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AIM: To investigate the protective effect of "DuZhong-DangGui" (DZ-DG) on the knee tissue of rats with osteoarthritis (OA) and its regulation role on NLPR3 inflammasome. METHODS: Twenty-four SPF male SD rats, eighteen rats were randomly selected to establish OA model by anteri- or cruciate ligament amputation (ACLT) method, and rats were divided into control group, OA model group, DZ-DG group and positive drug group, 6 in each group, treatment for 8 weeks. The peripheral blood were collected, ELISA was used to detect the levels of IL-1β, IL-6, IL-18, and TNF-α; cartilage tissue of knee joint were collected, HE staining was used to observe pathology changes, OARSI staining was used to observe cartilage calcification and preform quantitative OARSI scoring; immunohistochemistry and TUNEL staining were used to detect the contents of Caspase1 and Collagen II and the number of apoptosis in the tissue, respectively, and western blot was used to detect the protein expressions of matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1), p53, p21, NLPR3, apoptosis-associated speck-like protein containing a CARD (ASC) and Pro-Caspase-1. RESULTS: Compared to control group, OA model group rats serum levels of IL-1β, IL-6, IL-18, and TNF - α significantly increased (P<0.01), OARSI scores significantly increased (P<0.01), chondrocyte apoptosis was increased (P<0.01), Caspase-1 content and MMP-13, p53, p21, NLPR3, ASC, Pro-Caspase-1 protein expressions significantly increased (P <0.01), while Collagen II content and TIMP-1 protein expression significantly decreased (P<0.01). Compared with the OA model group, DZ-DG group and positive drug group rats serum levels of IL-1β, IL-6, IL -18 and TNF - α significantly decreased (P< 0.05), chondrocyte apoptosis were significantly decreased (P<0.01), Caspase1 content, MMP-13, p53, NLPR3, Pro-Caspase-1 significantly decreased (P< 0.05), Collagen Ⅱ content and TIMP- 1 protein expression (P<0.01); DZ-DG group rats protein expression of p21 and ASC were decreased (P<0.01). CONCLUSION: The DZ-DG have protection role on cartilage of OA rats, its effect may related to mediation of NLPR3/ASC/Pro-Caspase-1 pathway, to decrease of IL-1β, and inhibition of cell apoptosis.
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Objective To observe the expression of P21 and CDK6 in cervical squamous cell carcinoma,and to investigate the relationship between their expression and cervical squamous cell carcinoma.Methods The expression of CDK6 and P21 in 100 cases of cervical squamous cell carcinoma,20 cases of cervical CIN lesions and 20 cases of normal cervical tissues were detected by immunohistochemical ABC method,and the relationship between them and tumor differentiation,invasion depth,lymph node metastasis and clinical stage were analyzed.Results The expression rates of P21 and CDK6 in cervical squamous cell carcinoma tissues were significantly higher than those in CIN lesions and normal cervical tissues;The low expression of P21 was associated with the depth of tumor invasion,lymph node metastasis and clinical stage (P＜0.05),and was not associated with the degree of tumor differentiation (P＞0.05);The high expression of CDK6 was associated with tumor differentiation,invasion depth,lymph node metastasis and clinical stage (P＜0.05).Conclusion The abnormal expression of P21 and CDK6 may play an important role in the occurrence and development of cervical squamous cell carcinoma,and the two may have a certain significance in the prognosis of cervical squamous cell carcinoma.
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[Abstartc] Objetcive: To analyze the effect of p21 protein activated kinase 4( PAK4) on epithelial-mesenchymal transition of breast cancer cells.Met hods: The mRNA and protein expression of PAK4,E-Cadherin,N-Cadherin and Vimentin were detected by qRTP-CR and Western blot in breast cancer cells transfected with pcDNA -PAK4 or siRNA PAK4;The biology behaviors of MCF-7 cells and MDA-MB-231 cells transfected with pcDNA-PAK4 or siRNA PAK4 were analysed by cell migration assay ,invasion assay.Results:Overexpressing PAK4 could significant increase in the migration and invasion compared with vector-infected cells, decrease the expression of epithelial marker E-cadherin and upregulate the expression of mesenchymal markers N-cadherin and Vimentin in detached MCF7 cells.Silenced PAK4 gene in detached MDA-MB-231 cells could suppress the migration and invasion ,decrease the levels of the mesenchymal markers and increased the levels of the epithelial markers at both mRNA and protein levels .Conclusion:PAK4 plays a key role in EMT of breast cancer cells ,and its promoting EMT effect associated with upregulating the expression of Slug .
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Objective To investigate the effects of ionizing radiation on the expression of P21 protein in Jurkat cell line and p21 gene in thymocytes and splenocytes of mice.Methods Flow cytometry (FCM)was used to analyze the expression of P21 protein in Jurkat cells at 12 and 24 h after irradiation to 0,0.5,1.0,2.0,4.0,and 6.0 Gy.Real-time PCR was used to detect the expression of p21 gene in thymocytes and splenocytes of mice at4 and 24 h after irradiation to 0,0.5,1.0,2.0,4.0,and 6.0 Gy.Multi-staining was used to analyze the micronucleus rates of Rct in bone marrow.Results The expressions of P21 protein were increased in a dose-dependent manner during 0.5-4.0 Gy(t=-24.23--3.96,P<0.05),but decreased at 6.0 Gy at 12 and 24 h post-irradiation(t=-11.19,-14.50,P<0.05).The expressions of p2 1 gene in both thymocytes and splenocytes of mice were increased in dose-dependent manner in the range of 0-6.0 Gy(including 6.0 Gy)(t=-29.96-8.80,P<0.05),and reached to the peak at 6.0 Gy at 4 and 24 h post-irradiation(t=-11.84--3.42,P<0.05),except thymocytes at 4 h and 1.0 Gy post-irradiation(t=-3.42,P>0.05).Conclusions The expressions of P21 protein and p21 gene could be increased by X-ray irradiation.which shows good dosedependent manners in certain range of dose.
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Objective To investigate the role of P21 protein and XIAP on apoptosis-inducing effect by mevastatin against A549 cell line and its mechanisms. Methods The cells proliferation was detected by MTT assay. The cell cycle distribution and apoptosis induction were evaluated with flow cytometer and electron microscopy. The mRNA expression of P21 and XIAP was measured with reverse transcription-polymerase chain reaction.The protein expression of P21 and XIAP was tested with flow cytometer and Western Bolt respectively. Results Mevastatin inhibited A549 cell survival in a time-dependent and concentration-depended manner. Flow cytometry showed that mevastatin induced G0/G1 phase arrest and caused apoptosis. Mevastatin did not affect P21 expression at both mRNA and total protein level.Concomitantly,P21 protein localized on cellular membrane decreased.Both mRNA and protein expression in XIAP were down regulated by mevastatin. All these effects were reversed by mevalonate.Conclusion Mevastatin can inhibite proliferation,induce apoptosis and interfere with cell progression of A549 through mevalonate pathway. Its mechanisms are explained by decreasing the expression of XIAP mRNA and protern.Targeting HMG-CoA reductase,Mevastatin blocks the isoprenylation of P21 protein which affects its anchorage on the cellular membrane.
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Objective To investigate the expressions and significance of p21 protein and K-ras protein in congenital biliary dilatation(CBD),and their relationship.Methods Surgical specimens of CBD were obtained during operation on 36 children from Jan.2000 to Sep.2007.Nine normal bile ducts specimens from miscarried fetus and children's corpse.The specimens were fixed in 40 g?L-1 formalin and embeded by paraffin,slice-fixed.SP immunohistochemical method was used to detect the expressions of p21 protein and K-ras protein.Results The positive rate of K-ras protein was 13.8% in CBD,which was significantly higher than that in normal bile duct tissues.The positive rate of p21 protein was 55.6% in CBD,and 100% in normal bile duct tissues.p21 protein positive expression rate in CBD was significantly lower than that in normal bile duct tissues(P
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PURPOSE: The purpose of this study was to correlate the immunohistdegrees Chemical expressions of p53, p21 and bcl-2, with their loss of heterozygosity (LOH) and clinical significance. MATERIALS AND METHODS: Paraffin-embedded tissue sections from 30 patients with gastric car cinomas were examined for immunohistdegrees Chemical staining and LOH study. Primary antibodies used in immunohistdegrees Chemical staining were mouse mondegrees Clonal antibody to human p53, p21/ WAF1 and bcl-2. For PCR-LOH assays, D6S271, D6S105, D18S386, TP53, D17S796, and D17S786 microsatellite markers were used. RESULTS: The expression rates of p53, p21 and bcl-2 were 76.7%, 80% and 3.3%, respectively. The expression of p21 was correlated with lymph node metastasis. LOH were found in 20.8% at D6S271, 42.3% at D6S105, 31.6% at D18S386, 39.1% at TP53, 40.9% at D17S796, and 50.0% at D17S786. No correlation was found between the immunohistdegrees Chemical expression and the LOH in these gene sites. CONCLUSION: p53 and p21 were detected in high rate, whereas bcl-2 expression rate was very low in gastric adendegrees Carcinoma. Of them, overexpression of p21 was correlated with the tumor progression. High incidence rate of LOH may play an important role in gastric carcinogenesis. These findings suggest that the effects on apoptosis and cell cycle by p53 and p21 were important in development and progression of gastric cancer.
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Animals , Humans , Mice , Antibodies , Apoptosis , Carcinogenesis , Cell Cycle , Incidence , Loss of Heterozygosity , Lymph Nodes , Microsatellite Repeats , Neoplasm Metastasis , Stomach NeoplasmsABSTRACT
PURPOSE: p21/WAF1/CIP1, p27/KIP1 and p57/KIP2 are negative regulators of the cell division cycle. We evaluated the expressions of KIP CDK inhibitors and examined the relationship of clinicopathologic parameters and cell proliferation index in gastric adendegrees Carcinomas. MATERIALS AND METHODS: The study was carried by the TUNEL method for apoptosis, immuno histdegrees Chemical staining for PCNA, p53, p21/WAF1/CIP1, p27/KIP1, and p57/KIP2 proteins and Western blot for KIPs proteins of normal and cancer tissues of stomach. RESULTS: In normal gastric mucosa, p21/WAF1/CIP1 and p27/KIP1 proteins were expressed both mainly to the superficial portion of the glands with intestinal metaplasia and stromal cells. p57/KIP2 protein was also expressed normal crypt glandular epithelial and stromal cells. In gastric adendegrees Carcinoma, p21/WAF1/CIP1 was positive in 25 of 70 (35%) and showed significant decrease in deep tumor invasion (p=0.015) and the presence of angioinvasion (p=0.013). There was signi ficant inverse correlation between p27/KIP1 expression and cell proliferating index. p21/WAF1/ CIP1 expression was related to lower apoptotic index. Western blot analysis of KIP CDK inhibitors showed marked down-regulation of p21/WAF1/CIP1 protein in cancer than normal tissue, but alternative expression in p27/KIP1 and p57/KIP2 proteins. CONCLUSION: The above results indicated that the expression loss of KIP CDK inhibitors was contributed to tumor progression and aggressiveness in gastric adendegrees Carcinoma.
Subject(s)
Apoptosis , Blotting, Western , Cell Cycle , Cell Proliferation , Down-Regulation , Gastric Mucosa , In Situ Nick-End Labeling , Metaplasia , Proliferating Cell Nuclear Antigen , Stomach , Stomach Neoplasms , Stromal CellsABSTRACT
PURPOSE: The products of the ras oncogene are proteins of 188 or 189 amino acids and 21,000 molecular weights, termed simply p21 proteins. But the exact roles of c-ras proteins in cell proliferation and differentiation as well as in neoplastic transformation are little understood. The purpose of this study is to investigate the function of the p21 protein to the human colon cancer cell lines according to the exposure time and dosage of p21. METHODS: The authors divided tumor cell lines into 3 groups as follows; group 1 (control, colon cancer cell lines without administration of p21 or polyclonal antibody), group 2 (administration of p21 with labelling of 3H-thymidine and leucine), group 3 (simultaneous administration of p21 protein and polyclonal antibody with labelling of 3H-thymidine and 3H-leucine). After deciding the most effective dose of p21 protein and culture time with target cells in preliminary studies, the morphologic changes of target cells with administration of p21 protein and the p21 expression and interaction with anti-p21 polyclonal antibody were examined by phase contrast microscopy each other. RESULTS: The results were obtained as follows: 1. The most effective dose of the p21 with the colon cancer cell in increase uptake of 3H-thymidine and 3H-leucine were 50 ng but there were some differences in culture time of the 3H-leucine; 96 hours in SBA-1, 72 hours in HT-29 and 120 hours in SW-1116. 2. The increase uptakes of the 3H-thymidine by the different dosage of p21, 50 ng vs 200 ng were 131% (50 ng), 160% (200 ng) in SBA-1, 203% (50 ng), 123% (200 ng) in HT-29, and 127% (50 ng), 189% (200 ng) in SW-1116; and increase uptakes of 3H-leucine were 130% (50 ng), 159% (200 ng) in SBA-1, 113% (50 ng), 165% (200 ng), and 164% (50 ng), 169% (200 ng) in SW-1116. 3. There were some cellular proliferation and morphological changes of the colon cancer cells such as ruffling of the cell membrane, vesicle formation or rounding of the cell after administration of the mutant p21, but such changes were not observed after simultaneous administration of the mutant p21 and anti-p21 polyclonal antibody. CONCLUSIONS: The role of p21 protein has not been to make manifest wholly. In our study, the p21 protein induce the cell proliferation and morphological changes.
Subject(s)
Humans , Amino Acids , Cell Line , Cell Line, Tumor , Cell Membrane , Cell Proliferation , Colon , Colonic Neoplasms , DNA , Genes, ras , Microscopy, Phase-Contrast , Molecular Weight , Proto-Oncogene Proteins p21(ras)ABSTRACT
Objective:To evaluate p53,c erbB 2,p21 and nm23 oncoprotein expression in diagnosis and treatment of gastric cancer.Methods:The expression of p53,c erbB 2,p21 and nm23 oncoproteins was detected with immunohistochemical method in 63 surgically resected specimens and its endoscopic biopsy specimens of gastric cancer.Results:The positive rates of p53,c erbB 2,p21 and nm23 oncoprotein expression were 37 6%～46 2%,34 6%～56 8%,37 8%～61 5%,70 3%～30 8% respectively.Oncoprotein expression was not observed in non tumor endoscopic biopsy specimens.The expression of c erbB 2,p21 was correlated with grade of tumor differentiation and the expression of p21,nm23 oncoproteins was correlated with the depth of tumor invasion and clinical different stage,namely tumor metastasis.Positive rates of p53,c erbB 2,p21 and nm23 oncoproteins between biopsy and resected specimens was of coincidence.Conclusion:Determination of p53,c erbB 2,p21 and nm23 oncoprotein expression was might be useful in diagnosis and treatment of gastric cancer,differentiating benign and malignant tumor and clinical stages,of gastric cancer. [
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Objective:To study the expression of p73? and its clinical significance in breast carcinoma.Methods:The expression of p73? was detected by immunohistochemistry in 41 breast carcinoma tissues,13 benign breast tumor tissues and 8 normal breast tissues.Results:The positive expression of p73? was found in 20/41(48.8%) of breast carcinoma tissues,1/13(7.7%)of benign breast tumor tissues,0/8(0)of normal breast tissues.The positive expression rate of p73? in breast carcinoma tissues was significantly higher than that in benign breast tumor tissues or normal breast tissues(P