OBJECTIVES: To investigate the structural changes of rat thoracic aorta and changes in expression levels of Bmal1 and cyclins in thoracic aorta endothelial cells following heat stress. METHODS: Twenty male SD rats were randomized equally into control group and heat stress group. After exposure to 32 ℃ for 2 weeks in the latter group, the rats were examined for histopathological changes and Bmal1 expression in the thoracic aorta using HE staining and immunohistochemistry. In the cell experiments, cultured rat thoracic aortic endothelial cells (RTAECs) were incubated at 40 ℃ for 12 h with or without prior transfection with a Bmal1-specific small interfering RNA (si-Bmal1) or a negative sequence. In both rat thoracic aorta and RTAECs, the expressions of Bmal1, the cell cycle proteins CDK1, CDK4, CDK6, and cyclin B1, and apoptosis-related proteins Bax and Bcl-2 were detected using Western blotting. TUNEL staining was used to detect cell apoptosis in rat thoracic aorta, and the changes in cell cycle distribution and apoptosis in RTAECs were analyzed with flow cytometry. RESULTS: Compared with the control rats, the rats exposed to heat stress showed significantly increased blood pressures and lowered heart rate with elastic fiber disruption and increased expressions of Bmal1, cyclin B1 and CDK1 in the thoracic aorta (P<0.05). In cultured RTAECs, heat stress caused significant increase of Bmal1, cyclin B1 and CDK1 protein expression levels, which were obviously lowered in cells with prior si-Bmal1 transfection. Bmal1 knockdown also inhibited heat stress-induced increase of apoptosis in RTAECs as evidenced by decreased expression of Bax and increased expression of Bcl-2. CONCLUSIONS Heat stress upregulates Bmal1 expression and causes alterations in expressions of cyclins to trigger apoptosis of rat thoracic aorta endothelial cells, which can be partly alleviated by suppressing Bmal1 expression.
Animals ; ARNTL Transcription Factors/genetics* ; Male ; Aorta, Thoracic/metabolism* ; Rats ; Rats, Sprague-Dawley ; Endothelial Cells/metabolism* ; Apoptosis ; Cells, Cultured ; Heat-Shock Response ; Cyclin B1/metabolism* ; CDC2 Protein Kinase/metabolism* ; Cyclins/metabolism* ; RNA, Small Interfering ; bcl-2-Associated X Protein/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

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 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

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

OBJECTIVETo study the inhibitory effect of small interference RNA (siRNA) targeting cyclin A2 gene on the growth of osteosarcoma MG-63 and human normal skin fibroblast HSF cells and to explore whether cyclin A2 siRNAs could become a useful tool in the treatment of osteosarcoma.

METHODSThree pairs of siRNAs targeting cyclin A2 mRNA and a pair of nonsense siRNA were designed according to the current criteria. SiRNAs were chemically synthesized and purified. The siRNAs were transfected into MG-63 cells and HSF cells via oligofectamine. The cells transfected with nonsense siRNA served as negative control group and those only treated with PBS as blank control group. Quantitative fluorescence RT-PCR, Western-blot, MTT assay, reverse transcriptase (RT)-PCR, flow cytometry and clone forming test were employed to evaluate the efficacy of RNA interference. At the same time, the mRNA expression of PCNA and cyclin B1 in siRNA-treated MG-63 cells were examined.

RESULTSAlthough all three siRNAs could reduce the cyclin A2 expression, siRNA, appeared to be the most effective. After 48 h treatment with siRNA1, cyclin A2 mRNA and protein expression in MG-63 cells was significantly reduced by nearly 80% as compared with that of the blank control group, whereas the negative and blank control groups had similar expression levels. MG-63 cells treated with siRNA1 were arrested at G0/G1 phase by 80.1% and the proliferation of these tumor cells was suppressed 48 h after transfection. Furthermore, MG-63 cells showed a decreased colony forming ability after siRNA1 treatment. In addition, the cyclin A2-depleted MG-63 cells showed decreased levels of PCNA and cyclin B1. In contrast, although cyclin A2 expression in HSF reduced by nearly 60% after treatment by siRNA1 for 48h, these cells exhibited only a slight change in cell cycling, and neither clear inhibition of proliferation nor impaired colony forming ability was observed.

CONCLUSIONCyclin A2 is critical for proliferation of MG-63 cells. Cyclin A2-siRNAs can induce obvious inhibition of cyclin A2 mRNA and protein expression in MG-63 and HSF cells, which consequently down-regulate the proliferation of MG-63 cells. There is little effect on the proliferation of siRNA-treated HSF cells. Those results indicate that siRNAs against cyclin A2 may become a potential antiproliferative tool in future antitumor therapy.

Bone Neoplasms ; metabolism ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cyclin A2 ; genetics ; metabolism ; Cyclin B1 ; metabolism ; Fibroblasts ; cytology ; metabolism ; Gene Knockdown Techniques ; Humans ; Osteosarcoma ; metabolism ; pathology ; Proliferating Cell Nuclear Antigen ; metabolism ; RNA Interference ; RNA, Messenger ; RNA, Small Interfering ; Skin ; cytology ; Transfection

Cyclin B1, a positive regulator, controls mitosis occurrence, plays an important role in cell proliferation. To investigate the clinical significance of cyclin B1, the expression of cyclin B1 in acute leukemia (AL) patients was measured; the expression of cyclin B1 and p21(cipl), and their cell cycle distribution were assayed by flow cytometry in 136 adult patients with newly diagnosed AL, 10 continuous complete remission (CCR) AL and 17 normal controls; the mRNA of cyclin B1 and p21(cipl), and the proliferation cell nuclear antigen (PCNA) in patients and normal controls were detected with semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The results showed that the expression of cyclin B1 in newly diagnosed AL patients was significantly higher than that in normal controls. For the relapsed AL patients, the cyclin B1 expression was also higher than that in normal controls, but lower than that in newly diagnosed cases, there was no significant difference between the remission cases and normal controls, nor difference between CCR AL patients and normal controls. All patients with high cyclin B1 expression had an unscheduled expression manner, that cyclin B1 protein appeared in G(1) phase, and in some case it even higher than that of G(2) phase. The response rate (partial remission + complete remission) and survival rate in the cyclin B1 high expressed patients were higher than that of cyclin B1 low expressed patients. The relapse rate in cyclin B1 high expressed patients was higher than that in cyclin B1 normally expressed patients. The survival rate in cyclin B1 high expressed patients was higher than that in cyclin B1 low expression patients. A negative correlation between the expression of cyclin B1 and p21(cipl) was observed. Additionally, cyclin B1 protein expression was generally correlated with proliferation index (PI) and proliferation cell nuclear antigen (PCNA). It is concluded that this study demonstrates for the first time cyclin B1 overexpression and abnormally distribution in cell cyclin of newly diagnosed AL patients. It was considered that cyclin B1 may play an important role in leukemic pathogeneses and can be one of the factors influencing the prognosis of AL patients.
Acute Disease ; Adolescent ; Adult ; Aged ; Cell Proliferation ; Cyclin B ; genetics ; Cyclin B1 ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; Female ; HL-60 Cells ; Humans ; Kaplan-Meier Estimate ; Leukemia ; drug therapy ; genetics ; pathology ; Male ; Middle Aged ; Neoplasm Recurrence, Local ; Prognosis ; Proliferating Cell Nuclear Antigen ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo evaluate the effect of dynein inhibitor on mouse oocyte in vitro maturation and its cyclin B1 transcription level.

METHODSImmature mouse oocytes were cultured in vitro with a known dynein ATPase activity inhibitor-sodium orthovanadate (SOV) to detect the changes of maturation rate, and semi-quantitative RT-PCR and single cell RT-PCR were performed to detect the changes of cyclin B1 mRNA level.

RESULTSIn dose-dependent experiments, the maturation rates of oocytes were significantly different between 5 micromol/L SOV and control groups (P < 0.05), and decreased with SOV increasing doses. However, the elevation of cyclin B1 mRNA level of immatured oocytes cultured for 12 h depended on SOV concentrations ranging from 50 to 500 micromol/L. In incontinuity exposed SOV experiments, the maturation rates of oocytes markedly reduced after the first incubation with 400 micromol/L SOV at least for 1 h and were first cultured in SOV-free medium for 4 h or 8 h before exposure to SOV (P < 0.05). In time-course experiment, the opposite changes of cyclin B1 mRNA level in oocytes between SOV and control groups were observed.

CONCLUSIONDynein inhibitor might delay oocytes meiosis process, and cause ectopic expression of cyclin B1 in oocytes. Most Oocytes incubated with SOV blocked at germinal vesicles (GV) stage or M I to anaphase transition due to dynein dysfunction and ectopic transcription level of cyclin B1.

Animals ; Cells, Cultured ; Cyclin B ; genetics ; metabolism ; Cyclin B1 ; Dyneins ; antagonists & inhibitors ; Female ; Gene Expression Regulation ; Meiosis ; drug effects ; Mice ; Mice, Inbred BALB C ; Oocytes ; drug effects ; growth & development ; metabolism ; RNA, Messenger ; analysis ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors ; Vanadates ; pharmacology

The aim of this study was to investigate whether and how phosphodiesterase (PDE) inhibitors modulate the proliferation, cell cycle and apoptosis in lymphoma cells. The effects of aminophylline (AM), a non-specific PDE inhibitor, on Raji cells were explored in vitro. MTT assay, light and transmission electron microscopy and annexin V staining were used to observe cell proliferation, morphologic changes and apoptosis rate in AM-treated cells, and FCM and RT-PCR techniques were adopted to detect the effect on cell cycle, the expression of cyclin B1 and Bcl-2 and mitochondrial transmembrane potential in AM-treated cells. The results showed that AM inhibited the growth of Raji cells in a concentration-dependent manner. Morphologic observations showed apoptosis changes in AM-treated cells, including cytoplamic shrinkage, cytoplasmic bubbling, karyopyknosis and nuclear fragmentation. FCM and RT-PCR detection showed that AM intervention increased the fraction of annexin V(+) cells, reduced the value of mitochondrial transmembrane potential, induced S phase arrest, and down-regulated the expression of Bcl-2 at both mRNA and protein level and cyclin B1 protein in a concentration-dependent manner. It is concluded that PDE inhibitor aminophylline may induce Raji cell growth inhibition, S phase arrest, apoptosis via down-regulation of Bcl-2 and reduction of mitochondrial transmembrane potential.
Aminophylline ; pharmacology ; Apoptosis ; drug effects ; Burkitt Lymphoma ; drug therapy ; genetics ; pathology ; Cell Division ; drug effects ; Cyclin B ; genetics ; metabolism ; Cyclin B1 ; Dose-Response Relationship, Drug ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Intracellular Membranes ; drug effects ; physiology ; Membrane Potentials ; drug effects ; Mitochondria ; drug effects ; physiology ; Phosphodiesterase Inhibitors ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; RNA, Messenger ; drug effects ; genetics ; metabolism ; S Phase ; Tumor Cells, Cultured ; drug effects ; metabolism ; ultrastructure