p53 regulates primordial follicle activation through the mTOR signaling pathway / 生理学报

This paper aimed to investigate the role and potential mechanism of p53 on primordial follicle activation. Firstly, the p53 mRNA expression in the ovary of neonatal mice at 3, 5, 7 and 9 days post-partum (dpp) and the subcellular localization of p53 were detected to confirm the expression pattern of p53. Secondly, 2 dpp and 3 dpp ovaries were cultured with p53 inhibitor Pifithrin-μ (PFT-μ, 5 μmol/L) or equal volume of dimethyl sulfoxide for 3 days. The function of p53 in primordial follicle activation was determined by hematoxylin staining and whole ovary follicle counting. The proliferation of cell was detected by immunohistochemistry. The relative mRNA levels and protein levels of the key molecules involved in the classical pathways associated with the growing follicles were examined by immunofluorescence staining, Western blot and real-time PCR, respectively. Finally, rapamycin (RAP) was used to intervene the mTOR signaling pathway, and ovaries were divided into four groups: Control, RAP (1 μmol/L), PFT-μ (5 μmol/L), PFT-μ (5 μmol/L) + RAP (1 μmol/L) groups. The number of follicles in each group was determined by hematoxylin staining and whole ovary follicle counting. The results showed that the expression of p53 mRNA was decreased with the activation of primordial follicles in physiological condition. p53 was expressed in granulosa cells and oocyte cytoplasm of the primordial follicles and growing follicles, and the expression of p53 in the primordial follicles was higher than that in the growing follicles. Inhibition of p53 promoted follicle activation and reduced the primordial follicle reserve. Inhibition of p53 promoted the proliferation of the granulosa cells and oocytes. The mRNA and protein expression levels of key molecules in the PI3K/AKT signaling pathway including AKT, PTEN, and FOXO3a were not significantly changed after PFT-μ treatment, while the expression of RPS6/p-RPS6, the downstream effectors of the mTOR signaling pathway, was upregulated. Inhibition of both p53 and mTOR blocked p53 inhibition-induced primordial follicle activation. Collectively, these findings suggest that p53 may inhibit primordial follicle activation through the mTOR signaling pathway to maintain the primordial follicle reserve.

Targeting microRNA-125b inhibited the metastasis of Alisertib resistance cells through mediating p53 pathway / 中华肿瘤杂志

Objective: To clarify the mechanisms involvement in Alisertib-resistant colorectal cells and explore a potential target to overcome Alisertib-resistance. Methods: Drug-resistant colon cancer cell line (named as HCT-8-7T cells) was established and transplanted into immunodeficient mice. The metastasis in vivo were observed. Proliferation and migration of HCT-8-7T cells and their parental cells were assessed by colony formation and Transwell assay, respectively. Glycolytic capacity and glutamine metabolism of cells were analyzed by metabolism assays. The protein and mRNA levels of critical factors which are involved in mediating glycolysis and epithelial-mesenchymal transition (EMT) were examined by western blot and reverse transcription-quantitative real-time polymerase chain reaction(RT-qPCR), respectively. Results: In comparison with the mice transplanted with HCT-8 cells, which were survival with limited metastatic tumor cells in organs, aggressive metastases were observed in liver, lung, kidney and ovary of HCT-8-7T transplanted mice (P<0.05). The levels of ATP [(0.10±0.01) mmol/L], glycolysis [(81.77±8.21) mpH/min] and the capacity of glycolysis [(55.50±3.48) mpH/min] in HCT-8-7T cells were higher than those of HCT-8 cells [(0.04±0.01) mmol/L, (27.77±2.55) mpH/min and(14.00±1.19) mpH/min, respectively, P<0.05]. Meanwhile, the levels of p53 protein and mRNA in HCT-8-7T cells were potently decreased as compared to that in HCT-8 cells (P<0.05). However, the level of miRNA-125b (2.21±0.12) in HCT-8-7T cells was significantly elevated as compared to that in HCT-8 cells (1.00±0.00, P<0.001). In HCT-8-7T cells, forced-expression of p53 reduced the colon number (162.00±24.00) and the migration [(18.53±5.67)%] as compared with those in cells transfected with control vector [274.70±40.50 and (100.00±29.06)%, P<0.05, respectively]. Similarly, miR-125b mimic decreased the glycolysis [(25.28±9.51) mpH/min] in HCT-8-7T cells as compared with that [(54.38±12.70)mpH/min, P=0.003] in HCT-8-7T cells transfected with control. Meanwhile, in comparison with control transfected HCT-8-7T cells, miR-125b mimic also significantly led to an increase in the levels of p53 and β-catenin, in parallel with a decrease in the levels of PFK1 and HK1 in HCT-8-7T cells (P<0.05). Conclusions: Silencing of p53 by miR-125b could be one of the mechanisms that contributes to Alisertib resistance. Targeting miR-125b could be a strategy to overcome Alisertib resistance.

Effect of acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) on the expression of apoptosis-related factors in rats with premature ovarian insufficiency based on oxidative stress / 中国针灸

OBJECTIVE@#To explore the possible mechanism of acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) on premature ovarian insufficiency (POI) from the perspective of oxidative stress.@*METHODS@#Sixty female SD rats were randomly divided into a blank group, a model group, a sham acupuncture group, a medication group, and an acupuncture group, 12 rats in each group. Except the blank group, the rats in the remaining groups were intraperitoneally injected with cyclophosphamide to establish the POI model. After the model was successfully established, the rats in the acupuncture group were treated with acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28), with a depth of about 12 mm, and the needle was retained for 30 min; the acupuncture was given once a day, for a total of 4 weeks. The rats in the sham acupuncture group were treated with blunt-head needle to tap the skin surface of "Zhibian" (BL 54), without penetrating the skin, once a day for 4 weeks. The rats in the medication group were treated with estradiol valerate by gastric gavage for 4 weeks. After the intervention, the level of reactive oxygen species (ROS) in the ovarian tissue was detected by fluorescence probe; the expression of c-Jun N-terminal kinase (JNK), forkhead box O1 (FoxO1), tumor suppressor gene protein 53 (p53) and p53 up-regulated modulator of apoptosis (Puma) mRNA and protein in ovarian tissue were detected by real-time fluorescence quantitative PCR and Western blot.@*RESULTS@#Compared with the blank group, the level of ROS and the expression of JNK mRNA, p-JNK protein, FoxO1, p53, Puma mRNA and protein in the ovarian tissue in the model group were increased (P<0.01). Compared with the model group, the level of ROS and the expression of p-JNK protein, FoxO1, p53, Puma mRNA and protein in the ovarian tissue in the sham acupuncture group were slightly reduced, but the difference was not statistically significant (P>0.05). The level of ROS and the expression of JNK mRNA, p-JNK protein, FoxO1, p53, Puma mRNA and protein in the ovarian tissue in the acupuncture group and the medication group were reduced (P<0.01).@*CONCLUSION@#Acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) could improve the level of oxidative stress, down-regulate the expression of apoptosis-related factors JNK, FoxO1, p53 and Puma induced by oxidative stress, and inhibit the premature failure of ovarian reserve function caused by apoptosis of ovarian granulosa cells in POI rats.

Inhibitor of growth protein-2 silencing alleviates angiotensin Ⅱ-induced cardiac remodeling in mice by reducing p53 acetylation / 南方医科大学学报

OBJECTIVE@#To investigate the effect of inhibitor of growth protein-2 (Ing2) silencing on angiotensin Ⅱ (AngⅡ)-induced cardiac remodeling in mice and explore the underlying mechanism.@*METHODS@#An adenoviral vector carrying Ing2 shRNA or empty adenoviral vector was injected into the tail vein of mice, followed 48 h later by infusion of 1000 ng · kg-1 · min-1 Ang Ⅱ or saline using a mini-osmotic pump for 42 consecutive days. Transthoracic echocardiography was used to assess cardiac geometry and function and the level of cardiac hypertrophy in the mice. Masson and WGA staining were used to detect myocardial fibrosis and cross-sectional area of cardiomyocytes, and myocardial cell apoptosis was detected with TUNEL assay. Western blotting was performed to detect myocardial expressions of cleaved caspase 3, ING2, collagen Ⅰ, Ac-p53(Lys382) and p-p53 (Ser15); Ing2 mRNA expression was detected using real-time PCR. Mitochondrial biogenesis, as measured by mitochondrial ROS content, ATP content, citrate synthase activity and calcium storage, was determined using commercial assay kits.@*RESULTS@#The expression levels of Ing2 mRNA and protein were significantly higher in the mice with chronic Ang Ⅱ infusion than in saline-infused mice. Chronic infusion of AngⅡ significantly increased the left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) and reduced left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) in the mice. Ing2 silencing obviously alleviated AngⅡ-induced cardiac function decline, as shown by decreased LVEDD and LVESD and increased LVEF and LVFS, improved myocardial mitochondrial damage and myocardial hypertrophy and fibrosis, and inhibited cardiomyocyte apoptosis. Chronic AngⅡ infusion significantly increased myocardial expression levels of Ac-p53(Lys382) and p-p53(Ser15) in the mice, and Ing2 silencing prior to AngⅡ infusion lessened AngⅡ- induced increase of Ac-p53(Lys382) without affecting p53 (ser15) expression.@*CONCLUSION@#Ing2 silencing can inhibit AngⅡ-induced cardiac remodeling and dysfunction in mice by reducing p53 acetylation.

RITA selectively inhibits proliferation of BAP1-deficient cutaneous melanoma cells in vitro / 南方医科大学学报

OBJECTIVE@#To screen for small molecular compounds with selective inhibitory activity against cutaneous melanoma cells with BAP1 deletion.@*METHODS@#Cutaneous melanoma cells expressing wild-type BAP1 were selected to construct a BAP1 knockout cell model using CRISPR-Cas9 system, and small molecules with selective inhibitory activity against BAP1 knockout cells were screened from a compound library using MTT assay. Rescue experiment was carried out to determine whether the sensitivity of BAP1 knockout cells to the candidate compounds was directly related to BAP1 deletion. The effects of the candidate compounds on cell cycle and apoptosis were detected with flow cytometry, and the protein expressions in the cells were analyzed with Western blotting.@*RESULTS@#The p53 activator RITA from the compound library was shown to selectively inhibit the viability of BAP1 knockout cells. Overexpression of wild-type BAP1 reversed the sensitivity of BAP1 knockout cells to RITA, while overexpression of the mutant BAP1 (C91S) with inactivated ubiquitinase did not produce any rescue effect. Compared with the control cells expressing wild-type BAP1, BAP1 knockout cells were more sensitive to RITA-induced cell cycle arrest and apoptosis (P < 0.0001) and showed an increased expression of p53 protein, which was further increased by RITA treatment (P < 0.0001).@*CONCLUSION@#Loss of BAP1 results in the sensitivity of cutaneous melanoma cells to p53 activator RITA. In melanoma cells, the activity of ubiquitinase in BAP1 is directly related to their sensitivity to RITA. An increased expression of p53 protein induced by BAP1 knockout is probably a key reason for RITA sensitivity of melanoma cells, suggesting the potential of RITA as a targeted therapeutic agent for cutaneous melanoma carrying BAP1-inactivating mutations.

Lung Squamous Cell Carcinoma with EML4-ALK Fusion and TP53 Co-mutation Treated with Ensartinib: A Case Report and Literature Review / 中国肺癌杂志

Lung squamous cell carcinoma (LSCC) accounts for approximately 30% of non-small cell lung cancer (NSCLC) cases and is the second most common histological type of lung cancer. Anaplastic lymphoma kinase (ALK)-positive NSCLC accounts for only 2%-5% of all NSCLC cases, and is almost exclusively detected in patients with lung adenocarcinoma. Thus, ALK testing is not routinely performed in the LSCC population, and the efficacy of such treatment for ALK-rearranged LSCC remains unknown. Echinoderm microtubule associated protein like 4 (EML4)-ALK (V1) and TP53 co-mutations were identified by next generation sequencing (NGS) in this patient with advanced LSCC. On December 3, 2020, Ensatinib was taken orally and the efficacy was evaluated as partial response (PR). The progression-free survival (PFS) was 19 months. When the disease progressed, the medication was changed to Loratinib. To our knowledge, Enshatinib created the longest PFS of ALK-mutant LSCC patients treated with targeted therapy since literature review. Herein, we described one case treated by Enshatinib involving a patient with both EML4-ALK and TP53 positive LSCC, and the relevant literatures were reviewed for discussing the treatment of this rare disease.
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Mechanism of Huangjing Qianshi Decoction in treatment of prediabetic mice based on transcriptome sequencing / 中国中药杂志

Based on transcriptome sequencing technology, the mouse model of prediabetes treated with Huangjing Qianshi Decoction was sequenced to explore the possible mechanism of treating prediabetes. First of all, transcriptome sequencing was performed on the normal BKS-DB mouse group, the prediabetic model group, and the Huangjing Qianshi Decoction treatment group(treatment group) to obtain differentially expressed genes in the skeletal muscle samples of mice. The serum biochemical indexes were detected in each group to screen out the core genes of Huangjing Qianshi Decoction in prediabetes. Gene Ontology(GO) database and Kyoto Encyclopedia of Genes and Genomes(KEGG) database were used to conduct signaling pathway enrichment analysis of differentially expressed genes, and real-time quantitative polymerase chain reaction(RT-qPCR) was used to verify them. The results showed that the levels of fasting blood glucose(FBG), fasting insulin(FINS), insulin resistance index(HOMA-IR), total cholesterol(TC), triglycerides(TG), and low-density lipoprotein cholesterol(LDL-C) in the mouse model were significantly decreased after treatment with Huangjing Qianshi Decoction. In the results of differential gene screening, there were 1 666 differentially expressed genes in the model group as compared with the normal group, and there were 971 differentially expressed genes in the treatment group as compared with the model group. Among them, interleukin-6(IL-6) and NR3C2 genes, which were closely related to the regulation of insulin resis-tance function, were significantly up-regulated between the model group and the normal group, and vascular endothelial growth factor A(VEGFA) genes were significantly down-regulated between the model group and the normal group. However, the expression results of IL-6, NR3C2, and VEGFA genes were adverse between the treatment group and the model group. GO functional enrichment analysis found that the biological process annotation mainly focused on cell synthesis, cycle, and metabolism; cell component annotation mainly focused on organelles and internal components; and molecular function annotation mainly focused on binding molecular functions. KEGG pathway enrichment analysis found that it involved the protein tyrosine kinase 6(PTK6) pathway, CD28-dependent phosphoinositide 3-kinase/protein kinase B(PI3K/AKT) pathway, p53 pathway, etc. Therefore, Huangjing Qianshi Decoction can improve the state of prediabetes, and the mechanism may be related to cell cycle and apoptosis, PI3K/AKT pathway, p53 pathway, and other biological pathways regulated by IL-6, NR3C2, and VEGFA.

Effects and mechanism of p53 gene deletion on energy metabolism during the pluripotent transformation of spermatogonial stem cells / 生理学报

Previous studies have shown that long-term spermatogonial stem cells (SSCs) have the potential to spontaneously transform into pluripotent stem cells, which is speculated to be related to the tumorigenesis of testicular germ cells, especially when p53 is deficient in SSCs which shows a significant increase in the spontaneous transformation efficiency. Energy metabolism has been proved to be strongly associated with the maintenance and acquisition of pluripotency. Recently, we compared the difference in chromatin accessibility and gene expression profiles between wild-type (p53+/+) and p53 deficient (p53-/-) mouse SSCs using the Assay for Targeting Accessible-Chromatin with high-throughput sequencing (ATAC-seq) and transcriptome sequencing (RNA-seq) techniques, and revealed that SMAD3 is a key transcription factor in the transformation of SSCs into pluripotent cells. In addition, we also observed significant changes in the expression levels of many genes related to energy metabolism after p53 deletion. To further reveal the role of p53 in the regulation of pluripotency and energy metabolism, this paper explored the effects and mechanism of p53 deletion on energy metabolism during the pluripotent transformation of SSCs. The results of ATAC-seq and RNA-seq from p53+/+ and p53-/- SSCs revealed that gene chromatin accessibility related to positive regulation of glycolysis and electron transfer and ATP synthesis was increased, and the transcription levels of genes encoding key glycolytic enzymes and regulating electron transport-related enzymes were markedly increased. Furthermore, transcription factors SMAD3 and SMAD4 promoted glycolysis and energy homeostasis by binding to the chromatin of the Prkag2 gene which encodes the AMPK subunit. These results suggest that p53 deficiency activates the key enzyme genes of glycolysis in SSCs and enhances the chromatin accessibility of genes associated with glycolysis activation to improve glycolysis activity and promote transformation to pluripotency. Moreover, SMAD3/SMAD4-mediated transcription of the Prkag2 gene ensures the energy demand of cells in the process of pluripotency transformation and maintains cell energy homeostasis by promoting AMPK activity. These results shed light on the importance of the crosstalk between energy metabolism and stem cell pluripotency transformation, which might be helpful for clinical research of gonadal tumors.

Oenothein B inhibits proliferation and migration of breast cancer cells by regulating P53 / 中国中药杂志

The effects of oenothein B(OEB) on the proliferation, apoptosis, invasion, and migration of breast cancer MCF-7 and MDA-MB-231 cells were investigated by cell culture in vitro, network pharmacology, and molecular docking. In vitro cell experiments revealed that OEB inhibited the proliferation and colony formation ability, and promoted the apoptosis and formation of apoptotic bodies in breast cancer cells, as well as inhibited the invasion and migration of breast cancer cells. The targets of OEB were obtained using SwissTargetPrediction database and breast cancer targets were obtained from GeneCards. The targets of OEB and breast cancer were entered separately in Venny 2.1 software to obtain the Venn diagram of common targets of OEB and breast cancer. The common targets of OEB and breast cancer were input into STRING database to construct a protein-protein interaction(PPI) network, which was entered into Cytoscape 3.7.2 software for network topology analysis. Key targets were screened according to protein association strength, and analyzed for KEGG pathway enrichment. Molecular docking of OEB to key targets using AutoDock software revealed that OEB stably bound to the active pocket of P53, while OEB promoted the expression of P53 protein. MCF-7 and MDA-MB-231 cell viability and migration ability increased after silencing P53, and this change was reversed after treatment with OEB. Therefore, this study showed that OEB inhibited the proliferation, migration, and invasion of breast cancer MCF-7 and MDA-MB-231 cells, and promoted the apoptosis of breast cancer MCF-7 and MDA-MB-231 cells, which may be related to the targeted regulation of P53.

Effect and mechanism of Dahuang Zhechong Pills in improving liver aging in rats by regulating ROS-mediated PI3K/Akt/FoxO4 signaling pathway / 中国中药杂志

Recent studies have shown that the occurrence and development of common liver diseases, including non-alcoholic fatty liver disease, cirrhosis, and liver cancer, are related to liver aging(LA). Therefore, to explore the effect and mechanism of Dahuang Zhechong Pills(DHZCP), a traditional classic prescription in improving LA with multiple targets, the present study randomly divided 24 rats into a normal group, a model group, a DHZCP group, and a vitamin E(VE) group, with six rats in each group. The LA model was induced by continuous intraperitoneal injection of D-galactose(D-gal) in rats. For the LA model rats, the general situation was evaluated by aging phenotype and body weight(BW). LA was assessed by the pathological characteristics of hepatocyte senescence, hepatic function indexes, the staining characteristics of phosphorylated histone family 2A variant(γ-H2AX), and the expression levels of cell cycle arrest proteins(P21, P53, P16) and senescence-associated secretory phenotype(SASP) in the liver. The activation of the reactive oxygen species(ROS)-mediated phosphatidylinositol-3 kinase(PI3K)/protein kinase B(Akt)/forkhead box protein O4(FoxO4) signaling pathway was estimated by hepatic ROS expression feature and the protein expression levels of the key signaling molecules in the PI3K/Akt/FoxO4 signaling pathway. The results showed that after the treatment with DHZCP or VE for 12 weeks, for the DHZCP and VE groups, the characterized aging phenotype, BW, pathological characteristics of hepatocyte senescence, hepatic function indexes, relative expression of ROS in the liver, protein expression levels of key signaling molecules including p-PI3K, p-Akt, and FoxO4 in the liver, staining characteristics of γ-H2AX, and the protein expression levels of P16, P21, P53, interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) in the liver were improved, and the effects of DHZCP and VE were similar. Based on the D-gal-induced LA model in rats, this study demonstrates that DHZCP can ameliorate LA with multiple targets in vivo, and its effects and mechanism are related to regulating the activation of the ROS-mediated PI3K/Akt/FoxO4 signaling pathway in the liver. These findings are expected to provide new pharmacological evidence for the treatment of DHZCP in aging-related liver diseases.

Transition of autophagy and apoptosis in fibroblasts depends on dominant expression of HIF-1α or p53 / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

It has been revealed that hypoxia is dynamic in hypertrophic scars; therefore, we considered that it may have different effects on hypoxia-inducible factor-1α (HIF-1α) and p53 expression. Herein, we aimed to confirm the presence of a teeterboard-like conversion between HIF-1α and p53, which is correlated with scar formation and regression. Thus, we obtained samples of normal skin and hypertrophic scars to identify the differences in HIF-1α and autophagy using immunohistochemistry and transmission electron microscopy. In addition, we used moderate hypoxia in vitro to simulate the proliferative scar, and silenced HIF-1α or p53 gene expression or triggered overexpression to investigate the changes of HIF-1α and p53 expression, autophagy, apoptosis, and cell proliferation under this condition. HIF-1α, p53, and autophagy-related proteins were assayed using western blotting and immunofluorescence, whereas apoptosis was detected using flow cytometry analysis, and cell proliferation was detected using cell counting kit-8 (CCK-8) and 5-bromo-2'-deoxyuridine (BrdU) staining. Furthermore, immunoprecipitation was performed to verify the binding of HIF-1α and p53 to transcription cofactor p300. Our results demonstrated that, in scar tissue, HIF-1α expression increased in parallel with autophagosome formation. Under hypoxia, HIF-1α expression and autophagy were upregulated, whereas p53 expression and apoptosis were downregulated in vitro. HIF-1α knockdown downregulated autophagy, proliferation, and p300-bound HIF-1α, and upregulated p53 expression, apoptosis, and p300-bound p53. Meanwhile, p53 knockdown induced the opposite effects and enhanced HIF-1α, whereas p53 overexpression resulted in the same effects and reduced HIF-1α. Our results suggest a teeterboard-like conversion between HIF-1α and p53, which is linked with scar hyperplasia and regression.

Research Advances of EGFR-TP53 Co-mutation in Advanced Non-small Cell Lung Cancer / 中国肺癌杂志

With the rapid development and wide application of next generation sequencing (NGS) technology, a series of researches have revealed that concurrent genetic alterations play an important role in the response and resistance of epidermal growth factor receptor (EGFR)-mutant NSCLC to EGFR-tyrosine kinase inhibitor (TKI). Besides, TP53 mutation is the most common co-mutation gene in EGFR-mutant NSCLC, which has been proved to confer a worse prognosis in EGFR-mutated patients treated with first, second and third generation of EGFR-TKIs. Currently, it is still being explored how to select the best treatment strategies for patients with concomitant presence of TP53 mutation in EGFR-mutant NSCLC. Here, we review the literature on recent research progress of TP53 concurrent mutation in EGFR-mutant advanced NSCLC.
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Paired box 5 increases the chemosensitivity of esophageal squamous cell cancer cells by promoting p53 signaling activity / 中华医学杂志(英文版)

BACKGROUND@#Gene promoter methylation is a major epigenetic change in cancers, which plays critical roles in carcinogenesis. As a crucial regulator in the early stages of B-cell differentiation and embryonic neurodevelopment, the paired box 5 (PAX5) gene is downregulated by methylation in several kinds of tumors and the role of this downregulation in esophageal squamous cell carcinoma (ESCC) pathogenesis remains unclear.@*METHODS@#To elucidate the role of PAX5 in ESCC, eight ESCC cell lines, 51 primary ESCC tissue samples, and eight normal esophageal mucosa samples were studied and The Cancer Genome Atlas (TCGA) was queried. PAX5 expression was examined by reverse transcription-polymerase chain reaction and western blotting. Cell apoptosis, proliferation, and chemosensitivity were detected by flow cytometry, colony formation assays, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assays in ESCC cell lines with PAX5 overexpression or silencing. Tumor xenograft models were established for in vivo verification.@*RESULTS@#PAX5 methylation was found in 37.3% (19/51) of primary ESCC samples, which was significantly associated with age (P = 0.007) and tumor-node-metastasis stage (P = 0.014). TCGA data analysis indicated that PAX5 expression was inversely correlated with promoter region methylation (r = -0.189, P = 0.011 for cg00464519 and r = -0.228, P = 0.002 for cg02538199). Restoration of PAX5 expression suppressed cell proliferation, promoted apoptosis, and inhibited tumor growth of ESCC cell lines, which was verified in xenografted mice. Ectopic PAX5 expression significantly increased p53 reporter luciferase activity and increased p53 messenger RNA and protein levels. A direct interaction of PAX5 with the p53 promoter region was confirmed by chromatin immunoprecipitation assays. Re-expression of PAX5 sensitized ESCC cell lines KYSE150 and KYSE30 to fluorouracil and docetaxel. Silencing of PAX5 induced resistance of KYSE450 cells to these drugs.@*CONCLUSIONS@#As a tumor suppressor gene regulated by promoter region methylation in human ESCC, PAX5 inhibits proliferation, promotes apoptosis, and induces activation of p53 signaling. PAX5 may serve as a chemosensitive marker of ESCC.

Cancer cells corrupt normal epithelial cells through miR-let-7c-rich small extracellular vesicle-mediated downregulation of p53/PTEN / 国际口腔科学杂志·英文版

Tumor volume increases continuously in the advanced stage, and aside from the self-renewal of tumor cells, whether the oncogenic transformation of surrounding normal cells is involved in this process is currently unclear. Here, we show that oral squamous cell carcinoma (OSCC)-derived small extracellular vesicles (sEVs) promote the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of normal epithelial cells but delay their apoptosis. In addition, nuclear-cytoplasmic invaginations and multiple nucleoli are observed in sEV-treated normal cells, both of which are typical characteristics of premalignant lesions of OSCC. Mechanistically, miR-let-7c in OSCC-derived sEVs is transferred to normal epithelial cells, leading to the transcriptional inhibition of p53 and inactivation of the p53/PTEN pathway. In summary, we demonstrate that OSCC-derived sEVs promote the precancerous transformation of normal epithelial cells, in which the miR-let-7c/p53/PTEN pathway plays an important role. Our findings reveal that cancer cells can corrupt normal epithelial cells through sEVs, which provides new insight into the progression of OSCC.

Shexiang Baoxin Pill Regulates Intimal Hyperplasia, Migration, and Apoptosis after Platelet-Derived Growth Factor-BB-Stimulation of Vascular Smooth Muscle Cells via miR-451 / 中国结合医学杂志

OBJECTIVE@#To investigate the regulatory roles of Shexiang Baoxin Pill (SXBXW) in neointimal formation and vascular smooth muscle cells (VSMCs) invasion and apoptosis as well as the potential molecular mechanisms using cultured VSMCs model of vascular injury (platelet-derived growth factor (PDGF)-BB-stimulated) in vitro.@*METHODS@#VSMCs were randomly assigned to 5 groups: blank, PDGF-BB (20 ng/mL+ 0.1% DMSO), SXBXW-L (PDGF-BB 20 ng/mL + SXBXW low dose 0.625 g/L), SXBXW-M (PDGF-BB 20 ng/mL + SXBXW medium dose 1.25 g/L) and SXBXW-H (PDGF-BB 20 ng/mL+ SXBXW high dose 2.5 g/L) group. Cell proliferation was assessed using cell counting kit-8 (CCK-8) assay and bromodeoxyuridine (BrdU) incorporation assay, the migration effects were detected by Transwell assay, cell apoptosis rate was measured by the Annexin V/propidium iodide (PI) apoptosis kit. The markers of contractile phenotype of VSMCs were detected with immunofluorescent staining. To validate the effects of miR-451 in regulating proliferation, migration and apoptosis treated with SXBXW, miR-451 overexpression experiments were performed, the VSMCs were exposed to PDGF-BB 20 ng/mL + 0.1% DMSO and later divided into 4 groups: mimic-NC (multiplicity of infection, MOI=50), SXBXW (1.25 g/L) + mimic-NC, mimic-miR451 (MOI=50), and SXBXW (1.25 g/L) + mimic-miR451, and alterations of proteins related to the miR-451 pathway were analyzed using Western blot.@*RESULTS@#PDGF-BB induced VSMCs injury causes acceleration of proliferation and migration. SXBXW inhibited phenotypic switching, proliferation and migration and promoted cell apoptosis in PDGF-BB-induced VSMCs. In addition, miR-451 was shown to be down-regulated in the VSMCs following PDGF-BB stimulation. SXBXW treatment enhanced the expression of miR-451 in PDGF-BB-induced VSMCs (P<0.05). Compared with SXBXW + mimic-NC and mimic-miR451 groups, the expression of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (Ywhaz) and p53 was further reduced in SXBXW + mimic-miR451 group, while activating transcription factor 2 (ATF2) was increased in VSMCs (P<0.05).@*CONCLUSION@#SXBXW regulated proliferation, migration and apoptosis via activation of miR-451 through ATF2, p53 and Ywhaz in PDGF-BB-stimulated VSMCs.

Oxidative Damage to BV2 Cells by Trichloroacetic Acid: Protective Role of Boron via the p53 Pathway / 生物医学与环境科学（英文）

This study aimed to investigate the neurotoxicity induced by trichloroacetic acid (TCA) and the possible protective mechanisms of boron (B). Mouse BV2 cells were treated with TCA (0, 0.39, 0.78, 1.56, 3.12, 6.25, or 12.5 mmol/L) and B (0, 7.8, 15.6, 31.25, 62.5, 125, 500, or 1,000 mmol/L) for 3 h and 24 h, respectively. Then, reactive oxygen species, and supernatant proinflammatory cytokine and protein levels were analyzed after 24 h of combined exposure. Beyond the dose-dependent decrease in the cellular viability, it clearly increased after B supplementation ( P < 0.05). Moreover, B decreased oxidative damage, and significantly down-regulated IL-6 levels and up-regulated TNF-β production ( P < 0.05). B also decreased apoptosis via the p53 pathway. The present findings indicated that TCA may induce oxidative damage, whereas B mitigates these adverse effects by decreasing cell apoptosis.

p53 & Cyclin D1 expression in surgically resected clear margins of oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is one of the most well-known malignancies that affect the human population worldwide. The early diagnosis and early intervention of OSCC help improve the survival rate of the patients. The tumour free surgical margins are a positive prognostic factor for recurrence-free survival. The molecular markers can be used to detect the tumour free surgical margins. Aim: The aim of the study is to evaluate the expression of p53 & Cyclin D1 marker in resected surgical apparently clear margins and to correlate the p53 & Cyclin D1 expression with clinicopathological characteristics and patient outcome. Methods: The study population included retrospective cases of OSCC with apparently clear margins (2017-18) n=10 and Clinicopathological variables relevant to survival analysis were recorded. Finally, two margins were selected from each case, a total of 20 margins were included in this study. Paraffin-embedded wax blocks retrieved and tissue sections were made. Expression of cyclin D1 and p 53 was assessed by the immunohistochemical staining procedure Results: Positive expressions Cyclin D1 in 40% of mild dysplasia margins and 60% in clearance adequate margins were present. p53 expression was seen in 16% of mild dysplasia margins and 84% in clearance adequate margins. The expression of p53 and Cyclin D1 molecular markers are noted in the basal & parabasal layer of epithelium. Conclusion: Molecular markers could play a more reliable method for the assessment of dysplasia at the margins

Quercetin Inhibits ROS-p53-Bax-caspase-3 axis of apoptosis and augments gonadotropin and testicular hormones in chronic unpredictable stress-Induced testis injury / La quercetina inhibe el eje de apoptosis ROS-p53-Bax-caspasa-3 y aumenta la gonadotropina y las hormonas testiculares en la lesión testicular inducida por estrés crónico e impredecible

SUMMARY: A large body of evidence supports the protective role of the flavonol antioxidant compound quercetin in mammals. We tested the hypothesis that quercetin can protect against the hypothalamus-pituitary-gonadal (HPG) axis defect like a reduction in gonadotropins and testicular hormones and abnormal semen analysis induced by chronic unpredictable stress (CUS), possibly via the downregulation of oxidative stress (ROS) and p53-Bax-caspase-3 pathways. Rats were either exposed to a variety of unpredictable stressors daily before being sacrificed after 3 weeks (model group) or were treated with quercetin (50 mg/kg body weight/day) at the same time the CUS were induced (treated group). Harvested testicular tissues were stained with basic histological staining, and testis homogenates were assayed for the tumor suppressor p53, apoptosis regulator Bax, B-cell lymphoma 2 (Bcl-2), caspase-3, malondialdehyde (MDA), glutathione peroxidase (GPx), and superoxide dismutase (SOD). In addition, harvested epididymis tissues were used to assess semen analysis, and blood samples were assayed for the testicular hormone testosterone, the adrenal cortex hormone corticosterone, and the anterior pituitary gonadotropins, follicular stimulating hormone (FSH) and luteinizing hormone (LH). CUS induced profound testicular damage and significantly (p<0.05) induced p53, Bax, caspase-3, MDA, and corticosterone, which were significantly (p<0.05) inhibited by quercetin except corticosterone. Whereas, quercetin significantly (p<0.05) increased FSH, LH, testosterone, Bcl-2, GPx, and SOD levels that were inhibited by CUS. In addition, CUS induced oligozoospermia, asthenozoospermia, and teratozoospermia, which were significantly (p<0.05) protected by quercetin. Thus, Quercetin protects against CUS-induced HPG defects in rats, which is associated with the inhibition of ROS-p53-Bax-caspase-3 axis.

RESUMEN: El papel protector del compuesto antioxidante flavonol quercetina en los mamíferos ha sido ampliamente reportado. Probamos la hipótesis que la quercetina puede proteger contra el defecto del eje hipotálamo-hipofisiario- gonadal (HHG) como una reducción de gonadotropinas y hormonas testiculares y análisis de semen anormal inducido por estrés crónico impredecible (ECI), posiblemente a través de la regulación reducida del estrés oxidativo (REO) y las vías p53- Bax-caspasa-3. Las ratas fueron expuestas a una variedad de fac- tores estresantes impredecibles diariamente antes de ser sacrificadas después de 3 semanas (grupo modelo) o fueron tratadas con quercetina (50 mg / kg de peso corporal / día) al mismo tiempo que se indujo la ECI (grupo tratado). Los tejidos testiculares fueron teñidos con tinción histológica básica y los homogeneizados de testículo se analizaron para determinar el supresor de tumores p53, el regulador de apoptosis Bax, el linfoma de células B 2 (Bcl-2), la caspasa-3, el malondialdehído (MDA), la glutatión peroxidasa (GPx) y superóxido dismutasa (SOD). Además, se utilizaron tejidos del epidídimo recolectados para evaluar el análisis de semen y se analizaron muestras de sangre para determinar la hormona testicular testosterona, la hormona corticosterona de la corteza suprarrenal y las gonadotropinas de la hipófisis anterior, la hormona estimulante folicular (FSH) y la hormona luteinizante (LH). El ECI indujo daño testicular importante e indujo significativamente niveles de (p <0,05) p53, Bax, caspasa-3, MDA y corticosterona, que fueron inhibidos (p <0,05) por la quercetina. La quercetina aumentó significativamente (p <0,05) los niveles de FSH, LH, testosterona, Bcl-2, GPx y SOD que fueron inhibidos por ECI. Además, ECI indujo oligozoospermia, astenozoospermia y teratozoospermia, protegidos de manera significativa (p <0,05) por la quercetina. Por lo tanto, la quercetina protege contra los defectos de HHG inducidos por ECI en ratas, lo que está asociado con la inhibición del eje ROS-p53-Bax-caspasa-3.

Expression of Ki-67, Cyclin D1, P53, and P16 in patients with oral leukoplakia and leukoplakia cancerization with spicy diet in Chengdu / 华西口腔医学杂志

OBJECTIVES@#To investigate the expression of Ki-67, Cyclin D1, P53, and P16 in patients with oral leukoplakia (OLK) and OLK cancerization who have aspicy diet in Chengdu.@*METHODS@#Thirtypatients with OLK andspicy diet and 15 patients with OLK without spicy diet in Chengdu were divided into three groups: hyperplastic OLK (OLK-), OLK with mild to moderate dysplasia (OLK+), and severe dysplastic  OLK or oral squamous cell carcinoma (OSCC) transforming from OLK (OLK++/OSCC). The expression of Ki-67, Cyclin D1, P53, and P16 were detected by immunohistochemistry and statistically analyzed.@*RESULTS@#The expression of Ki-67 and P53 in patients with or without spicy diet in the OLK+and OLK++/OSCC groups were stronger than that of the OLK- group (@*CONCLUSIONS@#Spicy diet did not have an influence on the expression of Ki-67, Cyclin D1, P53, and P16 in patients with OLK and OSCC. The expression of Ki-67, Cyclin D1, and P53 increased with the development of OLK, whereas P16 showed opposite expression trend.

Effect of RITA on TP53 Mutant Human Mantle Cell Lymphoma Cell Line and Its Mechanism / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of RITA on TP53 mutant human mantle cell lymphoma (MCL) cell line Mino and its possible mechanism.@*METHODS@#Mino cells were cultured in RPMI-1640 and treated with RITA at a concentration of 0-16 μmol/L for 24,48,72 hours. Cell viability was assessed by CCK-8 assay. The cells were treated by RITA (0-8 μmol/L) for 48 h, the cell apoptosis induced by RITA was detected by annexin V/PI flow cytometry. Western blot was performed to evaluate the expression of protein BCL-2, Caspase-3, Cleaved Caspase-3, PARP, MDM2, and P53 in Mino cells.@*RESULTS@#After treatment with 0.5, 1, 2, 4, 8, and 16 μmol/L RITA for 48 h, the proliferation inhibition rate of Mino cells was (1.2±5.6)%, (14.9±4.9)%, (41.7±5.0)%, (61.8±2.4)%, (70.2±2.8)%, and (70.8±2.4)%, respectively. RITA could inhibit the proliferation of Mino cells significantly, and statistical analysis showed that the inhibition rate was increased with the increasing of RITA concentration (r=0.767). After the cells were treated by 4 μmol/L RITA for 24, 48, and 72 h, the proliferation inhibition rate was (25.2±3.8)%, (61.8±2.4)%, and (87.0±0.7)%, respectively. Satistical analysis showed that the inhibition rate was also increased with the increasing of treatment time (r=0.978). The apoptosis rate of Mino cells treated by 0, 2, 4, and 8 μmol/L RITA for 48 h was (5.4±0.4)%, (15.3±0.6)%, (38.7±1.7)%, and (50.8±1.1)%, respectively, and it showed dose-dependent manner (r=0.961). Western blot showed that with the increasing of RITA concentration, the BCL-2 protein expression was decreased in a dose-dependent manner (r=0.932), moreover, PARP cleavage and Caspase-3 activation were found, while the protein expression of MDM2 and P53 showed no change.@*CONCLUSION@#RITA can inhibit the proliferation and induce the apoptosis of Mino cells significantly. The mechanism may be dependent on the Caspase pathway, but independent on the P53 pathway.