Mechanism of Qizhu Kang'ai Prescription for Inhibiting Proliferation of Hepatocellular Carcinoma by Regulating Tumor Metabolic Reprogramming via PCK1/Akt/p21 Signal Axis / 中国实验方剂学杂志

ObjectiveTo study the effect of Qizhu Kang'ai prescription （QZAP） on the gluconeogenesis enzyme phosphoenolpyruvate carboxykinase 1 （PCK1） in the liver of mouse model of liver cancer induced by diethylnitrosamine （DEN） combined with carbon tetrachloride （CCl4） and Huh7 cells of human liver cancer， so as to explore the mechanism on regulating metabolic reprogramming and inhibiting cell proliferation of liver cancer cells. MethodDEN combined with CCl4 was used to construct a mouse model of liver cancer via intraperitoneal injection. A normal group， a model group， and a QZAP group were set up， in which QZAP （3.51 g·kg-1） or an equal volume of normal saline was administered daily by gavage， respectively. Serum and liver samples were collected after eight weeks of intervention. Serum alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， γ-glutamyltransferase （γ-GT）， and alpha-fetoprotein （AFP） in mice were detected to evaluate liver function changes of mice in each group. Hematoxylin-eosin （HE） staining and Sirius red staining were used to observe pathological changes in liver tissue. In the cell experiment， Huh7 cells were divided into blank group， QZAP low， medium， and high dose groups and/or PCK1 inhibitor （SKF-34288 hydrochloride） group， and Sorafenib group. The corresponding drug-containing serum and drug treatment were given， respectively. Cell counting kit-8 （CCK-8） method， colony formation experiment， Edu fluorescent labeling detection， intracellular adenosine triphosphate （ATP） content detection， and cell cycle flow cytometry detection were used to evaluate the proliferation ability， energy metabolism changes， and change in the cell cycle of Huh7 cells in each group. Western blot was used to detect the protein expression levels of PCK1， serine/threonine kinase （Akt）， phosphorylated Akt （p-Akt）， and cell cycle-dependent protein kinase inhibitor 1A （p21）. ResultCompared with the model group， the pathological changes such as cell atypia， necrosis， and collagen fiber deposition in liver cancer tissue of mice in the QZAP group were alleviated， and the number of liver tumors was reduced （P<0.01）. The serum ALT， AST， γ-GT， and AFP levels were reduced （P<0.01）. At the cell level， compared with the blank group， low， medium， and high-dose groups of QZAP-containing serum and the Sorafenib group could significantly reduce the survival rate of Huh7 cells （P<0.01） and the number of positive cells with Edu labeling （P<0.01） and inhibit clonal proliferation ability （P<0.01）. The QZAP groups could also reduce the intracellular ATP content （P<0.05） and increase the distribution ratio of the G0/G1 phase of the cell cycle （P<0.05） in a dose-dependent manner. Compared with the model group and blank group， PCK1 and p21 protein levels of mouse liver cancer tissue and Huh7 cells in the QZAP groups were significantly reduced （P<0.05，P<0.01）， and the p-Akt protein level was significantly increased （P<0.01）. Compared with the blank group， the ATP content and cell survival rate of Huh7 cells in the SKF-34288 hydrochloride group were significantly increased （P<0.05）， but there was no statistical difference in the ratio of Edu-positive cells and the proportion of G0/G1 phase distribution. Compared with the SKF-34288 hydrochloride group， the QZAP combined with the SKF-34288 hydrochloride group significantly reduced the ATP content， cell survival rate， and Edu-positive cell ratio of Huh7 cells （P<0.05） and significantly increased the G0/G1 phase distribution proportion （P<0.05）. ConclusionQZAP may induce the metabolic reprogramming of liver cancer cells by activating PCK1 to promote Akt/p21-mediated tumor suppression， thereby exerting an anti-hepatocellular carcinoma proliferation mechanism.

TP53 and p21 (CDKN1A) polymorphisms and the risk of systemic lupus erythematosus

Abstract Background The p53 and p21 proteins are important regulators of cell cycle and apoptosis and may contribute to autoimmune diseases, such as systemic lupus erythematosus (SLE). As genetic polymorphisms may cause changes in protein levels and functions, we investigated associations of TP53 and p21 (CDKN1A) polymorphisms (p53 72 G > C—rs1042522; p53 PIN3—rs17878362; p21 31 C > A—rs1801270; p21 70 C > T—rs1059234) with the development of systemic lupus erythematosus (SLE) in a Southeastern Brazilian population. Methods Genotyping of 353 female volunteers (cases, n = 145; controls, n = 208) was performed by polymerase chain reaction, restriction fragment length polymorphism and/or DNA sequencing. Associations between TP53 and p21 polymorphisms and SLE susceptibility and clinical manifestations of SLE patients were assessed by logistic regression analysis. Results Protective effect was observed for the genotype combinations p53 PIN3 A1/A1 -p21 31 C/A, in the total study population (OR 0.45), and p53 PIN3 A1/A2-p21 31 C/C, in non-white women (OR 0.28). In Whites, p53 72 C-containing (OR 3.06) and p53 PIN3 A2-containing (OR 6.93) genotypes were associated with SLE risk, and higher OR value was observed for the combined genotype p53 72 G/C-p53 PIN3 A1/A2 (OR 9.00). Further, p53 PIN3 A1/A2 genotype was associated with serositis (OR 2.82), while p53 PIN3 A2/A2 and p53 72 C/C genotypes were associated with neurological disorders (OR 4.69 and OR 3.34, respectively). Conclusions Our findings showed that the TP53 and p21 polymorphisms included in this study may have potential to emerge as SLE susceptibility markers for specific groups of patients. Significant interactions of the TP53 polymorphisms with serositis and neurological disorders were also observed in SLE patients. Highlights The polymorphisms TP53 rs1042522 (G > C) and TP53 rs17878362 (16 bp Del/Ins) were associated with SLE risk in whites. In whites, the combined genotype TP53 rs1042522 GC- TP53 rs17878362 A1A2 and the haplotype TP53 rs1042522 C-rs17878362 A2 represented higher SLE risk. Combination of TP53 rs17878362 (16 bp Del/Ins) and p21 rs1801270 (C > A) protected against SLE in non-white women. TP53 and p21 (CDKN1A) polymorphisms may be SLE susceptibility markers for specific groups.

Expression of p21-activated kinase 2 in laryngeal squamous cell carcinoma and its correlation with clinicopathological characteristics and chemosensitivity / 肿瘤研究与临床

Objective:To investigate the expression of p21-activated kinase 2 (PAK2) in laryngeal squamous cell carcinoma and its relationship with the clinicopathological characteristics and chemosensitivity of patients.Methods:Transcriptome sequencing (RNA-seq) data for laryngeal squamous cell carcinoma were downloaded from the Cancer Genome Atlas (TCGA) database, and 123 patients were included in the study (12 cases had cancer tissues and normal tissues data, and the remaining 111 only had cancer tissues data). Differential expression of PAK2 in cancer and para-cancer tissues was analyzed by using R software, and the potential function of PAK2 in laryngeal squamous cell carcinoma was investigated by using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database signaling pathway enrichment. A total of 34 patients with primary laryngeal squamous cell carcinoma tissues and corresponding para-carcinoma 34 tissue specimens who underwent surgical resection were retrospectively selected from Chaoyang Central Hospital between April 2016 and June 2021, and 20 cases of normal laryngeal mucosa tissues were selected as the controls. Immunohistochemistry was used to detect the expression of PAK2 in various tissues, and its correlation with clinicopathological factors was analyzed. A total of 35 supraglottic primary laryngeal squamous cell carcinoma patients were retrospectively collected before induction chemotherapy during the same period, including 20 patients sensitive to chemotherapy and 15 patients resistant to chemotherapy. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the relative expression level of PAK2 mRNA in cancer tissues.Results:Analysis of TCGA database data showed that PAK2 expression was increased in cancer tissues compared with that in para-cancer tissues ( P = 0.012); KEGG database signaling pathways showed that the high expression of PAK2 in laryngeal squamous cell carcinoma was related to signal transduction pathways, cell cycle, and cancer. Immunohistochemistry showed that the proportion of PAK2 positive in 34 cases of laryngeal squamous cell carcinoma tissues was higher than that in adjacent tissues and normal tissues [58.82% (20/34) vs. 0.03% (1/34), 0 (0/20), all P < 0.001]. There were statistically significant differences in the proportion of PAK2 positive patients stratified with different degrees of differentiation [high differentiation vs. low or middle differentiation: 33.33% (6/18)vs. 87.50% (14/16)], lymph node metastasis [presence vs. absence: 90.91% (10/11) vs. 43.48% (10/23)], TNM staging [stage Ⅲ-Ⅳ vs. stage Ⅰ-Ⅱ: 82.35% (14/17) vs. 35.29% (6/17)] (all P < 0.05), and PAK2 positive patients were not associated with clinical type, tumor size, smoking history, drinking history, and age (all P > 0.05). qRT-PCR showed that the relative expression level of PAK2 mRNA in the chemotherapy-resistant group was higher than that in the chemotherapy-sensitive group (3.89±0.12 vs. 0.78±0.23, P < 0.001). Conclusions:The expression level of PAK2 in laryngeal squamous cell carcinoma tissues is increased, and the high expression of PAK2 is closely related to the malignant clinical characteristics of patients with laryngeal squamous cell carcinoma. The high expression of PAK2 may indicate the insensitivity to traditional chemotherapy regimens, and PAK2 may be a potential gene that targets and regulates the chemosensitivity of laryngeal squamous cell carcinoma.

Up-regulation of p21 activated kinase 4 expression in the retina of diabetes mice and its effects on the behavior and mitochondrial function in retinal vascular endothelial cells / 中华眼底病杂志

Objective:To observe the effects of p21 activated kinase 4 (PAK4) on the mitochondrial function and biological behavior in retinal vascular endothelial cells.Methods:The experimental study was divided into two parts: in vivo animal experiment and in vitro cell experiment. In vivo animal experiments: 12 healthy C57BL/6J male mice were randomly divided into normal control group and diabetes group, with 6 mice in each group. Diabetes mice were induced by streptozotocin to establish diabetes model. Eight weeks after modeling, quantitative real-time polymerase chain reaction and Western blots were performed to detect the expression of PAK4 in diabetic retinas. In vitro cell experiments: the human retinal microvascular endothelial cells (hRMEC) were divided into three groups: conventional cultured cells group (N group), empty vector transfected (Vector group); pcDNA-PAK4 eukaryotic expression plasmid transfected group (PAK4 group). WB and qPCR were used to detect transfection efficiency, while scratching assay, cell scratch test was used to detect cell migration in hRMEC of each group. In vitro white blood cell adhesion experiment combined with 4 ', 6-diamino-2-phenylindole staining was used to detect the number of white blood cells adhering to hRMEC in each group. The Seahorse XFe96 cell energy metabolism analyzer measures intracellular mitochondrial basal respiration, adenosine triphosphate (ATP) production, maximum respiration, and reserve respiration capacity. The t-test was used for comparison between the two groups. Single factor analysis of variance was used for comparison among the three groups. Results:In vivo animal experiments: compared with normal control group, the relative expression levels of PAK4 mRNA and protein in retina of diabetic mice were significantly increased, with statistical significance ( t=25.372, 22.419, 25.372; P<0.05). In vitro cell experiment: compared with the N group and Vector group, the PAK4 protein, mRNA relative expression and cell mobility in the hRMEC of PAK4 group were significantly increased, with statistical significance ( F=36.821, 38.692, 29.421; P<0.05). Flow cytometry showed that the adhesion number of leukocytes on hRMEC in PAK4 group was significantly increased, and the difference was statistically significant ( F=39.649, P<0.01). Mitochondrial pressure measurement results showed that the capacity of mitochondrial basic respiration, ATP production, maximum respiration and reserve respiration in hRMEC in PAK4 group was significantly decreased, with statistical significance ( F=27.472, 22.315, 31.147, 27.472; P<0.05). Conclusion:Over-expression of PAK4 impairs mitochondrial function and significantly promotes leukocyte adhesion and migration in retinal vascular endothelial cells.

9p21 Locus Polymorphisms: Risk and Severity Factors of Coronary Artery Disease in Venezuelan Patients

Abstract Background: The 9p21 region is the most relevant locus associated with coronary heart disease in different populations. However, there are no studies that prove that this region is a risk factor in the Venezuelan population. Objectives: To analyze whether or not the 9p21 rs1333049 polymorphism is a risk factor for acute myocardial infarction (AMI) in Venezuelan patients, as well as to investigate its correlation with cardiovascular risk factors (CVRF), age of occurrence, type and severity of infarction, and the correlation of the rs10757274 polymorphism with severity of coronary artery disease. Methods: This was an association study, including 487 unrelated Venezuelan individuals, grouped in 354 patients with AMI and 133 controls. The rs1333049 and rs10757274 polymorphisms were determined using the polymerase chain reaction (PCR) technique with sequence-specific primers. The analysis of association was determined using the SNPStats tool. The continuous variable description and the correlations were performed using the SPSS statistical software. Significance was established at p<0.05. Results: A positive correlation was observed between the rs1333049 polymorphism and the presence of hypertension ( r: 0.145, p: 0.006), and between hypertension and heart infarction ( r: 0.318, p: <0.0001). A positive correlation was found between the rs10757274 polymorphism and the number of coronary vessels that presented obstructive lesions in patients aged ≤ 55 years ( r: 0.276, p: 0.0078). Conclusion: The rs1333049 polymorphism at the 9p21 locus is correlated with hypertension in Venezuelan patients, while the rs10757274 polymorphism is associated with the progression of coronary atherosclerosis, suggested by the correlation with the number of coronary vessels that presented significant obstructive lesions.

The regulation of ovarian reserve function by Zuogui pills combined with cetrorelix / 药学学报

The purpose of this study was to investigate how Zuogui pills from the Kidney-tonifying and Nourishing Yin formula, in combination with the gonadotrophin-releasing hormone antagonist cetrorelix, affected the ovarian local oxidative stress response in decreasing ovarian reserve (DOR) mice. All animal experiments were carried out in accordance with the guidelines and standards established by Jinan University's Experimental Animal Management Committee. Cyclophosphamide (CTX)-treated DOR mice were given Zuogui pills, cetrorelix, or a combination of the two drugs intragastrically. After treatment, there were changes in the estrous cycle, serum sex hormone levels, oxidative stress-related indexes, growth biochemical factor levels, and SIRT1/P53/P21 expression. In comparison to the model group, the Zuogui pills and the cetrorelix+Zuogui pills group had significantly prolonged estrous periods and shortened interestrous periods, and the cetrorelix+Zuogui pills group had a significantly shortened cycle length. Follicle-stimulating hormone (FSH) decreased and estradiol (E2) increased in all treatment groups compared to the model group, oxidative stress indexes nitric oxide synthase (NOS), nitric oxide (NO), and reactive oxygen species (ROS) decreased, growth biochemical factors brain derived neurotrophic factor (BDNF) and growth differentiation factor 9 (GDF-9) concentrations increased significantly, and leukemia inhibitory factor (LIF) showed no significant change. SIRT1/P53/P21 immunohistochemical results revealed that, when compared to the model group, the expression of SIRT1 increased while the expression of P53 and P21 proteins decreased in all treatment groups, with the cetrorelix+Zuogui pills group having the largest decrease, with significant differences in all indicators. We conclude that cetrorelix combined with Zuogui pills for kidney nourishing and Yin recipe improved the oxidative stress response in the follicle by regulating the SIRT1/P53/P21 pathway, reducing peroxide product production, protecting ovarian function, and regulating ovarian hormone secretion, and its efficacy is superior to that of cetrorelix or Zuogui pills alone.

Study on the mechanism of miRNA expression profile and enrichment analysis on p21 in oxygen-induced retinopathy mice / 中华眼底病杂志

Objective:To observe the expression of miRNA in retinal tissue of mice with oxygen-induced retinopathy (OIR), and screen miRNAs related to p21 and retinal neovascularization (RNV) formation.Methods:A experimental study. Forty healthy 7-day-old C57BL/6J mice were randomly divided into normal group and OIR group, with 20 mice in each group. The oxygen induced RNV model was constructed in the OIR group, and no treatment was performed in the normal group. At the age of 17 days, the mice were killed and the RNV of mice was observed by retinal fluorescence; the nuclei of vascular endothelium that broke through the inner limiting membrane of retina were counted under light microscope. The retinal tissues were taken for miRNA chip analysis to detect the differentially expressed miRNAs between the normal group and the OIR group. The resulting differential miRNA target genes were subjected to enrichment analysis based on gene annotation (GO) and Kyoto Encyclopedia of genes and genomes (KEGG); miRNAs and pathways that may be related to p21 were screened through Targetscan, MiRanda and MicroT-CDs database alignment. Independent sample t-test was used for pairwise comparison between groups. Results:Compared with the normal group, the area of nonperfusion area, RNV and the number of vascular endothelial nuclei that broke through the inner limiting membrane of the retina in the OIR group increased significantly, differences were statistically significant ( t=18.800, 9.025; P<0.05). Compared with the normal group, there were 54 miRNAs that were statistically differentially expressed in the OIR group, of which 47 were up-regulated and 7 were down-regulated. A total of 13 miRNAs related to p21 were screened from the alignment results of the three databases with the obtained differential miRNAs. According to the difference multiples, they were miR-7218-5p, miR-322-5p, miR-224-5p, miR-335-5p, miR-329-3p, miR-362-3p, miR-532-5p, miR-20b-5p, miR-20a-5p, miR-195a-5p, miR-423-5p, miR-497a-5p, and miR-129-5p. Differential miRNA target gene enrichment analysis yielded 1 112 go entries and 50 KEGG pathways, of which 50 go entries and 13 KEGG pathways were related to p21. Conclusion:13 miRNAs related to p21 were screened out in the OIR model.

Study on the mechanism of miRNA expression profile and enrichment analysis on p21 in oxygen-induced retinopathy mice / 中华眼底病杂志

Objective:To observe the expression of miRNA in retinal tissue of mice with oxygen-induced retinopathy (OIR), and screen miRNAs related to p21 and retinal neovascularization (RNV) formation.Methods:A experimental study. Forty healthy 7-day-old C57BL/6J mice were randomly divided into normal group and OIR group, with 20 mice in each group. The oxygen induced RNV model was constructed in the OIR group, and no treatment was performed in the normal group. At the age of 17 days, the mice were killed and the RNV of mice was observed by retinal fluorescence; the nuclei of vascular endothelium that broke through the inner limiting membrane of retina were counted under light microscope. The retinal tissues were taken for miRNA chip analysis to detect the differentially expressed miRNAs between the normal group and the OIR group. The resulting differential miRNA target genes were subjected to enrichment analysis based on gene annotation (GO) and Kyoto Encyclopedia of genes and genomes (KEGG); miRNAs and pathways that may be related to p21 were screened through Targetscan, MiRanda and MicroT-CDs database alignment. Independent sample t-test was used for pairwise comparison between groups. Results:Compared with the normal group, the area of nonperfusion area, RNV and the number of vascular endothelial nuclei that broke through the inner limiting membrane of the retina in the OIR group increased significantly, differences were statistically significant ( t=18.800, 9.025; P<0.05). Compared with the normal group, there were 54 miRNAs that were statistically differentially expressed in the OIR group, of which 47 were up-regulated and 7 were down-regulated. A total of 13 miRNAs related to p21 were screened from the alignment results of the three databases with the obtained differential miRNAs. According to the difference multiples, they were miR-7218-5p, miR-322-5p, miR-224-5p, miR-335-5p, miR-329-3p, miR-362-3p, miR-532-5p, miR-20b-5p, miR-20a-5p, miR-195a-5p, miR-423-5p, miR-497a-5p, and miR-129-5p. Differential miRNA target gene enrichment analysis yielded 1 112 go entries and 50 KEGG pathways, of which 50 go entries and 13 KEGG pathways were related to p21. Conclusion:13 miRNAs related to p21 were screened out in the OIR model.

The cell cycle inhibitor P21 promotes the development of pulmonary fibrosis by suppressing lung alveolar regeneration

The cell cycle inhibitor P21 has been implicated in cell senescence and plays an important role in the injury-repair process following lung injury. Pulmonary fibrosis (PF) is a fibrotic lung disorder characterized by cell senescence in lung alveolar epithelial cells. In this study, we report that P21 expression was increased in alveolar epithelial type 2 cells (AEC2s) in a time-dependent manner following multiple bleomycin-induced PF. Repeated injury of AEC2s resulted in telomere shortening and triggered P21-dependent cell senescence. AEC2s with elevated expression of P21 lost their self-renewal and differentiation abilities. In particular, elevated P21 not only induced cell cycle arrest in AEC2s but also bound to P300 and β-catenin and inhibited AEC2 differentiation by disturbing the P300-β-catenin interaction. Meanwhile, senescent AEC2s triggered myofibroblast activation by releasing profibrotic cytokines. Knockdown of P21 restored AEC2-mediated lung alveolar regeneration in mice with chronic PF. The results of our study reveal a mechanism of P21-mediated lung regeneration failure during PF development, which suggests a potential strategy for the treatment of fibrotic lung diseases.

The p21-activated kinases in neural cytoskeletal remodeling and related neurological disorders

The serine/threonine p21-activated kinases (PAKs), as main effectors of the Rho GTPases Cdc42 and Rac, represent a group of important molecular switches linking the complex cytoskeletal networks to broad neural activity. PAKs show wide expression in the brain, but they differ in specific cell types, brain regions, and developmental stages. PAKs play an essential and differential role in controlling neural cytoskeletal remodeling and are related to the development and fate of neurons as well as the structural and functional plasticity of dendritic spines. PAK-mediated actin signaling and interacting functional networks represent a common pathway frequently affected in multiple neurodevelopmental and neurodegenerative disorders. Considering specific small-molecule agonists and inhibitors for PAKs have been developed in cancer treatment, comprehensive knowledge about the role of PAKs in neural cytoskeletal remodeling will promote our understanding of the complex mechanisms underlying neurological diseases, which may also represent potential therapeutic targets of these diseases.

Effect of LncRNA-p21 Regulating Notch Signaling Pathway on Proliferation, Migration and Invasion of Non-small Cell Lung Cancer A549 Cells / 肿瘤防治研究

Objective To investigate the effect of LncRNA-p21 on the proliferation, migration and invasion of non-small cell lung cancer A549 cells by regulating Notch signaling pathway. Methods The pcDNA-lincRNA-p21 and empty plasmid pcDNA were transfected into A549 cells, and they were divided into overexpression group and empty vector group. Cells from the stably-transfected overexpression group were added with the Notch signaling pathway specific activator Jagged1 protein and set as the Notch activator group. In addition, the cells without treatment were taken as the control group. Cell proliferation, migration and invasion of each group were detected by MTT method, scratch test and Transwell cell test. The expressions of Notch 1, HES-1, NICD, E-cadherin, Vimentin mRNA and protein were detected by RT-qPCR and Western blot. Results The A value of MTT test at 24, 48 and 72 hours in the overexpression group was lower than those in the control group, empty vector group and Notch activator group, and the Notch activator group was higher than the control group and the empty vector group (P < 0.05). The cell migration rate, the number of transmembrane cells and the relative expressions of Notch1, HES-1, NICD, Vimentin mRNA and protein of overexpression group at 48 hours were lower than those of the control group, empty vector group and Notch activator group, and the Notch activator group was lower than the control group and the empty vector group (P < 0.05). The relative expressions of E-cadherin mRNA and protein of overexpression group were higher than those of control group, empty vector group and Notch activator group, and the Notch activator group was higher than the control group and the empty vector group (P < 0.05). Conclusion Overexpression of LncRNA-p21 gene could inhibit the proliferation, migration and invasion of non-small cell lung cancer A549 cells. Its regulatory mechanism may be related to inhibiting Notch signaling pathway, thereby blocking the epithelial-mesenchymal transition of A549 cells.

BRD4 inhibitor specifically inhibits the development of wild-type Kras differentiated thyroid carcinoma by regulating BRD4/miR-106b-5p/P21 axis / 国际肿瘤学杂志

Objective:To explore the influence of bromodomain-containing protein 4 (BRD4) inhibitor on wild-type Kras differentiated thyroid carcinoma (DTC) and its mechanism.Methods:The DTC cell line Kras WT TPC-1 was selected and the mutant Kras G12D TPC-1 cells were constructed. CCK-8 assay was used to detect the effect of BRD4 inhibitor JQ-1 on the proliferation activity of Kras WT TPC-1 cells. Kras WT TPC-1 cells were treated with 0.2 μmol/L JQ-1 (JQ-1 group), and a negative control group (NC group) was set. Transwell invasion assay and flow cytometry were used to detect the effect of JQ-1 on the invasion and apoptosis of Kras WT TPC-1 cells. The effect of JQ-1 on the expressions of BRD4, miR-106b-5p and P21, and the effect of P21 inhibitor UC2288 on the expressions of P21 and BRD4 were detected. Kras WT TPC-1 cells were divided into JQ-1+ NC-OE group, JQ-1+ p21-OE group (overexpression of p21) and JQ-1+ p21-OE+ miR-106b-5p mimic group (overexpression of p21 and miR-106b-5 at the same time), and the proliferation, invasion and apoptosis of cells in each group were detected. TPC-1 cells were divided into Kras WT group, Kras WT+ JQ-1 group, Kras G12D group and Kras G12D+ JQ-1 group, and the cell proliferation, invasion and apoptosis of each group were detected. Results:JQ-1 inhibited the proliferation activity of Kras WT TPC-1 cells in a dose-dependent and time-dependent manner. In the NC group and JQ-1 group, the numbers of cell invasion were 124.67±9.61 and 82.67±8.02, and the apoptosis rates were (5.91±0.34)% and (10.33±1.10)%, respectively, with statistically significant differences ( t=5.812, P=0.004; t=6.653, P=0.003). JQ-1 significantly inhibited the expressions of BRD4 and miR-106b-5p, and promoted the expression of P21 in Kras WT TPC-1 cells. UC2288 significantly inhibited P21 expression, but had no significant effect on BRD4 expression. In the JQ-1+ NC-OE group, JQ-1+ p21-OE group and JQ-1+ p21-OE+ miR-106b-5p mimic group, the proliferation activities at 24 h of Kras WT TPC-1 cells was 0.46±0.03, 0.35±0.04 and 0.44±0.03 ( F=8.720, P=0.017), and the proliferation activity of JQ-1+ p21-OE group was significantly lower than that of the JQ-1+ NC-OE group ( P<0.05). The numbers of cell invasion in the three groups were 83.00±9.17, 56.67±6.03 and 79.67±10.07 ( F=8.347, P=0.018), and the number of cell invasion in the JQ-1+ p21-OE group was significantly lower than that in the JQ-1+ NC-OE group ( P=0.009). The apoptosis rates of the three groups were (10.00±0.49)%, (15.39±1.14)% and (10.32±0.80)% ( F=37.764, P<0.001), and the apoptosis rate of the JQ-1+ p21-OE group was significantly higher than that in the JQ-1+ NC-OE group ( P<0.001). There were no significant differences in cell proliferation activity, invasion number and apoptosis rate between JQ-1+ p21-OE+ miR-106b-5p mimic group and JQ-1+ NC-OE group (all P>0.05). In Kras WT group, Kras WT+ JQ-1 group, Kras G12D group and Kras G12D+ JQ-1 group, the cell proliferation activities at 24 h were 0.50±0.05, 0.39±0.04, 0.68±0.08 and 0.64±0.05 ( F=17.776, P<0.001). Compared with the Kras WT group, cell proliferation activity in the Kras WT+ JQ-1 group was significantly decreased, while that in the Kras G12D group was significantly increased (both P<0.05). The numbers of cell invasion in the four groups were 129.33±11.50, 86.00±9.54, 161.67±13.01 and 146.33±13.20 ( F=22.598, P<0.001). Compared with the Kras WT group, the number of cell invasion in the Kras WT+ JQ-1 group was significantly decreased ( P=0.002), and that in the Kras G12D group was significantly increased ( P=0.010). The apoptosis rates in the four groups were (6.17±0.50)%, (10.42±0.73)%, (3.43±0.47)% and (3.41±0.32)% ( F=119.170, P<0.001). Compared with the Kras WT group, the apoptosis rate in the Kras WT+ JQ-1 group was significantly increased ( P<0.001), and that in the Kras G12D group was significantly decreased ( P<0.001). There were no significant differences in cell proliferation activity, invasion number and apoptosis rate between Kras G12D+ JQ-1 group and Kras G12D group (all P>0.05). Conclusion:BRD4 inhibitor can specifically inhibit the development of wild-type Kras DTC via regulating the molecular axis of BRD4/miR-106b-5p/P21, but has no significant effect on the proliferation, invasion and apoptosis of mutant Kras DTC tumor cells.

Effect of Huluan Decoction on SIRT1/NF-κB/p53/p21 Pathway in Model Mice with Premature Ovarian Insufficiency / 中国实验方剂学杂志

Objective:To observe the effects of Huluan decoction on the expression of apoptosis-related protein silence information regulator 1 （SIRT1）， tumor suppressor gene （p53）， acetylated p53 （Ac-p53）， cyclin-dependent kinase inhibitor （p21）， and nuclear factor-<italic>κ</italic>B （NF-<italic>κ</italic>B） p65 and changes in ovarian tissue structure of a mouse model with premature ovarian insufficiency （POI） induced by tripterygium glycosides. Method:Fifty female C57BL/6J mice were randomly divided into the blank group， model group， low- and high-dose Huluan decoction groups， and western medicine （estradiol valerate） group. After intragastric administration of tripterygium glycosides at 80 mg·kg^-1·d^-1 for 14 successive days， mice in the low- and high-dose Huluan decoction groups and western medicine group were treated with Huluan decoction at 1.6 g·kg^-1·d^-1 and 6.2 g·kg^-1·d^-1 and estradiol valerate at 0.13 mg·kg^-1·d^-1， respectively， by gavage since the 15^th day， while those in the blank group were provided with an equal amount of distilled water for 21 consecutive days. Following the last administration， the blood was sampled for detecting the levels of serum anti-Müllerian hormone （AMH）， follicle-stimulating hormone （FSH）， and luteinizing hormone （LH） by ELISA and the ovary was harvested for observing the morphological changes by HE staining. The mRNA expression levels of p53 and p21 were measured by Real-time polymerase chain reaction （Real-time PCR）， and the protein expression levels of SIRT1， p53， p21， Ac-p53， NF-<italic>κ</italic>B were assayed by Western blot. Result:Compared with the blank group， the model group exhibited a disordered estrous cycle， diminished ovarian volume， decreased number of follicles at various developmental stages， reduced AMH， and elevated FSH and LH， and elevated p53，p21，Ac-p53，NF-<italic>κ</italic>B protein expression and p53，p21 mRNA， reduced SIRT1 protein expression（<italic>P</italic><0.01）. As revealed by the comparison with the model group， each medication group displayed an increased number of follicles， elevated AMH， reduced FSH and LH （<italic>P</italic><0.01）， up-regulated SIRT1 protein expression （<italic>P</italic><0.01）， and significantly down-regulated mRNA and protein expression of aging-related genes p53， p21， and down-regulated Ac-p53，NF-<italic>κ</italic>B protein expression （<italic>P</italic><0.01）. Conclusion:Huluan decoction significantly reverses the aging process and improves ovarian function possibly by boosting the activity of SIRT1/NF-<italic>κ</italic>B/p53/p21 pathway in ovarian cells， changing the apoptotic state， increasing the growing and mature follicles， and reducing the atretic follicles.

Hop-derived Xanthohumol Induces HL-60 Leukemia Cells Death

Background : Acute promyelocytic leukemia (APL) affects both kids and adults, however it is more prevalent in younger population. Although APL has a favorable prognostic, patients that relapse often do not respond positively to additional chemotherapy. Therefore, there is a need to further identify ways to overcome these challenges. Hypothesis: In this study, we examined antileukemic effects of xanthohumol (XN), a prenylated flav onoid derived from hops ( Humulus lupulus L ), on human promyelocytic HL - 60 cells. Materials and Methods : HL - 60 cells were exposed to different concentrations of XN (?M) for 24 h. Cell viability, cell morphology, chromatin condensation, cPARP - 1 level, and caspase - 3 activation, and the expression of p21 WAF1/Cip1 were analyzed. Results : XN reduced HL - 60 cell viability in a dose - dependent manner. XN induced a dose - dependent morphological changes including cell shrinkage and b lebbing , and significantly increased the number of cells with condensed chromatin. XN significantly increased the level of cPARP - 1, active caspase - 3, and the expression of p21WAF/CIP mRNA. Conclusion : These data indicate that XN induces HL - 60 cell death by regula ting cell cycle progression and apoptosis. This study suggests that XN may have antileukemic preventive effects.

Comparison of senescence progression in mesenchymal cells from human umbilical cord walls measured by immunofluorescence and flow cytometry of p16 and p21 / Comparação da progressão da senescência em células mesenquimais de parede de cordão umbilical humano medida por imunofluorescência e citometria de fluxo de p16 e p21

ABSTRACT Objective To follow the expansion of mesenchymal stem cells from umbilical cords by two classic senescence markers, p16 (INK4A) and p21 (CDKN1A), using practical, fast, and less expensive methods than the gold standard Western blotting technique, to evaluate its applicability in the laboratory. Methods Mesenchymal stem cells from umbilical cords were isolated from Wharton's jelly and, after quality control, morphological and immunophenotypic characterization by flow cytometry, were expanded in culture until coming close to cell cycle arrest (replicative senescence). Results A comparison was made between young cells, at passage 5, and pre-senescent cells, at passage 10, evaluating the protein expression of the classic cell senescence markers p16 and p21, comparing the results obtained by Western blotting with those obtained by flow cytometry and indirect immunofluorescence. Conclusion Follow-up of cell cultures, through indirect p16 immunofluorescence, allows the identification of mesenchymal stem cells from umbilical cord cultures at risk of reaching replicative senescence.

RESUMO Objetivo Acompanhar a expansão de células-tronco mesenquimais de cordão umbilical por dois marcadores clássicos de senescência, p16 (INK4A) e p21 (CDKN1A), usando métodos práticos, rápidos e com custo menor do que a técnica padrão-ouro de Western blotting, para avaliar sua aplicabilidade em laboratório. Métodos Células-tronco mesenquimais de cordão umbilical foram isoladas da geleia de Wharton e, após controle de qualidade e caracterização morfológica e imunofenotípica por citometria de fluxo, foram expandidas em cultura, até chegarem próximas à parada do ciclo celular (senescência replicativa). Resultados Foi feita a comparação entre células jovens, na passagem 5, e células pré-senescentes, na passagem 10, avaliando a expressão proteica dos marcadores clássicos de senescência celular p16 e p21, comparando os resultados obtidos por Western blotting com os obtidos por citometria de fluxo e imunofluorescência indireta. Conclusão O seguimento de culturas celulares, por meio da imunofluorescência indireta de p16, permite identificar as culturas de células-tronco mesenquimais de cordão umbilical em risco de atingirem a senescência replicativa.

Epigallocatechin gallate induces gene demethylation to promote acute myeloid leukemia cell apoptosis by regulating p19-p53-p21 signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the mechanism by which epigallocatechin gallate (EGCG) induces gene demethylation and promotes the apoptosis of acute myeloid leukemia KG-1 and THP-1 cell lines.@*METHODS@#KG-1 and THP-1 cells treated with 25, 50, 75, 100 or 150 μg/mL EGCG for 48 h were examined for gene methylation using MSP and for cell proliferation using MTT assay. The changes in cell cycle and apoptosis of the two cell lines after treatment with EGCG for 48 h were detected using flow cytometry. The mRNA and protein expressions of DNMT1, CHD5, p19, p53 and p21 in the cells were detected using RT-quantitative PCR and Western blot.@*RESULTS@#EGCG dose-dependently reversed hypermethylation of gene and reduced the cell viability in both KG-1 and THP-1 cells ( < 0.05). EGCG treatment caused obvious cell cycle arrest in G1 phase, significantly increased cell apoptosis, downregulated the expression of DNMT1 and upregulated the expressions of CHD5, p19, p53 and p21 in KG-1 and THP-1 cells ( < 0.05).@*CONCLUSIONS@#EGCG reduces hypermethylation of gene in KG-1 and THP-1 cells by downregulating DNMT1 to restore its expression, which results in upregulated expressions of p19, p53 and p21 and induces cell apoptosis.

Role of P21 in Resistance of Lung Cancer / 中国肺癌杂志

Lung cancer is the most common malignant tumor in the world with the highest incidence of deaths. In recent years, the treatment of lung cancer has made a significant breakthrough. However, as the tumor progresses, lung cancer cells inevitably acquire resistance and the efficacy of the treatment are greatly reduced. P21 protein plays a dual role in tumors, which not only regulates the cell cycle, induces apoptosis, inhibits cell proliferation, but also protects cells against apoptosis and promotes tumor cell resistance. This article reviews the research on P21 and lung cancer resistance, to provide new ideas for individualized treatment of lung cancer and overcoming lung cancer resistance.

The cell cycle inhibitor p21 promote mouse lung fibrosis by activating alveolar macrophages / 药学学报

This study was to determine the expression of the cell cycle inhibitor p21 in alveolar macrophages (AMs) and the role of p21 in activation of AMs in bleomycin (BLM) injury-induced lung fibrosis. The expression of CD206 in AMs was measured by immunofluorescence staining. Reverse transcription-polymerase chain reaction (RT-PCR) assay was used to detect the expression of macrophage activation markers. The coculture assay for macrophage and fibroblast was employed to explore the effect of macrophage on fibroblast activation. Immunofluorescence staining and western blotting assay were adopted to detect the expression of p21 in fibrotic tissues. AMs were treated with p21 knockdown or overexpression virus, RT-PCR and the co-culture system were used to explore the effect of p21 expression on macrophage activation. The Experimental Animal Welfare Ethics Committee of the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College approved all of the protocols for this research. Our results showed that the expression of CD206 and macrophage activation markers was increased in AMs from fibrotic mice, indicating that AMs from fibrotic mice were associated with a profibrotic phenotype. Moreover, the expression of p21 was upregulated in AMs after BLM treatment. Depletion of p21 suppressed macrophage activation, while overexpression of p21 promoted the profibrotic phenotype of AMs from healthy mice. In summary, BLM injury causes the progressive accumulation of p21 in AMs, which induces the production of a number of profibrotic factors promoting the development of pulmonary fibrosis.

Celastrol inhibits PAK1 kinase and inhibits the proliferation of pancreatic cancer cells / 药学学报

The p21-activated kinase 1 (PAK1) is a member of the P21-activated protein kinase family that plays an important role in the proliferation and on cogenesis of pancreatic cancer. PAK1 is an important target for the treatment of pancreatic cancer. At present, akinase inhibitor targeting PAK1 is still in the preclinical research stage. Therefore, screening for new PAK1 kinase inhibitors is of great significance. In this study the natural compound celastrol was found to have a significant inhibitory effect on PAK1, with an IC50 value of 3.614 μmol·L-1. Molecular docking results showed that celastrol had good binding to PAK1. An MTT assay indicated that celastrol inhibited the proliferation of pancreatic cancer cells BxPC-3 and PANC-1. Mechanistic studies revealed that the inhibition of pancreatic cancer cells by celastrol was reversed by PAK1 siRNA. Celastrol inhibited PAK1 and the subsequent activation of downstream signaling pathways, thereby activating apoptosis signaling pathways and triggering apoptosis in pancreatic cancer cells. These findings suggested that celastrol induced apoptosis in pancreatic cancer cells by suppressing the PAK1 kinase signaling pathway and has potential value for the treatment of pancreatic cancer.

Effect of Yanghetang on Apoptosis of Triple Negative Breast Cancer Cell Line MDA-MB-231 Based on Egr1/p21 Signaling Pathway / 中国实验方剂学杂志

Objective::To investigate the effect of ancient recipe Yanghetang and its drug-contained serum on apoptosis of triple negative breast cancer cell line MDA-MB-231 based on the signal pathway of early growth response gene 1 (Egr1)/cyclin dependent inhibitor (p21). Method::The drug-containing serum of Yanghetang was prepared from rats, and MDA-MB-231 cells were cultured in vitro. The blank control group was set, and MDA-MB-231 cells in Yanghetang high, middle, and low dose groups (23.4, 11.7, 5.85 g·kg-1) were intervened for 24, 48, 72 h, respectively. After that, the cell counting kit-8(CCK-8) method was used to detect the cell proliferation of each group. The blank control group was set, while MDA-MB-231 cells in Yanghetang high, middle, and low dose groups (23.4, 11.7, 5.85 g·kg-1) were treated for 48 h, and then flow cytometry was used to detect the apoptosis of each group and the distribution of cell cycle. The expression of Egr1 and p21 mRNA in each group was detected by quantificational Real-time polymerase chain reaction (Real-time PCR), while the expression of Egr1 and p21 protein in each group was detected by Western blot. Result::After MDA-MB-231 cells were intervened by Yanghetang for 24, 48, 72 h, MDA-MB-231 cell proliferation was significantly inhibited in Yanghetang high and middle dose groups as compared with the blank control group (P<0.01). After MDA-MB-231 cells were intervened by Yanghetang for 48 h, the apoptosis was significantly increased in Yanghetang high and middle dose groups as compared with the blank control group (P<0.05, P<0.01). In the Yanghetang high, middle dose groups, the proportion of cell cycle G0/G1 phase decreased, and the proportion of G2/M phase increased (P<0.05, P<0.01). The mRNA and protein expressions of Egr1, p21 were increased in Yanghetang high and middle groups (P<0.05, P<0.01). Conclusion::Yanghetang can activate Egr1/p21 signaling pathway in MDA-MB-231 cells, increase the expression of Egr1 and p21, and cause G2/M cell cycle arrest, thereby inducing apoptosis of MDA-MB-231 cells.