .001); (7) mRNA expression of POMC and PTTG1 were significantly lower (P < .001); (8) mRNA expression of caspase-3 was significantly higher (P < .001); (9) concentration of ACTH was lower (P < .001); and (10) percentage of cells in the G0/G1 phase was significantly higher, while the percentage of cells in the S phase was significantly lower (P < .05). Conclusions: Imatinib combined with everolimus can affect the AtT-20 cell cycle through the signaling pathway of the phosphatidylin-ositol-3-kinase (PI3K)/Akt/ protein kinase A (PKA) system and can inhibit cell proliferation and induce cell apoptosis. Therefore, Imatinib and everolimus may be an effective combination of candidates for drugs for mouse pituitary tumor.

Evaluation of the anticancer activity of Origanum Marjoram as a safe natural drink for daily use.

BACKGROUND: Chemotherapeutic agents have numerous side effects. There is a major interest in using natural and safe plants as food or drink to prevent from cancer. Origanum marjoram (OMAE) is a medicinal plant that can be used as a tea, food, and additive in traditional medicine. OBJECTIVE: This study aimed to evaluate the potential anticancer effects of OMAE as a soft drink for daily use against a model cancer, prevention and treatment. METHOD: MCF-7 cells were chosen as model cancer cells. The MTT assay was used to assess the in vitro inhibitory effects of OMAE on cell growth. Moreover, quantitative real-time PCR (qRT-PCR) was used to detect specific genes associated with cancer, such as ESR1, Bax, Bcl-2, and p53. Furthermore, the DNA damage was evaluated using the comet assay. RESULTS: OMAE has IC50 of 53.1 and IC90 of 97.5 µg/ml dependent inhibition of cell proliferation after 48 h of treatment toward MCF-7. Also, a significant decrease in the expression level of the ESR1 gene in the MCF-7 cell line. Furthermore, there was a significant increase in the comet length and comet-positive cells after treatment with OMAE (88.7%) compared with those in the untreated control cells (9.5%), suggesting a high induction of DNA damage by OMAE. Also, OMAE showed a modification in bcl-2, tumor suppressor gene (p53), and Bax levels and influenced the BAX/BCL-2 ratio via releasing the cytochrome C. CONCLUSION: The results of the study were promising, suggesting that the reduced apoptotic rate of MCF-7 breast cancer cells in this work was correlated to the potential anticancer effect of OMAE which would be a suitable preventable drink against cancer. However, further studies are needed to fully understand the potential of OMAE as a cancer treatment.

Schisandrin B promotes senescence of activated hepatic stellate cell via NCOA4-mediated ferritinophagy.

CONTEXT: Schisandrin B (Sch B), an active ingredient from Schisandrae chinensis (Turcz.) Baill. (Schisandraceae) Fructus, possesses diverse pharmacological activities including antitumor, anti-inflammation, and hepatoprotection. OBJECTIVE: To explore the effect of Sch B on activated HSCs senescence in hepatic fibrosis and the mechanisms implicated. MATERIALS AND METHODS: ICR mice with CCl4-induced hepatic fibrosis were supplemented with Sch B (40 mg/kg) for 30 d and LX2 cells were treated with Sch B (5, 10 and 20 µM) for 24 h. Cellular senescence was assessed by senescence-related indicators senescence-associated ß-galactosidase (SA-ß-gal) activity and the expression of p16, p21, p53, γ-H2AX, H3K9me3, TERT, TRF1, and TRF2. Ferric ammonium citrate (FAC) and NCOA4 siRNA were used to evaluate the mechanisms underlying Sch B's regulation of cellular senescence. RESULTS: Sch B (40 mg/kg) reduced serum levels of AST and ALT (53.2% and 63.6%), alleviated hepatic collagen deposition, and promoted activated HSCs senescence in mice. Treatment with Sch B (20 µM) decreased cell viability to 80.38 ± 4.87% and elevated SA-ß-gal activity, with the levels of p16, p21 and p53 increased by 4.5-, 2.9-, and 3.5-fold and the levels of TERT, TRF1 and TRF2 decreased by 2.4-, 2.7-, and 2.6-fold in LX2 cells. FAC (400 µM) enhanced Sch B's effect mentioned above. NCOA4 siRNA weakened the effects of Sch B on iron deposition and HSCs senescence. CONCLUSIONS: Sch B could ameliorate hepatic fibrosis through the promotion of activated HSCs senescence, which might be attributed to its induction of NCOA4-mediated ferritinophagy and subsequent iron overload.

Proanthocyanidins inhibit the apoptosis and aging of nucleus pulposus cells through the PI3K/Akt pathway delaying intervertebral disc degeneration.

BACKGROUND: Low back pain is a common symptom of intervertebral disc degeneration (IDD), which seriously affects the quality of life of patients. The abnormal apoptosis and senescence of nucleus pulposus (NP) cells play important roles in the pathogenesis of IDD. Proanthocyanidins (PACs) are polyphenolic compounds with anti-apoptosis and anti-aging effects. However, their functions in NP cells are not yet clear. Therefore, this study was performed to explore the effects of PACs on NP cell apoptosis and aging and the underlying mechanisms of action. METHODS: Cell viability was evaluated by cell counting kit-8 (CCK-8) assay. The apoptosis rate was determined TUNEL assays. Levels of apoptosis-associated molecules (Bcl-2, Bax, C-caspase-3 and Caspase-9) were evaluated via western blot. The senescence was observed through SA-ß-gal staining and western blotting analysis was performed to observe the expression of senescence-related molecules (p-P53, P53, P21 and P16). RESULTS: Pretreatment with PACs exhibited protective effects against IL-1ß-induced NP cell apoptosis including apoptosis rate, expressions of proapoptosis and antiapoptosis related genes and protein. PACs could also alleviate the increase of p-p53, P21, and P16 in IL-1ß-treated NP cells. SA-ß-gal staining showed that IL-1ß-induced senescence of NP cells was prevented by PACs pertreatment. In addition, PACs activated PI3K/Akt pathway in IL-1ß-stimulated NP cells. However, these protected effects were inhibited after LY294002 treatment. CONCLUSION: The results of the present study showed that PACs inhibit IL-1ß-induced apoptosis and aging of NP cells by activating the PI3K/Akt pathway, and suggested that PACs have therapeutic potential for IDD.

Synthesis of new thienylnicotinamidines: Proapoptotic profile and cell cycle arrest of HepG2 cells.

Fourteen new thienylnicotinamidines and their analogs 5a-5k, 12, 13a, and 13b were prepared and their antiproliferative potential was evaluated against the growth of 60 cancer cell lines. The tested compounds had a strong antiproliferative efficacy against almost all cancer cell lines, with the average GI50 at ~2.20 µM. The effect of the thienylnicotinamidines on the growth of normal lung fibroblast cells (WI-38) indicated that these derivatives are safe to the normal cells. The selectivity index (SI) ranges from 5.5- to 42.0-fold. The conceivable mechanisms of action of the effective compounds 5d, 5f, 5g, 5i, 5j, and 5k with high SI were investigated. Although the thienylnicotinamidines are similar in structure, they could be divided into three groups as per their effects on gene expression: The first group (5d and 5f) elevated p53 and caspase 3 expression, the second group (5g and 5i) elevated p53 expression, and the last group (5j and 5k) elevated p53 and reduced topoII expression. Many thienylnicotinamides inhibited the vascular endothelial growth factor receptor-2 (VEGFR-2) in cell lysates at concentrations comparable to or better than pazopanib. The data of caspase 3 expression were confirmed by measuring the protein level by Western blot and the activity of the cleaved active enzyme. The ability to arrest the cell cycle and induce apoptosis was confirmed by flow cytometry. Taken together, two derivatives, 5d and 5f, with a distinctive VEGFR-2 inhibitory activity and a proapoptotic and cell cycle arrest profile merit further investigations.

Magnetic Targeting of 5-Fluorouracil-Loaded Liposome-Nanogels for In Vivo Breast Cancer Therapy and the Cytotoxic Effects on Liver and Kidney.

In our previous paper, we demonstrated the ex vivo studies of non-toxic liposome-nanogel systems by which the long-term drug release could be provided from hybrid systems for the 5-fluorouracil (5-FU) drug molecule. The aim of this study was the in vivo magnetic targeting of 5-FU-loaded Fe3O4 nanoparticles including DPPC liposome-based PEGylated nanogels (5-FU loaded Fe3O4LPN) to breast cancer tissue and the investigation of the treatment and cytotoxic effects of that hybrid system to the liver and kidney in CD-1 mice using an external magnetic field. The effectiveness of the control, 5-FU group, Fe3O4LPN, and 5-FU-loaded Fe3O4LPN systems was evaluated using histopathology in terms of p53, ESR, PRG and C-erB-2, and qRT-PCR in terms of TYMS, ESR-1, RPG, and EGRF. Also, the cytotoxicity was analyzed by histopathological evaluation of kidney and liver tissues. Caspase-3 and caspase-9 evaluations were performed by qRT-PCR. The creatinine and ALT levels were also evaluated by comparing the blood samples of all groups. A total of 300-nm TEM-sized Fe3O4LNP hybrid system was successfully prepared. That system significantly decreased the TYMS and ESR1 levels after treatment process and increased the levels of p53 expression. The levels of caspase-3 mRNA did not change during the treatment, but the level of caspase-9 mRNA level was significantly decreased. The magnetically targeted liposome-based nanogel hybrid system is promising an effective therapy for the breast tumor with less liver and kidney damage. This Fe3O4LNP hybrid system could be useful for the similar small molecules.

p53 pulse modulation differentially regulates target gene promoters to regulate cell fate decisions.

The p53 tumor suppressor regulates distinct responses to cellular stresses. Although different stresses generate different p53 dynamics, the mechanisms by which cells decode p53 dynamics to differentially regulate target genes are not well understood. Here, we determined in individual cells how canonical p53 target gene promoters vary in responsiveness to features of p53 dynamics. Employing a chemical perturbation approach, we independently modulated p53 pulse amplitude, duration, or frequency, and we then monitored p53 levels and target promoter activation in individual cells. We identified distinct signal processing features-thresholding in response to amplitude modulation, a refractory period in response to duration modulation, and dynamic filtering in response to frequency modulation. We then showed that the signal processing features not only affect p53 target promoter activation, they also affect p53 regulation and downstream cellular functions. Our study shows how different promoters can differentially decode features of p53 dynamics to generate distinct responses, providing insight into how perturbing p53 dynamics can be used to generate distinct cell fates.

Mathematical modelling of core regulatory mechanism in p53 protein that activates apoptotic switch.

The p53 protein, a tumour suppressor, is a key player in the DNA damage response. The activation of apoptosis by p53 involves the intrinsic apoptotic pathway to eliminate stressed cells that contain DNA lesions. Recent experiments have found that apoptosis happen in an all-or-none switch like manner  (Albeck et al., 2008; Rehm et al., 2002). We focus on modelling the mechanism of p53 activation of apoptosis in response to sustained high DNA double-strand breaks. The aim of the research is to investigate the design principles behind the regulation of p53 activation of apoptotic switch. Building on previous models (Chong et al., 2015; Zhang et al., 2009a), we developed a mathematical model that incorporated the molecular interactions in the core regulation of p53 and the apoptosis initiation module involving Puma, Bcl2 and Bax. Activation of Bax was assumed to be an indicator of apoptosis initiation. Chen et al. (2013) suggested that one of the components in the p53 pathway may control a threshold activation of apoptosis. We hypothesized that ATM auto-activation is the component that controls p53 threshold activation of apoptosis with ATM's multi-site autophosphorylation depending on damage intensity. The constructed model demonstrated how molecular interactions and stress signalling molecule ATM's auto-activation of the p53 network dictate cell fate decisions. Our simulation results are qualitatively consistent with the experimental findings of all-or-none activation of apoptosis and predicted overexpression of Bcl2 as a factor in causing malfunction of the apoptotic switch. We present a simplified yet plausible model of molecular mechanism that controls p53 activation of apoptotic switch.

Δ133p53 decreases the chemosensitivity of carcinoma cell line H1299.

The research evaluated the effect of Δ133p53 on the chemosensitivity of lung adenocarcinoma cell line H1299. By this study, the drug-resistant molecular marker and a new target for cancer therapy could be provided. Δ133p53 or negative control plasmid were transferred into H1299 cells by lentivirus vector. The expression of Δ133p53 in transfected cells was examined using immunofluorescence. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method and colony formation test were applied to detect drug sensitivity after cisplatin or 5-fluorouracil (5-FU) treatment. After cisplatin (CDDP)/FU treatment, MTT assay demonstrated that the inhibition rate of H1299/Δ133p53 cell was reduced compared with that of the H1299 and H1299/NEG cells at the same concentration of drug. The 50% inhibitory concentrations (IC 50 ) of CDDP and 5-FU rose by 36.1 and 30.2%, respectively (P < 0.05). The colony formation assay suggested that the cell proliferation ability of H1299/Δ133p53 cell was prominently increased when compared with that of control group H1299 and H1299 /NEG cells (P < 0.05). The present study demonstrated that the transfection of the Δ133p53 gene in H1299 cells led to the reduction of chemosensitivity.

Expression and role of p53 in oral lichen planus patients.

PURPOSE: Oral lichen planus (OLP) is an autoimmune skin and mucosal disorder. The range of malignant transformation in OLP varies between 0.1-3%. p53 is a tumor suppressor protein. Defective p53 could allow abnormal cells to proliferate, resulting in cancer. p53 plays an important role in cell cycle control and apoptosis and loss of p53 function has been demonstrated in about half of all human cancers. The purpose of the study was to investigate the malignant potential of OLP on the basis of p53 expression and to correlate p53 expression with clinical and histopathological features of OLP. METHODS: 40 patients with OLP underwent biopsy. All tissue samples were treated immunohistochemicaly using avidin-biotin peroxidase complex method. RESULTS: In 80% of OLP specimens the nuclei of basal and parabasal keratinocytes were p53-positive, but in low numbers. Low percentage of p53-positive cells in older and medium percentage of p53-positive cells in younger group of OLP patients were noted. Higher intensity of p53 stained keratinocytes, no matter their low number, could represent mutant and more stable form of p53 protein, and at the same time signal for monitoring of disease due to potential malignant transformation. Low percentage and weak intensity of p53-positive cells was detected mostly in OLP specimens with highly expressed civatte bodies (CB). Upregulation of apoptosis didn't correspond with the expression of CB. CONCLUSION: We believe that low percentage of p53-positive and well-marked keratinocytes in OLP represent the influence of mutant p53 protein, and that increasing expression of this protein could serve as a valuable diagnostic sign of early carcinogenesis. According to our results intensity of p53 coloration of keratinocytes could help assessing the malignant potential of OLP.

Design of p53-derived peptides with cytotoxicity on breast cancer.

The tumor suppressor p53 plays essential role in conserving stability by preventing genome mutation, which is inactivated naturally by its negative regulator MDM2. Thus, targeting p53-MDM2 protein-protein interaction has been raised as a new cancer therapy in the medicinal community. In the current study, we report a successful application of an integrative protocol to design novel p53-derived peptides with cytotoxicity on human breast cancer cells. A quantitative structure-activity relationship-improved statistical potential was used to evaluate the binding potency of totally 24,054 single- and dual-point mutants of p53 peptide to MDM2 in a high-throughput manner, from which 46 peptide mutants with high predicted affinity and typical helical feature were involved in a rigorous modeling procedure that employed molecular dynamics simulations and post-binding energy analysis to systematically investigate the structural, energetic and dynamic aspects of peptide interactions with MDM2. Subsequently, a biological analysis was performed on a number of promising peptide candidates to determine their cytotoxic effects on human breast cancer cell line MDF-7. Six dual-point mutants were found to have moderate or high activities with their IC50 values ranging from 16.3 to 137.0 µM, which are better than that of wild-type p53 peptide (IC50 = 182.6 µM) and close to that of classical anticancer agent cis-platin (IC50 = 4.3 µM). Further, the most active peptide ETFSDWWKLLAE was selected as parent to further derive new mutants on the basis of the structural and energetic profile of its complex with MDM2. Consequently, three triple-point mutants (LTFSDWWKLLAE, ESFSDWWKLLAE and ETFADWWKLLAE) were obtained, and their biological activities (IC50 = 15.1, 27.0 and 8.7 µM, respectively) were determined to be comparable or better than the parent (IC50 = 16.3 µM).

Inhibition of p53-MDM2 binding reduces senescent cell abundance and improves the adaptive responses of skeletal muscle from aged mice.

Skeletal muscle adaptation to external stimuli, such as regeneration following injury and hypertrophy in response to resistance exercise, are blunted with advanced age. The accumulation of senescent cells, along with defects in myogenic progenitor cell (MPC) proliferation, have been strongly linked as contributing factors to age-associated impairment in muscle adaptation. p53 plays an integral role in all these processes, as upregulation of p53 causes apoptosis in senescent cells and prevents mitotic catastrophe in MPCs from old mice. The goal of this study was to determine if a novel pharmaceutical agent (BI01), which functions by upregulating p53 through inhibition of binding to MDM2, the primary p53 regulatory protein, improves muscle regeneration and hypertrophy in old mice. BI01 effectively reduced the number of senescent cells in vitro but had no effect on MPC survival or proliferation at a comparable dose. Following repeated oral gavage with 2 mg/kg of BI01 (OS) or vehicle (OV), old mice (24 months) underwent unilateral BaCl2 injury in the tibialis anterior (TA) muscle, with PBS injections serving as controls. After 7 days, satellite cell number was higher in the TA of OS compared to OV mice, as was the expression of genes involved in ATP production. By 35 days, old mice treated with BI01 displayed reduced senescent cell burden, enhanced regeneration (higher muscle mass and fiber cross-sectional area) and restoration of muscle function relative to OV mice. To examine the impact of 2 mg/kg BI01 on muscle hypertrophy, the plantaris muscle was subjected to 28 days of mechanical overload (MOV) in OS and OV mice. In response to MOV, OS mice had larger plantaris muscles and muscle fibers than OV mice, particularly type 2b + x fibers, associated with reduced senescent cells. Together our data show that BI01 is an effective senolytic agent that may also augment muscle metabolism to enhance muscle regeneration and hypertrophy in old mice.