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1.
Einstein (Säo Paulo) ; 18: eAO5236, 2020. graf
Article in English | LILACS | ID: biblio-1133772

ABSTRACT

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.


Subject(s)
Humans , Umbilical Cord/physiology , Fluorescent Antibody Technique/methods , Cellular Senescence , Mesenchymal Stem Cells/physiology , Flow Cytometry/methods , Biomarkers/blood , Cells, Cultured , Blotting, Western , Cyclin-Dependent Kinase Inhibitor p16 , Cyclin-Dependent Kinase Inhibitor p21
2.
Article in Chinese | WPRIM | ID: wpr-772129

ABSTRACT

OBJECTIVE@#To investigate the interaction between interleukin-17 (IL-17) and interferon-γ (IFN-γ) and how their interaction affects the growth of mouse hepatoma Hepa1-6 cells.@*METHODS@#Hepa1-6 cells treated with IL-17 and IFN-γ either alone or in combination were examined for changes in cell proliferation using MTT assay and in cell cycle distribution using flow cytometry. Western blotting was used to detect the protein expression levels of proliferating cell nuclear antigen (PCNA), cyclin D1, P21 and P16 and the phosphorylation of p38MAPK, ERK1/2 and Stat1 in the cells.@*RESULTS@#Compared with control group, IFN-γ treatment obviously inhibited the growth and proliferation of Hepa1-6 cells, induced cell cycle arrest at G0/G1 phase, reduced the protein expression of PCNA and cyclin D1, and increased the protein expression of P21. IL-17 alone had no effect on the growth of Hepa1-6 cells. In the combined treatment, IL-17 significantly antagonized the effects of IFN-γ. Compared with those treated with IFN-γ alone, the cells with the combined treatment showed significantly decreased G0/G1 cell population, increased the protein expressions of PCNA and cyclin D1, and decreased the protein expression of P21. IL-17 significantly inhibited IFN-γ-induced phosphorylation of p38MAPK and ERK1/2 without affecting the phosphorylation of Stat1.@*CONCLUSIONS@#IL-17 obviously reverses the antitumor effects of IFN-γ to promote the proliferation of mouse hepatoma cells and accelerate the development of hepatocellular carcinoma.


Subject(s)
Animals , Carcinoma, Hepatocellular , Metabolism , Pathology , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Cyclin D1 , Metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Metabolism , Interferon-gamma , Interleukin-17 , Pharmacology , Liver Neoplasms , Metabolism , Pathology , Mice , Neoplasm Proteins , Metabolism , Proliferating Cell Nuclear Antigen , Metabolism
3.
Journal of Experimental Hematology ; (6): 1001-1007, 2019.
Article in Chinese | WPRIM | ID: wpr-771848

ABSTRACT

OBJECTIVE@#To investigate the methylation status of CHD5 gene promoter in bone marrow from acute myeloid leukemia (AML) patients, and the underlying mechanism for initiating the pathogenesis of AML via p19/p53/p21 pathway.@*METHODS@#Methylation status of the CHD5 gene promoter was detected by using methylation-specific polymerase chain reaction (MSPCR) in bone marrow from AML patients, and the iron-deficiency anemia (IDA) samples were served as control. The expression of CHD5, p19, p53 and p21 was determined by real-time quantitative reverse transcriptase PCR and Western blot.@*RESULTS@#The methylation of CHD5 gene in bone marrow from AML patients increased significantly (39.06%) as compared with control group (6.67%). The methylation of CHD5 gene significantly correlated with chromosome karyotype differentiation (P<0.01), but did not correlate with the patient's sex, age and clinical classification (P>0.05). The mRNA expression of CHD5 gene in AML decreased, compared with control group, the mRNA and protein expression of p19, p53 and p21 in AML with CHD5 methylation promoter decreased.@*CONCLUSION@#The hypermeltylation of CHD5 gene promoter in AML patients can lead to decrease of CHD5, p19, p53 and p21 expression levels which may reduce the inhibitory effect on proliferation of leukemia cells through the regulation of p19, p53 and p21 pathway, thus promotes the occurence of AML.


Subject(s)
Cyclin-Dependent Kinase Inhibitor p16 , Cyclin-Dependent Kinase Inhibitor p21 , DNA Helicases , DNA Methylation , Humans , Leukemia, Myeloid, Acute , Nerve Tissue Proteins , Promoter Regions, Genetic , Tumor Suppressor Protein p53
4.
Article in Chinese | WPRIM | ID: wpr-771533

ABSTRACT

The aim of this paper was to explore the effects and possible mechanisms in vitro of tea polyphenols (TP) delaying human glomerular mesangial cells (HGMCs) senescence induced by high glucose (HG). HGMCs were cultured in vitro and divided into the normal group (N, 5.5 mmol·L⁻¹ glucose), the mannitol group(MNT, 5.5 mmol·L⁻¹ glucose plus 24.5 mmol·L⁻¹ mannitol), the high dose of D-glucose group (HG, 30 mmol·L⁻¹ glucose), the low dose of TP group (L-TP, 30 mmol·L⁻¹ glucose plus 5 mg·L⁻¹ TP) and the high dose of TP group (H-TP, 30 mmol·L⁻¹ glucose plus 20 mg·L⁻¹ TP), which were cultured in 5% CO₂ at 37 °C, respectively. Firstly, the effects of TP on the cell morphology of HGMCs were observed after 72 h-intervention. Secondly, the cell cycle, the positive rate of senescence-associated-β-galactosidase (SA-β-gal) staining and the telomere length were detected, respectively. Finally, the protein expressions of p53, p21 and Rb in the p53-p21-Rb signaling pathway were investigated, respectively. And the expressions of p-STAT3 and miR-126 were examined severally. The results indicated that HG not only arrested the cell cycle in G₁ phase but also increased the positive rate of SA-β-gal staining, and shortened the telomere length. HG led to the protein over-expressions of p53, p21 and Rb and HGMCs senescence by activating the p53-p21-Rb signaling pathway. In addition, L-TP delayed HGMCs senescence by improving the cell cycle G₁ arrest, reducing SA-β-gal staining positive rate and lengthening the telomere length. L-TP reduced the protein over-expressions of p53, P21 and Rb induced by HG and inhibited the telomere-p53-p21-Rb signaling pathway. Moreover, the expression of p-STAT3 was increased and the expression of miR-126 was decreased in HGMCs induced by HG. L-TP reduced the expression of p-STAT3 and increased the expression of miR-126 in HGMCs. In conclusion, HG could induce HGMCs senescence by activating the telomere-p53-p21-Rb signaling pathway in vitro. L-TP could delay HGMCs senescence through regulating STAT3/miR-126 expressions and inhibiting the telomere-p53-p21-Rb signaling pathway activation. These findings could provide the effective interventions in clinic for preventing and treating renal cell senescence in diabetic kidney disease.


Subject(s)
Cells, Cultured , Cellular Senescence , Cyclin-Dependent Kinase Inhibitor p21 , Glucose , Humans , Mesangial Cells , MicroRNAs , Polyphenols , STAT3 Transcription Factor , Tea , Telomere , Tumor Suppressor Protein p53
5.
Article in Chinese | WPRIM | ID: wpr-771460

ABSTRACT

OBJECTIVE@#To study the effect of knocking down fascin on cervical cancer cell proliferation and tumorigenicity in nude mice.@*METHODS@#Cervical cancer CaSki cells were infected with a lentiviral vector carrying fascin siRNA or with a negative control lentivirus, and fascin mRNA and protein expressions in the cells were detected using qRT-PCR and Western blotting. MTT assay was used to determine the proliferation of CaSki cells with fascin knockdown. CaSki cells transfected with fascin siRNA or the control lentiviral vector and non-transfected CaSki cells were inoculated subcutaneously in nude mice, and the volume and weight of the transplanted tumor were measured; Western blotting was used to detect the expressions of proliferating cell nuclear antigen (PCNA), survivin, cyclin dependent kinase 4 (CDK4) and p21 proteins in the tumor xenograft.@*RESULTS@#Infection with the lentiviral vector carrying fascin siRNA, but not the negative control vector, caused significant reductions in the expression levels of fascin mRNA and protein in CaSki cells ( < 0.05). Fascin knockdown resulted in significantly reduced proliferation of CaSki cells ( < 0.05). The nude mice inoculated with CaSki cells with fascin knockdown showed reduced tumor volume and weight, lowered levels of PCNA, survivin and CDK4, and increased expression of p21 protein in the tumor xenograft compared with the control mice. The negative control lentivirus did not affect the proliferation or tumorigenicity of CaSki cells in nude mice or the expression levels of PCNA, survivin, CDK4 or p21 proteins in the xenografts.@*CONCLUSIONS@#Knocking down fascin can inhibit the growth and tumorigenicity of cervical cancer cells in nude mice.


Subject(s)
Animals , Apoptosis , Carrier Proteins , Genetics , Metabolism , Cell Line, Tumor , Cell Proliferation , Cyclin-Dependent Kinase 4 , Metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Metabolism , Female , Gene Knockdown Techniques , Genetic Vectors , Humans , Mice , Mice, Nude , Microfilament Proteins , Genetics , Metabolism , Proliferating Cell Nuclear Antigen , Metabolism , RNA, Messenger , Metabolism , RNA, Small Interfering , Survivin , Metabolism , Transfection , Tumor Burden , Uterine Cervical Neoplasms , Pathology
6.
Article in English | WPRIM | ID: wpr-719222

ABSTRACT

PURPOSE: Growth hormone transduction defect (GHTD) is characterized by severe short stature, impaired STAT3 (signal transducer and activator of transcription-3) phosphorylation and overexpression of the cytokine inducible SH2 containing protein (CIS) and p21/CIP1/WAF1. To investigate the role of p21/CIP1/WAF1 in the negative regulation of the growth hormone (GH)/GH receptor and Epidermal Growth Factor (EGF)/EGF Receptor pathways in GHTD. METHODS: Fibroblast cultures were developed from gingival biopsies of 1 GHTD patient and 1 control. The protein expression and the cellular localization of p21/CIP1/WAF1 was studied by Western immunoblotting and immunofluorescence, respectively: at the basal state and after induction with 200-μg/L human GH (hGH) (GH200), either with or without siRNA CIS (siCIS); at the basal state and after inductions with 200-μg/L hGH (GH200), 1,000-μg/L hGH (GH1000) or 50-ng/mL EGF. RESULTS: After GH200/siCIS, the protein expression and nuclear localization of p21 were reduced in the patient. After successful induction of GH signaling (control, GH200; patient, GH1000), the protein expression and nuclear localization of p21 were reduced. After induction with EGF, p21 translocated to the cytoplasm in the control, whereas in the GHTD patient it remained located in the nucleus. CONCLUSIONS: In the GHTD fibroblasts, when CIS is reduced, either after siCIS or after a higher dose of hGH (GH1000), p21’s antiproliferative effect (nuclear localization) is also reduced and GH signaling is activated. There also appears to be a positive relationship between the 2 inhibitors of GH signaling, CIS and p21. Finally, in GHTD, p21 seems to participate in the regulation of both the GH and EGF/EGFR pathways, depending upon its cellular location.


Subject(s)
Biopsy , Blotting, Western , Cell Cycle , Cyclin-Dependent Kinase Inhibitor p21 , Cyclin-Dependent Kinases , Cytoplasm , Epidermal Growth Factor , Fibroblasts , Fluorescent Antibody Technique , Growth Hormone , Humans , Phosphorylation , RNA, Small Interfering , Transducers
7.
Journal of Experimental Hematology ; (6): 1116-1121, 2018.
Article in Chinese | WPRIM | ID: wpr-689519

ABSTRACT

<p><b>OBJECTIVE</b>To study the effect of HDAC inhibitor Scriptaid on multiple myeloma IM9 cells and preliminarily clarify the mechanism of Scriptaid-induced cell apoptosis.</p><p><b>METHODS</b>The cell viability, cell cycle and cell apoptosis were measured by CCK8 assay and flow cytometry respectively, the relative target gene expression levels were detected by RT-PCR, the effect of Scriptaid on p21 promoter activity was detected by using luciferase reporter assay.</p><p><b>RESULTS</b>Scriptaid inhibited IM9 cell viability in a dose-dependent manner. Scriptaid induced IM9 cell cycle arrest at G/M phase in a dose-dependent manner. Scriptaid triggered IM9 cell apoptosis was obviously, the mRNA levels of apoptosis-related proteins Caspase 9, Caspase 3 and PARP1 were also activated. The apoptosis-associated factors BAD, PTEN and p21 increased following treatment with different dose of Scriptaid, meanwhile, p21 promoter activity was also activated significantly.</p><p><b>CONCLUSION</b>HDAC inhibitor Scriptaid can promote IM9 cell apoptosis by transcriptional activation of p21 promoter in concentration-dependent manner.</p>


Subject(s)
Apoptosis , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p21 , Histone Deacetylase Inhibitors , Pharmacology , Humans , Hydroxylamines , Pharmacology , Quinolines , Pharmacology
8.
Acta Physiologica Sinica ; (6): 33-40, 2017.
Article in Chinese | WPRIM | ID: wpr-331596

ABSTRACT

Senescence-associated secretory phenotype (SASP) is often a concomitant result of cell senescence, embodied by the enhanced function of secretion. The SASP factors secreted by senescent cells include cytokines, proteases and chemokines, etc, which can exert great influence on local as well as systemic environment and participate in the process of cell senescence, immunoregulation, angiogenesis, cell proliferation and tumor invasion, etc. Relative to the abundance of SASP models in human cells, the in vitro SASP model derived from mouse cells is scarce at present. Therefore, the study aimed to establish a mouse SASP model to facilitate the research in the field. With this objective, we treated the INK4a-deficient mouse NIH-3T3 cells and the wildtype mouse embryonic fibroblasts (MEF) respectively with mitomycin C (MMC), an anticarcinoma drug which could induce DNA damage. The occurring of cell senescence was evaluated by cell morphology, β-gal staining, integration ratio of EdU and Western blot. Quantitative RT-PCR and ELISA were used to detect the expression and secretion of SASP factors, respectively. The results showed that, 8 days after the treatment of NIH-3T3 cells with MMC (1 μg/mL) for 12 h or 24 h, the cells became enlarged and the ratios of β-gal-positive (blue-stained) cells significantly increased, up to 77.4% and 90.4%, respectively. Meanwhile, the expression of P21 protein increased and the integration ratios of EdU significantly decreased (P < 0.01). Quantitative RT-PCR detection showed that the mRNA levels of several SASP genes, including IL-6, TNF-α, IL-1α and IL-1β increased evidently. ELISA detection further observed an enhanced secretion of IL-6 (P < 0.01). On the contrary, although wildtype MEF could also be induced into senescence by MMC treatment for 12 h or 24 h, embodied by the enlarged cell volume, increased ratios of β-gal-positive cells (up to 71.7% and 80.2%, respectively) and enhanced expression of P21 protein, the secretion of IL-6 displayed no significant change. Our study indicated that, although MMC could induce senescence in both mouse NIH-3T3 cells and wildtype MEF, only senescent NIH-3T3 cells displayed the canonical SASP phenomena. Current study suggested that senescent NIH-3T3 cells might be an appropriate in vitro SASP model of mouse cells.


Subject(s)
Animals , Cell Proliferation , Cellular Senescence , Cyclin-Dependent Kinase Inhibitor p21 , Genetics , Metabolism , Cytokines , Genetics , Metabolism , DNA Damage , Fibroblasts , Interleukin-6 , Bodily Secretions , Mice , Mitomycin , Pharmacology , NIH 3T3 Cells , Phenotype
10.
Article in Chinese | WPRIM | ID: wpr-328233

ABSTRACT

<p><b>OBJECTIVE</b>To explore the inhibition and molecular mechanism of icaritin (ICT) combined doxorubicin (DOX) on human osteosarcoma MG-63 cells in vitro.</p><p><b>METHODS</b>The control group, ICT groups (10, 20, 40, 80, and 160 µmol/L), DOX groups (1, 2, 4, 8, and 16 µg/mL), and combination groups (20 µmol/ L ICT +1 µg/mL DOX, 20 µmol/L ICT +2 µg/mL DOX, 20 µmol/L ICT +4 µg/mL DOX, 40 µmol/L ICT +1 µg/mL DOX, 40 µmol/L ICT +2 µg/mL DOX, 40 µmol/L ICT +4 µg/mL DOX, 80 µmol/L ICT +1 µg/mL DOX, 80 µmol/L ICT +2 µg/mL DOX, 80 µmol/L ICT +4 µg/mL DOX) were set up. Human osteosarcoma MG-63 cells were respectively cultured and their effects on morphological changes were observed using inverted phase contrast microscope after 24-and 48-h intervention. The cell proliferation inhibition rate of each group was de- termined using CCK-8, and IC50 calculated. The MG-63 apoptosis rate was detected using Annexin V-FITC/ PI double dye flow cytometry. Expression levels of bcl-2, caspase-3, and p21 were detected using RT-PCR.</p><p><b>RESULTS</b>ICT and DOX could obviously inhibit the proliferation of MG-63 cell. Along with ICT concentration increasing from 10 µmol/L to 160 µmol/L, the cell proliferation inhibition rate also increased gradually from 9.67% ± 3.62% to 89.18% ± 9.66%. The IC50 was 46.93 µmol/L and 3.87 µg/mL respectively. ICT and DOX could cause either early or late stage apoptosis, down-regulate Bcl-2 gene expression, and up-regulate gene expressions of Caspase-3 and p21 respectively (P < 0.05). Aforesaid changes were more obviously seen in combination groups than in lCT groups and DOX groups (P < 0.05).</p><p><b>CONCLUSION</b>CT combined DOX had additive or synergistic inhibition effect for the proliferation of osteosarcoma MG-63 cells, which might be related with regulating gene expressions of bcl-2, caspase-3, and p21.</p>


Subject(s)
Apoptosis , Bone Neoplasms , Metabolism , Pathology , Caspase 3 , Metabolism , Cell Line, Tumor , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p21 , Metabolism , Down-Regulation , Doxorubicin , Pharmacology , Drug Synergism , Flavonoids , Pharmacology , Humans , Osteosarcoma , Metabolism , Pathology , Proto-Oncogene Proteins c-bcl-2 , Metabolism
11.
Article in Chinese | WPRIM | ID: wpr-272481

ABSTRACT

<p><b>OBJECTIVE</b>To study the effects of LIF combined with bFGF on the proliferation, stemness and senescence of hUC-MSC.</p><p><b>METHODS</b>Experiments were divided into 4 groups: control group, in which the cells were treated with complete medium (α-MEM containing 10% FBS); group LIF, in which the cells were treated with complete medium containing 10 ng/ml LIF; group bFGF, in which the cells were treated with complete medium containing 10 ng/ml bFGF; combination group, in which the cells were treated with complete medium containing 10 ng/ml LIF and 10 ng/ml bFGF. The growth curves of hUC-MSC at passage 4 in different groups were assayed by cell counting kit 8. Cellular morphologic changes were observed under inverted phase contrast microscope; hUC-MSC senescence in different groups was detected by β-galactosidase staining. The expression of PCNA, P16, P21, P53, OCT4 and NANOG genes was detected by RT-PCR.</p><p><b>RESULTS</b>The cell growth curves of each group were similar to the S-shape; the cell proliferation rate from high to low as follows: that in the combination group > group bFGF > group LIF > control group. Senescence and declining of proliferation were observed at hUC-MSC very early in control group; the cells in group LIF maintained good cellular morphology at early stage, but cell proliferation was slow and late senescence was observed; a few cells in group bFGF presented signs of senescence, but with quick proliferation; the cells in combination group grew quickly and maintained cellular morphology of hUC-MSC for long time. The LIF and bFGF up-regulated the expression of PCNA, OCT4 and NANOG, while they down-regulated the expression of P16, P21, P53, and their combinative effects were more significant.</p><p><b>CONCLUSION</b>LIF combined with bFGF not only can promote the proliferation and maintenance of stemness of hUC-MSC, but also can delay the senescence of hUC-MSC.</p>


Subject(s)
Cell Cycle , Cell Differentiation , Cell Proliferation , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p16 , Metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Metabolism , Fibroblast Growth Factor 2 , Pharmacology , Genes, Homeobox , Humans , Leukemia Inhibitory Factor , Pharmacology , Mesenchymal Stem Cells , Cell Biology , Octamer Transcription Factor-3 , Metabolism , Organic Chemicals , Proliferating Cell Nuclear Antigen , Metabolism , Tumor Suppressor Protein p53 , Metabolism , Umbilical Cord , Cell Biology
12.
Chinese Journal of Hematology ; (12): 144-148, 2016.
Article in Chinese | WPRIM | ID: wpr-234015

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effect of monoamine oxidase inhibitor phenelzine on proliferation, apoptosis and histone modulation in acute lymphoblastic leukemia cell line Molt-4 cells.</p><p><b>METHODS</b>The effect of Phenelzine on cell proliferation was detected by MTT. Apoptotic rate was measured by flow cytometry. The variation of apoptosis associated proteins Caspase-3, Bcl-2 and Bax, cyclin-dependent kinase inhibitor p21, tumor suppressor protein p15, and the expression level of histone methylation of H3K4, H3K9 and histone acetylation of H3, DNMT1 were detected by Western Blot.</p><p><b>RESULTS</b>① Molt-4 cell proliferation rates were (87.68±3.54)%, (67.84±3.24)%, (51.48±3.37)%, (28.72±2.56)% respectively after exposured to phenelzine at 5, 10, 15, 20 μmol/L for 24 h, P<0.05. ② After 10 μmol/L of phenelzine exposure for 24, 48, 72 h, cell proliferation rates were (67.84±3.24)%, (50.24±2.01)%, (40.31±2.25)%, P<0.05. ③ The apoptotic rates were (13.64±2.58)%, (31.24±3.42)%, (56.37±4.26)% after phenelzine treatment at 5, 10, 20 μmol/L for 24 h, which was concentration dependent. ④ Phenelzine could upregulate the expression of Bax, caspase-3, p21, and downregulate Bcl-2 expression. Phenelzine upregulated the methylation level of histone H3K4me1, H3K4me2 and histone acetylated H3, while it didn't change the level of histone H3K4me3, H3K9me1, H3K9me2. ⑤ Phenelzine inhibited DNMT1 expression and promoted p15 expression.</p><p><b>CONCLUSIONS</b>Phenelzine increased the methylation of histone H3K4me1, H3K4me2, acetylation of histone H3 and p21, and decreased the expression of DNMT1 and p15, and ultimately inhibited the proliferation and apoptosis of Molt-4 cells.</p>


Subject(s)
Acetylation , Apoptosis , Apoptosis Regulatory Proteins , Metabolism , Cell Line, Tumor , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p15 , Metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Metabolism , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases , Metabolism , Histones , Metabolism , Humans , Methylation , Phenelzine , Pharmacology
13.
Article in Chinese | WPRIM | ID: wpr-815121

ABSTRACT

To investigate the effect of jianpi-jiedu (JPJD) prescription-contained serum on colorectal cancer SW48 cell proliferation and the underlying mechanisms.
 Methods: Crude extract from JPJD was made by water extract method and the main components of crude extract from JPJD were analyzed by ultra-performance liquid phase high resolution time of flight mass spectrometry (UPLC-Q-TOF/MS). The low, medium, and high-concentration of JPJD-contained serum were prepared by the serum pharmacological method. The effect of serum containing JPJD on SW48 cell proliferation was determined by MTT assay. The cell cycle was detected by flow cytometric method. The protein levels of mammalian target of rapamycin (mTOR), phospho-mTOR, P-P53, and -P21, and the mRNA level of mTOR were examined by Western blot and RT-PCR, respectively.
 Results: Seven compounds including calycosin-7-glucoside, astragaloside, ginsenoside-Re, ginsenoside-Rb1, glycyrrhizinic acid, apigenin, atractylenolide-II were identified. MTT assays demonstrated that the SW48 cell proliferation was inhibited by medium and high concentration of JPJD-contained serum and the percentages of cells at G1 phase in SW48 cell cultured in the medium and high concentration of JPJD serum group were significantly higher than those in the control group (P<0.05). Meanwhile, the levels of mTOR mRNA and phospho-mTOR protein in the medium and high concentration of JPJD serum groups were substantially lower than those in the control group (P<0.05). Conversely, the expressions of phospho-P53 and P21 protein were significantly increased in the medium and high concentration of JPJD serum group compared with those in the control group.
 Conclusion: JPJD prescription-contained serum can inhibit SW48 cell proliferation, which may be related to mTOR-P53-P21 signaling pathways.


Subject(s)
Animals , Apigenin , Blotting, Western , Cell Cycle , Cell Division , Cell Proliferation , Genetics , Colorectal Neoplasms , Cyclin-Dependent Kinase Inhibitor p21 , Drugs, Chinese Herbal , Pharmacology , Flow Cytometry , Ginsenosides , Glycyrrhizic Acid , Humans , Lactones , Phosphorylation , Genetics , RNA, Messenger , Saponins , Sesquiterpenes , Signal Transduction , TOR Serine-Threonine Kinases , Triterpenes , Tumor Suppressor Protein p53
14.
Article in Chinese | WPRIM | ID: wpr-749663

ABSTRACT

OBJECTIVE@#To explore the relation between the expression of p53, p21, PCNA and COX-2 and local recurrence in resection margins of laryngeal carcinoma operation.@*METHOD@#SElect 98 patients with laryngeal carcinoma, who came to our hospital from Nov, 2005 to Dec, 2010. Diagnosed with early laryngeal squamous cell carcinoma by a pathological examination, all these patients received CO2 laser surgery to cut the entire tumors. Keep the primary lesion and resection margin tissues after that and take 5 mm peritumoral tissue as a sample of operation resection margin. With the chemical method of SP, record in detail the local recurrence condition by clinical diagnosis every 3-6 months or telephone follow-up for 2 years.@*RESULT@#(1)The positive rate of p53, p21, PCNA and COX-2 in the tumor tissues of the 98 patients with laryngeal carcinoma are 65. 3%, 52. 0%, 70. 4% and 69. 4%. The positive rate of p53, p21, PCNA and COX-2 in their resection margin tissues are 23. 5%, 39. 8%, 32. 7% and 30. 6%. So there is no relation between the expression of p53, p21, PCNA and COX-2 in tumor tissue and tumorous grading and TNM staging. (2)There appeared 17 cases of local recurrence in two years, with a recurrence rate of 17. 3%. The recurrence rate of patients with a positive expression of p53, p21, PCNA and COX-2 in resection margin tissue is higher than those with a negative expression(P0. 05).@*CONCLUSION@#Through the discussion on the effect of p53, p21, PC- NA and COX-2 in the process of laryngeal carcinoma cell proliferation and local recurrence, the study proposes that biochemical metabolism and molecular structure level abnormal expression occur before the change of cell morphology apparent, and suggests that positive index should be examined regularly and effective foreseeability intervention can be applied to patients with positive expression.


Subject(s)
Carcinoma, Squamous Cell , Metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Metabolism , Cyclooxygenase 2 , Metabolism , Head and Neck Neoplasms , Metabolism , Humans , Laryngeal Neoplasms , Metabolism , Neoplasm Recurrence, Local , Neoplasm Staging , Proliferating Cell Nuclear Antigen , Metabolism , Squamous Cell Carcinoma of Head and Neck , Tumor Suppressor Protein p53 , Metabolism
15.
Article in English | WPRIM | ID: wpr-296578

ABSTRACT

<p><b>OBJECTIVE</b>To explore the role of p21 in ionizing radiation-induced changes in protein levels during the G2/M transition and long-term G2 arrest.</p><p><b>METHODS</b>Protein expression levels were assessed by western blot in the human uveal melanoma 92-1 cells after treatment with ionizing radiation. Depletion of p21 was carried out by employing the siRNA technique. Cell cycle distribution was determined by flow cytometry combined with histone H3 phosphorylation at Ser28, an M-phase marker. Senescence was assessed by senescence- associated-β-galactosidase (SA-β-gal) staining combined with Ki67 staining, a cell proliferation marker.</p><p><b>RESULTS</b>Accompanying increased p21, the protein levels of G2/M transition genes declined significantly in 92-1 cells irradiated with 5 Gy of X-rays. Furthermore, these irradiated cells were blocked at the G2 phase followed by cellular senescence. Depletion of p21 rescued radiation-induced G2 arrest as demonstrated by the upregulation of G2/M transition kinases, as well as the high expression of histone H3 phosphorylated at Ser28. Knockdown of p21 resulted in entry into mitosis of irradiated 92-1 cells. However, cells with serious DNA damage failed to undergo cytokinesis, leading to the accumulation of multinucleated cells.</p><p><b>CONCLUSION</b>Our results indicated that p21 was responsible for the downregulation of G2/M transition regulatory proteins and the bypass of mitosis induced by irradiation. Downregulation of p21 by siRNA resulted in G2-arrested cells entering into mitosis with serious DNA damage. This is the first report on elucidating the role of p21 in the bypass of mitosis.</p>


Subject(s)
Cell Cycle Checkpoints , Radiation Effects , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21 , Genetics , Metabolism , DNA Damage , Down-Regulation , Fibroblasts , Metabolism , Radiation Effects , Gene Expression Regulation , Radiation Effects , Humans , Mitosis , Radiation Effects , RNA Interference , RNA, Small Interfering , Radiation, Ionizing , Up-Regulation
16.
Article in English | WPRIM | ID: wpr-290161

ABSTRACT

The present study evaluated the expression of key molecules and the status of DNA in both oral squamous cell carcinoma (OSCC) and adjacent tissues to establish a molecular surgical boundary and provide a cancer progression model. Biopsy samples from 50 OSCC patients were divided into T (cancer), P1 (0-0.5 cm), P2 (0.5-1 cm), P3 (1-1.5 cm) and P4 (1.5-2 cm) groups based on the distances from the visible boundary of the primary focus. Twenty samples of normal mucosa were used as controls. We used immunohistochemical staining and flow cytometry to evaluate p53, p21(CIP1/WAF1), eIF4E and Ki-67 expression and to determine DNA status, respectively. Sub-mucosal invasion was present in the P1 and P2 groups as determined by haematoxylin and eosin staining. Mutant p53 expression decreased gradually from cancerous to normal mucosae, whereas p21(CIP1/WAF1) expression displayed an opposite trend. eIF4E expression decreased from cancerous to normal mucosae. Ki-67 expression, the heteroploidy ratio, S-phase fraction and proliferative index decreased gradually with the distance from the tumour centre. Based on these results, we suggest that the resection boundary in OSCC surgery should be beyond 2 cm from the tumour. Additionally, the adjacent tissues of the primary focus could be used as a model for assessing cancer progression.


Subject(s)
Adult , Aged , Carcinoma, Squamous Cell , Metabolism , Pathology , Cyclin-Dependent Kinase Inhibitor p21 , Metabolism , Disease Progression , Eukaryotic Initiation Factor-4E , Metabolism , Female , Humans , Male , Middle Aged , Mouth Neoplasms , Metabolism , Pathology , Tumor Suppressor Protein p53 , Metabolism
17.
Chinese Journal of Oncology ; (12): 512-516, 2015.
Article in Chinese | WPRIM | ID: wpr-286789

ABSTRACT

<p><b>OBJECTIVE</b>The aim of this study was to investigate the prognostic value of combined expression of Aurora A, Ki-67, p53 and p21 WAF1 in patients after curative resection of non-small cell lung cancer (NSCLC).</p><p><b>METHODS</b>Expressions of Aurora A, Ki-67, p53 and p21 WAF1 in 58 tumor samples from resected primary NSCLCs were detected by immunohistochemistry. The correlation of proteins, survival and clinicopathological characteristics was analyzed.</p><p><b>RESULTS</b>The positive rates of Aurora A, Ki-67, p53 and p21 WAF1 expression were 89.7% (52/58), 53.4% (31/58), 46.6% (27/58) and 34.5% (20/58), respectively. Aurora A expression was positively correlated with nodal metastasis (69.2% vs. 37.8%, P = 0.045). The univariable analysis showed that the overall survival (OS) was 75.0%in patients with low Aurora A expression and 46.0% in patients with high Aurora A expression (P = 0.039). The 3-year survival rate was 40.0% in patients with positive expression of Aurora A and p53, 65.0% in the patients with positive expression of Aurora A or p53, and 82.1% in the patients with negative expression of Aurora A and p53 (P = 0.039). The Cox regression model showed that combined expression of Aurora and p53 is an independent factor affecting the prognosis of NSCLC patients (P = 0.015).</p><p><b>CONCLUSIONS</b>Our findings suggest that the positive expression of Aurora A, Ki-67 and p53 proteins is an unfavorable factor affecting the prognosis for NSCLC patients, and the overexpression of Aurora A is an independent unfavorable factor association with shorter OS in NSCLC patients. Detection of positive Aurora A and p53 expression may be a useful predictive prognostic indicator for NSCLC patients.</p>


Subject(s)
Aurora Kinase A , Metabolism , Carcinoma, Non-Small-Cell Lung , Metabolism , Mortality , General Surgery , Cyclin-Dependent Kinase Inhibitor p21 , Metabolism , Humans , Immunohistochemistry , Ki-67 Antigen , Metabolism , Lung Neoplasms , Metabolism , Mortality , General Surgery , Prognosis , Survival Analysis , Survival Rate , Tumor Suppressor Protein p53 , Metabolism
18.
Article in English | WPRIM | ID: wpr-250391

ABSTRACT

Senescence is an important obstacle to cancer development. Engaging a senescent response may be an effective way to cure acute myeloid leukemia (AML). The aim of this study was to examine the effect of resveratrol-downregulated phosphorylated liver kinase B1 (pLKB1) on the senescence of acute myeloid leukemia (AML) stem cells. The protein expressions of pLKB1 and Sirtuin 1 (SIRT1), a regulator of pLKB1, were measured in CD34(+)CD38(-) KG1a cells treated with resveratrol (40 μmol/L) or not by Western blotting. Senescence-related factors were examined, including p21 mRNA tested by real-time PCR, cell morphology by senescence-associated β-galactosidase (SA-β-gal) staining, cell proliferation by MTT assay and cell cycle by flow cytometry. Besides, apoptosis was flow cytometrically determined. The results showed that pLKB1 was highly expressed in CD34(+)CD38(-) KG1a cells, and resveratrol, which could downregulate pLKB1 through activation of SIRT1, induced senescence and apoptosis of CD34(+)CD38(-) KG1a cells. It was concluded that resveratrol-downregulated pLKB1 is involved in the senescence of AML stem cells.


Subject(s)
Apoptosis , Cell Line, Tumor , Cell Proliferation , Cellular Senescence , Cyclin-Dependent Kinase Inhibitor p21 , Genetics , Down-Regulation , Gene Expression Regulation, Neoplastic , Humans , Leukemia, Myeloid, Acute , Genetics , Pathology , Neoplastic Stem Cells , Phosphorylation , Protein-Serine-Threonine Kinases , Genetics , Metabolism , Sirtuin 1 , Genetics , Metabolism , Stilbenes , Pharmacology
19.
Article in English | WPRIM | ID: wpr-250349

ABSTRACT

In this study, one immortalized human normal prostatic epithelial cell line (BPH) and four human prostate cancer cell lines (LNCaP, 22Rv1, PC-3, and DU-145) were treated with Ganoderma Lucidum triterpenoids (GLT) at different doses and for different time periods. Cell viability, apoptosis, and cell cycle were analyzed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR and Western blotting. It was found that GLT dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. GLT-induced apoptosis was due to activation of Caspases-9 and -3 and turning on the downstream apoptotic events. GLT-induced cell cycle arrest (mainly G1 arrest) was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4 (CDK4) and E2F1 expression at the late time. These findings demonstrate that GLT suppresses prostate cancer cell growth by inducing growth arrest and apoptosis, which might suggest that GLT or Ganoderma Lucidum could be used as a potential therapeutic drug for prostate cancer.


Subject(s)
Antineoplastic Agents, Phytogenic , Pharmacology , Apoptosis , Caspase 3 , Genetics , Metabolism , Caspase 9 , Genetics , Metabolism , Cell Line, Tumor , Cell Survival , Cyclin D1 , Genetics , Metabolism , Cyclin-Dependent Kinase 4 , Genetics , Metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Genetics , Metabolism , Dose-Response Relationship, Drug , E2F1 Transcription Factor , Genetics , Metabolism , G1 Phase Cell Cycle Checkpoints , Genetics , Gene Expression Regulation, Neoplastic , Humans , Male , Nucleosomes , Metabolism , Pathology , Plant Extracts , Chemistry , Prostate , Metabolism , Pathology , Reishi , Chemistry , Signal Transduction , Triterpenes , Pharmacology
20.
Article in English | WPRIM | ID: wpr-250343

ABSTRACT

Neural stem cells (NSCs) proliferation can be influenced by repetitive transcranial magnetic stimulation (rTMS) in vivo via microRNA-106b-25 cluster, but the underlying mechanisms are poorly understood. This study investigated the involvement of microRNA-106b-25 cluster in the proliferation of NSCs after repetitive magnetic stimulation (rMS) in vitro. NSCs were stimulated by rMS (200/400/600/800/1000 pulses per day, with 10 Hz frequency and 50% maximum machine output) over a 3-day period. NSCs proliferation was detected by using ki-67 and EdU staining. Ki-67, p21, p57, cyclinD1, cyclinE, cyclinA, cdk2, cdk4 proteins and miR-106b, miR-93, miR-25 mRNAs were detected by Western blotting and qRT-PCR, respectively. The results showed that rMS could promote NSCs proliferation in a dose-dependent manner. The proportions of ki-67+ and Edu+ cells in 1000 pulses group were 20.65% and 4.00%, respectively, significantly higher than those in control group (9.25%, 2.05%). The expression levels of miR-106b and miR-93 were significantly upregulated in 600-1000 pulses groups compared with control group (P<0.05 or 0.01 for all). The expression levels of p21 protein were decreased significantly in 800/1000 pulses groups, and those of cyclinD1, cyclinA, cyclinE, cdk2 and cdk4 were obviously increased after rMS as compared with control group (P<0.05 or 0.01 for all). In conclusion, our findings suggested that rMS enhances the NSCs proliferation in vitro in a dose-dependent manner and miR-106b/p21/cdks/cyclins pathway was involved in the process.


Subject(s)
Animals , Animals, Newborn , Biomarkers , Metabolism , Cell Proliferation , Genetics , Cyclin-Dependent Kinase 2 , Genetics , Metabolism , Cyclin-Dependent Kinase 4 , Genetics , Metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Genetics , Metabolism , Cyclin-Dependent Kinase Inhibitor p57 , Genetics , Metabolism , Cyclins , Genetics , Metabolism , Gene Expression Regulation , Hippocampus , Cell Biology , Metabolism , Ki-67 Antigen , Genetics , Metabolism , Magnetic Fields , MicroRNAs , Genetics , Metabolism , Neural Stem Cells , Cell Biology , Metabolism , Primary Cell Culture , Rats , Rats, Sprague-Dawley , Signal Transduction
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