OBJECTIVE: To investigate the effects of methionine restriction on proliferation, cell cycle and apoptosis of human acute leukemia cells. METHODS: Cell Counting Kit-8 (CCK-8) assay was used to detect the effect of methionine restriction on HL-60 and Jurkat cells proliferation. The effect of methionine restriction on cell cycle of HL-60 and Jurkat cells was examined by PI staining. Annexin V-FITC / PI double staining was applied to detect apoptosis of HL-60 and Jurkat cells following methionine restriction. The expression of cell cycle-related proteins cyclin B1, CDC2 and apoptosis-related protein Bcl-2 was evaluated by Western blot assay. RESULTS: Methionine restriction significantly inhibited the proliferation of HL-60 and Jurkat cells in a time-dependent manner (HL-60: r =0.7773, Jurkat: r =0.8725), arrested the cells at G2/M phase (P < 0.001), and significantly induced apoptosis of HL-60 and Jurkat cells (HL-60: P < 0.001; Jurkat: P < 0.05). Furthermore, Western blot analysis demonstrated that methionine restriction significantly reduced the proteins expression of Cyclin B1 (P < 0.05), CDC2 (P < 0.01) and Bcl-2 (P < 0.001) in HL-60 and Jurkat cells. CONCLUSION Acute leukemia cells HL-60 and Jurkat exhibit methionine dependence. Methionine restriction can significantly inhibit the proliferation, promote cell cycle arrest and induce apoptosis of HL-60 and Jurkat cells, which suggests that methionine restriction may be a potential therapeutic strategy for acute leukemia.
Humans ; Cyclin B1/pharmacology* ; Cell Proliferation ; Methionine/pharmacology* ; Cell Cycle ; Apoptosis ; Leukemia, Myeloid, Acute ; Cell Division ; Cell Cycle Proteins ; Jurkat Cells ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; HL-60 Cells

Glioblastoma is a common brain tumor and the overall survival rate of the patients is very low, so it is an effective way to develop the potential chemotherapy or adjuvant chemotherapy drugs in glioblastoma treatment. As a well-known antimalarial drug, artesunate(ARTs) has clear side effects, and recently it has been reported to have antitumor effects, but rarely reported in glioblastoma. Different concentrations of ARTs were used to treat the glioblastoma cells, and then the inhibitory effect of ARTs on glioblastoma proliferation was detected by MTT assay; Ki67 immunofluorescence assay was used to detect the proliferation of cells; Soft agar experiment was used to explain the clonal formation abilities ; Flow Cytometry was used to detect the cell cycle; and Western blot assay was used to determine the expression of key cell cycle protein. MTT assay results indicated that ARTs-treated glioblastoma cell A172, U251, U87 were significantly inhibited in a time-and-dose dependent manner as compared to the control group(DMSO treatment group). Soft agar experiment showed that ARTs could significantly reduce the clonal formation ability of glioblastoma. Furthermore, Flow cytometry analysis showed that ARTs could obviously increase the cell proportion in G₀/G₁ phase and reduce the cell proportion in S phase. Western blot results showed that the expressions of cell cycle-related proteins CDK2, CDK4, cyclin D1 and cyclin B1 were all obviously down-regulated. Above all, ARTs may inhibit the proliferation of glioblastoma cells by arresting cell cycle in G₀/G₁ phase through down-regulating the expression of CDK2, CDK4, cyclin D1, cyclin B1. These results may not only provide a novel method for rediscovering and reusing ARTs but also provide a new potential drug for treating glioblastoma.
Antineoplastic Agents ; pharmacology ; Apoptosis ; Artesunate ; pharmacology ; Cell Cycle Checkpoints ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin B1 ; metabolism ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 2 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Glioblastoma ; drug therapy ; pathology ; Humans

Although previous reports showed drug-eluting stent (DES) could effectively inhibit neointima formation, in-stent restenosis (ISR) remains an important obstacle. The purpose of this study was to investigate different effects of paclitaxel on proliferation and cell cycle regulators between vascular smooth muscle cells (VSMCs) and vascular endothelial cells (VECs) of rats in vitro. The cultured VSMCs and VECs of rats from the same tissues were examined by using immunohistochemistry, flow cytometry and Western blotting in control and paclitaxel-treated groups. The results showed paclitaxel could effectively inhibit proliferation of VSMCs and VECs. However, as compared with VECs, proliferation of VSMCs in paclitaxel-treated group decreased less rapidly. The percentage of cells in G0-G1 and G2-M phases was reduced, and that in S phase increased after treatment for 72 h. The expression of cyclin D1 and B1, p27 and PCNA in VSMCs of paclitaxel-treated group was up-regulated, but that of p21 down-regulated as compared with VECs. It is concluded that there are significant differences in the expression of cell cycle regulators and proliferation rate between paclitaxel-treated VSMCs and paclitaxel-treated VECs, suggesting that the G1-S checkpoint regulated by paclitaxel may play a critical role in the development of complications of DES, which provides new strategies for treatments of ISR.
Animals ; Blotting, Western ; Cell Cycle ; drug effects ; Cell Cycle Proteins ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cyclin B1 ; metabolism ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; Endothelial Cells ; drug effects ; metabolism ; Flow Cytometry ; G1 Phase Cell Cycle Checkpoints ; drug effects ; Immunohistochemistry ; Microscopy, Fluorescence ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Paclitaxel ; pharmacology ; Proliferating Cell Nuclear Antigen ; metabolism ; Rats ; Tubulin Modulators ; pharmacology

Fucoidan is one of the main bioactive components of polysaccharides. The current study was focused on the anti-tumor effects of fucoidan on human heptoma cell line HepG2 and the possible mechanisms. Fucoidan treatment resulted in cell cycle arrest and apoptosis of HepG2 cells in a dose-dependent manner detected by MTT assay, flow cytometry and fluorescent microscopy. The results of flow cytometric analysis revealed that fucoidan induced G2/M arrest in the cell cycle progression. Hoechst 33258 and Annexin V/PI staining results showed that the apoptotic cell number was increased, which was associated with a dose-dependent up-regulation of Bax and down-regulation of Bcl-2 and p-Stat3. In parallel, the up-regulation of p53 and the increase in reactive oxygen species were also observed, which may play important roles in the inhibition of HepG2 growth by fucoidan. In the meantime, Cyclin B1 and CDK1 were down-regulated by fucoidan treatment. Down-regulation of p-Stat3 by fucoidan resulted in apoptosis and an increase in ROS in response to fucoidan exposure. We therefore concluded that fucoidan induces apoptosis through the down-regulation of p-Stat3. These results suggest that fucoidan may be used as a novel anti-cancer agent for hepatocarcinoma.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Blotting, Western ; CDC2 Protein Kinase ; genetics ; metabolism ; Cyclin B1 ; genetics ; metabolism ; Dose-Response Relationship, Drug ; Down-Regulation ; drug effects ; Flow Cytometry ; G2 Phase Cell Cycle Checkpoints ; genetics ; Gene Expression Regulation, Neoplastic ; drug effects ; Hep G2 Cells ; Hepatoblastoma ; genetics ; metabolism ; pathology ; Humans ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Microscopy, Fluorescence ; Polysaccharides ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; Reactive Oxygen Species ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; STAT3 Transcription Factor ; genetics ; metabolism ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; bcl-2-Associated X Protein ; genetics ; metabolism

OBJECTIVETo evaluate the effect of combined targeting of MEK and PI3K signaling pathways on K-ras mutated non-small cell lung cancer cell line A549 cells and the relevant mechanisms.

METHODSA549 cells were treated with different concentrations of two inhibitors. Growth inhibition was determined by MTT assay. According to the results of MTT test, the cells were divided into four groups: the control group, PI3K inhibitor group (GDC-0941,0.5 and 5.0 µmol/L), combination group I (0.5 µmol/L AZD6244+0.5 µmol/L GDC-0941) and combination group II (5.0 µmol/L AZD6244+5.0 µmol/L GDC-0941). The cell cycle and apoptosis were analyzed by flow cytometry. The expression of proteins related to apoptosis was tested with Western blot.

RESULTSBoth GDC-0941 and AZD6244 inhibited the cell proliferation. The combination group II led to a stronger growth inhibition. The combination group I showed an antagonistic effect and combination group II showed an additive or synergistic effect. Compared with the control group, the combination group I led to reduced apoptotic rate [(20.70 ± 0.99)% vs. (18.65 ± 0.92 )%, P > 0.05]; Combination group II exhibited enhanced apoptotic rate [(37.85 ± 3.18)% vs. (52.27 ± 4.36)%, P < 0.01]. In addition, in the combination group II, more A549 cells were arrested in G0/G1 phase and decreased S phase (P < 0.01), due to the reduced expressions of CyclinD1 and Cyclin B1, the increased cleaved PARP and the diminished ratio of Bcl-2/Bax.

CONCLUSIONSFor single K-ras mutated NSCLC cell line A549 cells, combination of RAS/MEK/ERK and PI3K/AKT/mTOR inhibition showed synergistic effects depending on the drug doses. Double pathways targeted therapy may be beneficial for these patients.

Apoptosis ; Benzimidazoles ; Carcinoma, Non-Small-Cell Lung ; genetics ; metabolism ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cyclin B1 ; Drug Synergism ; Enzyme Inhibitors ; Humans ; Phosphatidylinositol 3-Kinases ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins p21(ras) ; Signal Transduction ; TOR Serine-Threonine Kinases ; ras Proteins ; metabolism

OBJECTIVETo characterize the human primary cyclins (D1, E, A, B1) expressed in gastric carcinoma, and to clarify the relationship between the types of expressed primary cyclins and clinicopathological features of gastric carcinoma.

METHODSPrimary cyclins (D1, E, A, B1) expressed in single cells separated from 68 cases gastric carcinoma tissues were analyzed by flow cytometry. We classified the gastric carcinomas by different types of the expressed primary cyclins, and explore the roles of primary cyclins expressed in cell cycle and the expression patterns of the cyclins. The results were analyzed together with clinicopathological features.

RESULTSThe patterns of expressed primary cyclins could be classified into five types. The proportion was 10.3% (7/68), 22.1% (15/68), 25.0% (17/68), 29.4% (20/68), and 13.2% (9/68), respectively, from type I to type V. Each type could be, according to the degree of in-cycle cyclins expressed, divided into different sub-types. The types of primary cyclins expressed were strongly linked to invasive depth and lymph node metastasis of the gastric carcinoma (P < 0.01). The rates of lymph node metastasis were 26.6%, 43.8%, 82.3%, 95.0%, and 100.0%, respectively, from type I to type V. The type of primary cyclins expressed was also significantly associated with disease stage (TNM stage). The proportion of stage IV disease was 0, 6.7%, 17.6%, 25.0% and 55.6%, respectively, from type I to type V. It was shown that there were relationships between the sub-types of primary cyclins expressed and different growth-types, degree of cell differentiation, or, the tumor gross types (P < 0.01).

CONCLUSIONSThe types of primary cyclins expression are different in the process of the occurrence, development and metastasis of gastric carcinoma, and are correlated with clinicopathological features of gastric carcinoma.

Adult ; Aged ; Aged, 80 and over ; Cell Differentiation ; Cyclin A1 ; metabolism ; Cyclin B1 ; metabolism ; Cyclin D1 ; metabolism ; Cyclin E ; metabolism ; Cyclins ; classification ; metabolism ; Female ; Humans ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Invasiveness ; Neoplasm Staging ; Oncogene Proteins ; metabolism ; Stomach Neoplasms ; metabolism ; pathology

OBJECTIVETo investigate the effect of the selective PI3K inhibitor and MEK inhibitor on KRAS and PTEN co-mutated non-small cell lung cancer cell line NCI-H157 and the relevant mechanisms.

METHODSNCI-H157 was cultured routinely and treated with different concentrations of the two inhibitors. Cell proliferation was detected by MTT cell cycle assay. Based on the MTT results the cells were divided into four groups: the control group, PI3K inhibitor group (GDC-0941, 0.5 and 5.0 µmol/L), combination group I (0.5 µmol/L AZD6244 + 0.5 µmol/L GDC-0941) and combination group II (5.0 µmol/L AZD6244 + 5.0 µmol/L GDC-0941). Colony formation assay was performed to detect colony formation efficiency. The cell cycle and apoptosis were analyzed by flow cytometry. The expression of protein related to apoptosis was tested with Western blot.

RESULTSCell growth was inhibited by the two inhibitors. Combination groups led to stronger cell proliferation inhibition: combination group Ishowed synergistic effect of their actions and combination group II showed an additive effect; in both groups, there were decreased colony number [(77.2 ± 1.54)/well vs (61.50 ± 2.12)/well, P < 0.01] and [(51.00 ± 4.00)/ well vs (22.50 ± 3.53)/well, P < 0.01]; and enhanced apoptotic ratios [(18.30 ± 0.82)% vs (21.32 ± 0.56)%, P < 0.01] and [(27.14 ± 1.58)% vs (42.45 ± 4.42)%, P < 0.01]. In addition, compared to the PI3K inhibitor alone group, the NCI-H157 cells in the combination groups showed increased G0/G1 phase and decreased S phase (P < 0.01). Western blotting showed that the combination groups demonstrated significantly decreased expression of cyclin D1 and cyclin B1, increased p21 and cleaved PARP and decreased bcl-2/bax ratio, compared to the PI3K inhibitor only group.

CONCLUSIONThe combined inhibition of PI3K (AZD6244) and MEK (GDC-0941) has synergistic effects on the proliferation of NCI-H157 cells, but such effects appear to be in a dose-dependent manner.

Apoptosis ; drug effects ; Benzimidazoles ; administration & dosage ; pharmacology ; Carcinoma, Non-Small-Cell Lung ; genetics ; pathology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin B1 ; metabolism ; Cyclin D1 ; metabolism ; Dose-Response Relationship, Drug ; Drug Synergism ; Humans ; Indazoles ; administration & dosage ; pharmacology ; Lung Neoplasms ; genetics ; pathology ; Mitogen-Activated Protein Kinase Kinases ; antagonists & inhibitors ; metabolism ; Mutation ; PTEN Phosphohydrolase ; genetics ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; metabolism ; Poly(ADP-ribose) Polymerases ; metabolism ; Proto-Oncogene Proteins ; genetics ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Proto-Oncogene Proteins p21(ras) ; metabolism ; Signal Transduction ; Sulfonamides ; administration & dosage ; pharmacology ; bcl-2-Associated X Protein ; metabolism ; ras Proteins ; genetics

OBJECTIVE: To investigate the expression of eIF3P170, cdc2, cyclinB1 and cyclinD1 in developing cardiac myocytes, and the correlation between eIF3P170 with cdc2, cyclin D1, and cyclin B1 in mice. METHODS: Mouse cardiac myocytes were obtained at different time points. RT-PCR was employed to detect the expression of eIF3P170, cdc2, cyclin D1 and cyclin B1 mRNA. RESULTS: Expressions of eIF3P170, cdc2, cyclinD1 and cyclinB1 mRNA were higher in the embryonic Day 13, 15, 18 and postnatal Day 1, 2, 3, 5. Expressions at postnatal Day 5 reached the highest (all P values<0.05 vs other time points), and then the expressions of these genes gradually decreased to the weakest at postnatal Day 30 (all P values<0.05 vs other time points). The mRNA expression of eIF3P170 was positively correlated with cdc2, cyclin D1 and cyclin B1 mRNA expression respectively. CONCLUSION The mRNA expressions of eIF3 P170, cdc2, cyclin D1 and cyclin B1 in the embryo and the early life after birth are high. They reach the maximum at postnatal Day 5, then gradually decreased.
Animals ; CDC2 Protein Kinase ; metabolism ; Cell Cycle ; Cyclin B1 ; metabolism ; Cyclin D1 ; metabolism ; Eukaryotic Initiation Factor-3 ; metabolism ; Mice ; Myocytes, Cardiac ; cytology ; metabolism ; RNA, Messenger

PURPOSE: The molecular mechanisms that are responsible for the initiation and progression of breast cancers are largely unknown. This study was to analyze the cyclin B1, cdc2, p53 and p16 tumor suppressor genes in human breast cancer. MATERIALS AND METHODS: To investigate the role of cyclin B1, cdc2, p53 and p16 in the pathogenesis and progression of breast carcinomas, 98 cases of breast cancers were examined by immunohistochemical method. The correlations of cyclin B1, cdc2, p53 and p16 expression with various clinico-pathologic findings were analysed. RESULTS: In the normal breast tissues, cyclin B1, cdc2 and p16 were weakly expressed, while p53 was not expressed. On the other hand, cyclin B1, cdc2, p53 and p16 were overexpressed in breast cancer, showing correlation between the expression of cyclin B1 and cdc2 and breast cancers (p=0.00). The overexpressions of cdc2 and p16 were correlated with an infiltrative tumor border pattern and this was statistically significant (p<0.05). In addition, the overexpression of cdc2 was correlated with histologic high grade carcinomas (p=0.00). CONCLUSION: Cyclin B1 and cdc2 appeared to be involved in the genesis or progression of breast cancers. In addition, the overexpressions of p16 and p53 may play important roles in more aggressive tumor and the overexpression of cdc2 is associated with progression of tumor to a higher grade of breast carcinomas. The deranged overexpressions of cyclin B1, cdc2, p16 and p53 may play an important role in human breast carcinogenesis.
Adult ; Aged ; Breast Neoplasms/*genetics/metabolism/pathology ; Cyclin B/*genetics/metabolism ; Cyclin B1/*genetics/metabolism ; Cyclin-Dependent Kinase Inhibitor p16/*genetics/metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Middle Aged ; Tumor Suppressor Protein p53/*genetics/metabolism

PURPOSE: Surgical therapy is the primary treatment for oral cancer, but it can cause facial distortion. Therefore, if anticancer drugs are effective against oral cancer, they may be used preferentially. However, oral squamous carcinoma cells (OSCCs) are resistant to these drugs, so finding a way to enhance the sensitivity of these cells to anticancer drugs is important. The bacterial protein azurin is known to selectively enter cancer cells and induce apoptosis. In this study, we show the anticancer effect of azurin in OSCC. MATERIALS AND METHODS: OSCC cell line (YD-9) was subjected to azurin treatment. Cell viability, morphology and protein expression levels were monitored after treatment of azurin. Cells were also subjected to combination treatment of azurin with either 5-fluorouracil or etopside. RESULTS: Azurin-treated cells showed decreased cell viability accompanied by apoptotic phenotypes including morphological change, DNA breakage, and increases in p53 and cyclin B1 protein levels. Combination treatment of azurin with other anti-tumor agents caused an increase in sensitivity to anticancer drugs in azurin-treated YD-9 cells. CONCLUSION: Azurin has a strong synergistic anticancer effect on oral cancer cells when it is used along with anticancer drugs.
Antineoplastic Agents/*administration & dosage ; Apoptosis/drug effects ; Azurin/*administration & dosage/genetics ; Carcinoma, Squamous Cell/*drug therapy/metabolism/pathology ; Cell Line, Tumor ; Cyclin B1/metabolism ; Drug Synergism ; Etoposide/administration & dosage ; Fluorouracil/administration & dosage ; Humans ; Mouth Neoplasms/*drug therapy/metabolism/pathology ; Tumor Suppressor Protein p53/metabolism