Humans ; Models, Molecular ; Repressor Proteins ; chemistry ; metabolism ; Retinoblastoma-Binding Protein 4 ; chemistry ; metabolism

OBJECTIVETo investigate the effect of RbAp48 knockdown on the migration and invasion of human cervical cancer cells and explore the mechanism.

METHODSA small interference RNA (siRNA) was used to knock down the expression of RbAp48 in MS751 cells. The changes in cell migration and invasion were evaluated using wound healing assay and Transwell assay, respectively, and the expressions of RbAp48, vimentin, N-cadherin, E-cadherin, Snail, Twist, MMP-2 and TIMP-2 were determined with Western blotting.

RESULTSAfter siRNA-mediated RbAp48 knockdown, MS751 cells showed a significantly reduced expression of RbAp48 with significantly suppressed cell migration and invasion (P<0.01). RbAp48 knockdown induced obvious down-regulation of the expressions of interstitial cell phenotype proteins vimentin, N-cadherin, and MMP-2 and up-regulation of epithelial cell phenotype proteins E-cadherin and TIMP-2, suggesting the inhibition of epithelial- mesenchymal transition of the cells. The expressions of Snail and Twist were significantly down-regulated in the cells following RbAp48 knockdown.

CONCLUSIONKnockdown of RbAp48 can significantly inhibit epithelial-mesenchymal transition and suppress the migration and invasion of cervical cancer cell line MS751, the mechanism of which may involve the down-regulation of Snail and Twist expressions.

Antigens, CD ; metabolism ; Cadherins ; metabolism ; Cell Line, Tumor ; Cell Movement ; Down-Regulation ; Epithelial-Mesenchymal Transition ; Female ; Gene Knockdown Techniques ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Neoplasm Invasiveness ; Nuclear Proteins ; metabolism ; RNA, Small Interfering ; Retinoblastoma-Binding Protein 4 ; genetics ; Snail Family Transcription Factors ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism ; Transcription Factors ; metabolism ; Twist-Related Protein 1 ; metabolism ; Up-Regulation ; Uterine Cervical Neoplasms ; pathology ; Vimentin ; metabolism

The latest findings of our laboratory showed that Angelica sinensis polysaccharide (ASP) showed a definite effect in regulating the aging of hematopoietic stem cells. Leukemia is a type of malignant hematopoietic tumor in hematopoietic stem cells. There have been no relevant reports about ASP's effect in regulating the aging of leukemia cells. In this study, human acute myeloid leukemia (AML) KG1alpha cell lines in logarithmic growth phase were taken as the study object, and were divided into the ASP group, the cytarabine (Ara-C) group, the ASP + Ara-C group and the control group. The groups were respectively treated with different concentration of ASP, Ara-C and ASP + Ara-C for different periods, with the aim to study the effect of ASP combined with Ara-C in regulating the aging of human acute myeloid leukemia KG1alpha cell lines and its relevant mechanism. The results showed that ASP, Ara-C and ASP + Ara-C could obviously inhibit KG1alpha cell proliferation in vitro, block the cells in G0/G1 phase. The cells showed the aging morphological feature. The percentage of positive stained aging cells was dramatically increased, and could significantly up-regulate the expression of aging-related proteins P16 and RB, which were more obvious in the ASP + Ara-C group. In conclusion, the aging mechanism of KG1alpha cell induced by ASP and Ara-C may be related to the regulation of the expression of aging-related proteins, suggesting that the combined administration of ASP and anticancer drugs plays a better role in the treatment of leukemia .
Aging ; drug effects ; genetics ; metabolism ; Angelica sinensis ; chemistry ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; metabolism ; Humans ; Leukemia ; drug therapy ; genetics ; metabolism ; physiopathology ; Polysaccharides ; pharmacology ; Retinoblastoma Protein ; genetics ; metabolism ; Tumor Cells, Cultured

OBJECTIVETo observe the effect of extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chuanxiong (EXT) on delaying vascular smooth muscle cells (VSMCs) aging in aged rats.

METHODSVSMCs were obtained by the modified tissue explants technique and were shown to be positive for smooth muscle α-actin (SM-α-actin) by immunohistochemistry staining. VSMCs obtained from the young rats were served as the young control group; VSMCs obtained from the old rats were treated with no drug (the old group), with low dose extracts (20 mg/L, the EXT low-concentration group) and high dose extracts (40 mg/L, the EXT high concentration group), and with Probucal (10(-6) mol/L, the Probucal group) as a positive control. All groups were cultured for 24 h in the medium with 10% serum for 24 h followed by another 24 h in the serum-free medium. At the end of the 48-h culture, the following analyses were performed including determination of senescence-associated β-galactosidase (SAβ-Gal) activity, flow cytometry analysis of cell cycle, real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) analyses of p16, Cyclin D1, cyclin-dependent kinase 4 (CDK4) and retinoblastoma (Rb) mRNA expression, and Western blotting analyses of p16, cyclin D1, CDK4 and phosphoretinoblastoma (pRb) protein expressions.

RESULTS(1) In comparison to the younger rats, VSMCs from aged rats had significantly more SAβ-Gal positive cells (P<0.01) and more cells in S phase (P<0.05). VSMCs from the all treated groups showed a significant decrease in both SAβ-Gal positive cells (P<0.05) and S phase (P<0.05) compared to the old rats. (2) Compared with the young group, VSMCs in the old group had a significant decrease in p16 and Rb mRNA expression and a significant increase in Cyclin D1 and CDK4 mRNA expression. Compared with the old group, VSMCs in the treated groups had a significant increase in p16 and Rb mRNA expression and a significant decrease in Cyclin D1 and CDK4 mRNA expression (P<0.05). (3) Compared with the young group, VSMCs in the old group had a significant decrease in p16 protein expression and a significant increase in Cyclin D1, CDK4 and pRb protein expressions (P<0.05). Compared with the old group, VSMCs in the treated groups had a significant increase in p16 protein expression and a significant decrease in cyclinD1, CDK4 and pRb protein expressions (P<0.05).

CONCLUSIONSVSMCs obtained from old rats showed typical signs of cellular senescence and vascular aging. EXT had an effect on delaying senescence of VSMCs in vitro by altering the p16-cyclinD/CDK-Rb pathway.

Aging ; drug effects ; Animals ; Aorta ; cytology ; Cell Cycle ; drug effects ; Cellular Senescence ; drug effects ; Cyclin D1 ; genetics ; metabolism ; Cyclin-Dependent Kinase 4 ; genetics ; metabolism ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Flow Cytometry ; Gene Expression Regulation ; drug effects ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; metabolism ; Panax ; Plant Extracts ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Retinoblastoma Protein ; genetics ; metabolism ; beta-Galactosidase ; metabolism

OBJECTIVETo investigate the effect of EBV immediate-early protein Zta on cell cycle of Daudi cells and the involved mechanisms.

METHODSThe expression vector encoding Zta was constructed and electroporated into Daudi cells. Flow cytometric analysis was used to detect the cell cycle, Western blot to the protein levels of p21, Rb and E2F-1.

RESULTSThe vector was constructed successfully, the expression of Zta protein inhibited the proliferation of Daudi cells and promoted cell cycle from G(0)/G(1) phase \[(30.0 ± 3.4)%\] to S phase \[(47.7 ± 1.1)%\]. Meanwhile, Rb expression was significantly downregulated, E2F-1 and p21 expression upregulated by Zta.

CONCLUSIONZta could promote G(0)/G(1) phase to S phase transition in Daudi cells, which might be associated with the reduced expression of Rb and increased expression of E2F-1 and p21 protein.

Cell Cycle ; genetics ; Cell Division ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; E2F1 Transcription Factor ; metabolism ; Genetic Vectors ; Herpesvirus 4, Human ; genetics ; Humans ; Immediate-Early Proteins ; genetics ; Retinoblastoma Protein ; metabolism ; Trans-Activators ; genetics ; Transcriptional Activation ; Viral Proteins ; genetics

This study is to investigate the mechanism of human promyelocytic leukemia HL-60 cells proliferation induced by alteronol in vitro. Human promyelocytic leukemia HL-60 cells cultured in vitro were treated with different concentrations of alteronol. Inhibition rate was detected by SRB assay. Cellular morphological changes were observed by Hoechst and AO/EB (acridine orange/ethidium bromide dye) staining. The apoptosis rate was determined by Annexin V-FITC/PI assay. Cell cycle distribution was determined by flow cytometry. Western blotting analysis was carried out to determine the cell cycle related proteins. The proliferation of HL-60 cells treated with alteronol was inhibited in a concentration-dependent manner. Based on cell viability assay, observation on cell morphology and apoptosis rate, it confirmed that alteronol played an obvious role in proliferation inhibition of human promyelocytic leukemia HL-60 cells, but it did not induce apoptosis in human promyelocytic leukemia HL-60 cells in different concentrations groups. Alteronol could effectively inhibit the proliferation of human promyelocytic leukemia HL-60 cells inducing cell cycle arrest at G1 phase, as well as, alteration expression of cell cycle proteins level of CyclinD1 and pRb.
Antineoplastic Agents ; administration & dosage ; pharmacology ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cyclin D1 ; metabolism ; Dose-Response Relationship, Drug ; HL-60 Cells ; Humans ; Naphthoquinones ; administration & dosage ; pharmacology ; Phosphorylation ; Retinoblastoma Protein ; metabolism

OBJECTIVE: To investigate the expression of MCM2, Ki-67 and Rb and its biological characteristic in human laryngeal squamous cell carcinomas(LSCC). METHOD: The expression of MCM2 protein and Rb protein were detected in 60 cases of LSCC, 10 cases of precarcinoma, 10 cases of vocal cord polyps and 10 cases of normal laryngeal tissues, and Ki-67 protein were detected in 60 cases of LSCC and 10 cases of normal laryngeal tissues by Elivision plus immunohistochemical staining, and analyze their relations with clinicopathological characteristics. RESULT: The positive expression rate of MCM2 in LSCC was significantly higher than that in precarcinoma and normal laryngeal tissues (P < 0.05), and was positively correlated with pathological grades, clinical stages and lymph node metastases (P < 0.05) of LSCC. The positive expression rate of Rb protein in LSCC was significantly lower than that in precarcinoma and normal laryngeal tissues (P < 0.05). The expression level of MCM2 in LSCC was negatively corelated with Rb (r = -0.542, P < 0.05), the expression level of Ki-67 in LSCC (76.67%) was significantly higher than that in normal laryngeal tissues (30.00%) (P < 0.01) and the expression level of MCM2 in LSCC was positively corelated with Ki-67(r = 0.596, P < 0.01). The LI of MCM2 in the 3-year survival rate of LSCC was significantly lower than that in Ki-67 (P < 0.05). CONCLUSION Over expression of MCM2 and loss of Rb protein were related to the carcinogenesis and development of LSCC. The determination of MCM2 can be an index for estimating the level of malignancy and prognosis of LSCC.
Carcinoma, Squamous Cell ; metabolism ; mortality ; pathology ; Cell Cycle Proteins ; Humans ; Ki-67 Antigen ; metabolism ; Laryngeal Neoplasms ; metabolism ; mortality ; pathology ; Larynx ; metabolism ; Lymphatic Metastasis ; Minichromosome Maintenance Complex Component 2 ; metabolism ; Neoplasm Proteins ; metabolism ; Polyps ; metabolism ; Precancerous Conditions ; metabolism ; mortality ; pathology ; Retinoblastoma Protein ; metabolism ; Survival Rate ; Vocal Cords ; metabolism

Cellular senescence is a tumor-suppressive process instigated by proliferation in the absence of telomere replication, by cellular stresses such as oncogene activation, or by activation of the tumor suppressor proteins, such as Rb or p53. This process is characterized by an irreversible cell cycle exit, a unique morphology, and expression of senescence-associated-beta-galactosidase (SA-beta-gal). Despite the potential biological importance of cellular senescence, little is known of the mechanisms leading to the senescent phenotype. p41-Arc has been known to be a putative regulatory component of the mammalian Arp2/3 complex, which is required for the formation of branched networks of actin filaments at the cell cortex. In this study, we demonstrate that p41-Arc can induce senescent phenotypes when it is overexpressed in human tumor cell line, SaOs-2, which is deficient in p53 and Rb tumor suppressor genes, implying that p41 can induce senescence in a p53-independent way. p41-Arc overexpression causes a change in actin filaments, accumulating actin filaments in nuclei. Therefore, these results imply that a change in actin filament can trigger an intrinsic senescence program in the absence of p53 and Rb tumor suppressor genes.
Actin Cytoskeleton/metabolism ; Actin-Related Protein 2-3 Complex/*metabolism ; *Cell Aging ; Cell Cycle Proteins/metabolism ; Cell Line, Tumor ; Cell Nucleus/metabolism ; Fibroblasts/physiology ; Humans ; Recombinant Proteins/genetics/*metabolism ; Retinoblastoma Protein/*deficiency/genetics ; Tumor Suppressor Protein p53/*deficiency/genetics

To explore the potential of the anti-sense nucleic acid of CyclinD1 in lung cancer therapy, the expression vector containing the anti-sense nucleic acid of CyclinD1 was constructed and named pcDNA3.1-CyclinD1. The A549 cells were transfected with pcDNA3.1-CyclinD1 vectors. After being screened by G418, the stable expression positive clones were obtained. MTT method and flow cytometry technique were used to detect cell proliferation and apoptosis, respectively. The results showed the transfected cells exhibited significantly increased apoptosis and inhibited cell growth, compared with negative control and empty vector groups. To investigate the mechanism for anti-sense nucleic acid of CyclinD1 inducing A549 cells apoptosis, the expression levels of retinoblastoma protein (pRb), adenovirus E2 factor-1 (E2F-1), vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2 and MMP-9 were detected by Western blot, and the results showed the expressions of these proteins were all decreased significantly in anti-sense nucleic acid of CyclinD transfected group, compared with those in negative control and empty vector groups. In a word, anti-sense nucleic acid of CyclinD1 induces the apoptosis of lung adenocarcinoma cancer cells, and the depressions of pRb, E2F-1, VEGF, MMP-2 and MMP-9 expressions may be the possible mechanism.
Adenocarcinoma ; pathology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cyclin D1 ; genetics ; DNA, Antisense ; pharmacology ; Genetic Vectors ; Humans ; Lung Neoplasms ; pathology ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Recombination, Genetic ; Retinoblastoma Protein ; metabolism ; Transfection ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo study the effect of binding activities of the NH(2) terminus of E1A to the proteins regulating cell growth on ras-induced cell senescence and explore the mechanism of E1A-mediated escape from ras-induced senescence by E1A in human fibroblast.

METHODSIn primary human fibroblasts, the proteins regulating cell growth in association with E1A NH(2) terminus, including the Rb family proteins, p300/CBP, and p400, were inactivated or interfered. The effect of alterations in the binding activities of these proteins on cell senescence bypass mediated by E1A was evaluated by cell growth curve.

RESULTSThe Inactivation of Rb family proteins alone was not sufficient to rescue ras-induced cell senescence, whereas inactivation of both the Rb proteins and p300/CBP blocked ras-induced senescence of human fibroblasts.

CONCLUSIONRb and p300/CBP binding activities are both required for E1A to bypass ras-induced senescence in human fibroblasts.

Adenovirus E1A Proteins ; pharmacology ; Cellular Senescence ; drug effects ; Fibroblasts ; cytology ; Humans ; Primary Cell Culture ; Retinoblastoma Protein ; metabolism ; Skin ; cytology ; p300-CBP Transcription Factors ; metabolism ; ras Proteins ; antagonists & inhibitors ; pharmacology