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

AIMTo investigate the radiosensitizing effect and mechanism of action of staurosporine (STP) in human colon carcinoma HT-29 and breast cancer MCF-7/ADR cells.

METHODSThe effect of STP on the cytotoxicity of X-ray was determined by clonogenic assay. The effect of STP on cell cycle arrest induced by X irradiation was studied in two cell lines by using flow cytometry, Western Blotting was performed to indicate the changes of cyclin B1 and cdc2 protein levels.

RESULTSSTP sensitized the two cell lines to X-ray by clonogenic assay. STP potentiated the cytotoxicity of X-ray by 2.10- and 2.09-fold in HT-29 and MCF-7/ADR cells. Flow cytometry assay showed that exposure of HT-29 and MCF-7/ADR cells to X-ray caused cells arrest in G2 phase. The percentage of arrest G2 phase cells were 56% and 52.7%, respectively. The addition of STP after irradiation resulted in a dose-dependent reduction of G2 phase arrest induced by X-ray. Furthermore, the results showed that STP blocked decrease of cyclin B1 expression induced by X-ray, while mitotic index measurement indicated that X-ray-irradiated cells treated with STP entered mitosis. The data suggested that the potentiation of cytotoxicity of X-ray by STP is associated with the suppression of cyclin B1 expression, which result in the abrogation of G2 arrest, before the cells entered into M phase, they had not enough time to repair.

CONCLUSIONSTP is a potent G2 checkpoint abrogator and markedly enhanced the cytotoxicity of X irradiation in the p53 mutant cancer cells.

Breast Neoplasms ; pathology ; Cyclin B ; biosynthesis ; Cyclin B1 ; Enzyme Inhibitors ; pharmacology ; Female ; G2 Phase ; drug effects ; HT29 Cells ; Humans ; Mitotic Index ; Particle Accelerators ; Radiation Tolerance ; drug effects ; Radiation-Sensitizing Agents ; pharmacology ; Staurosporine ; pharmacology ; Tumor Cells, Cultured

BACKGROUNDCyclin B1 (CLB1) is necessary for mitotic initiation in mammalian cells and plays important roles in cancer development. Therefore, a potential strategy in cancer therapy is to suppress the activity of CLB1 by delivering antisense constructs of CLB1 into tumor cells. In previous CLB1 studies, antisense constructs with a short half life were often used and these constructs might not persistently inhibit CLB1.

METHODSWe successfully created a recombinant plasmid encoding the full-length antisense cDNA of mouse cyclin B1 (AS-mCLB1) and transfected this construct to the murine Lewis lung carcinoma (LL/2) and CT-26 colon carcinoma (CT-26) cells. We isolated clones of LL/2 and CT-26 transfectants with stable expression of AS-mCLB1. Reverse transcriptional polymerase chain reaction (RT-PCR) and Western blot were applied to detect the expression of the mRNA and protein levels of CLB1. To further test the efficacy of this strategy in vivo, AS-mCLB1-expressing LL/2 and CT-26 transfectants were implanted into mice.

RESULTSWe found the expression of the mRNA and protein levels of CLB1 decrease in these transfectants. The inhibition of CLB1 caused prominent G1 arrest, abnormal morphology, retarded cell growth and an increase in apoptosis. In AS-mCLB1-expressing LL/2 and CT-26 transfectants implanted mice, tumorigenicity was effectively suppressed compared with the controls. In addition, the expression of AS-mCLB1 also significantly increases the survival duration of implanted animals.

CONCLUSIONAS-mCLB1 is likely to be useful in future cancer therapy, which may be associated with its ability to down-regulate the expression of CLB1 and then induce G1arrest and apoptosis in tumor cells.

Animals ; Apoptosis ; Cell Proliferation ; Cell Survival ; Cyclin B ; antagonists & inhibitors ; genetics ; Cyclin B1 ; DNA, Antisense ; pharmacology ; DNA, Complementary ; pharmacology ; G1 Phase ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Neoplasms, Experimental ; pathology ; therapy

In order to investigate the role and the mechanism of protein tyrosine phosphatase (PTPase) signaling pathway in the regulation of proliferation, cell cycle and apoptosis in lymphoma cells, the effects of sodium orthovanadate, Na(3)VO(4), a specific PTPase inhibitor, were explored on Raji lymphoblast-like cell line by MTT assay and CFU-Raji culture, morphologic observation, DNA gel electrophoresis, FCM and RT-PCR. Results showed that MTT assay and CFU-Raji culture demonstrated that sodium or thovanadate inhibited the growth of Raji cells in a concentration-dependent fashion; morphologic observations showed that Raji cells exhibited cytoplasm shrinkage, cytoplasm membrane blebbing, nuclear fragmentation and chromatin condensation forming crescents along nuclear membrane characteristic of apoptosis in the presence of Na(3)VO(4); DNA gel electrophoresis revealed typical DNA ladder reminiscent of DNA cleavage at internucleosomal sites in Na(3)VO(4) treated cells; FCM and RT-PCR indicated that Na(3)VO(4) intervention increased the fraction of annexin V(+) PI(-) cells, reduced the value of mitochondrial transmembrane potential, induced G(2)/M arrest and down-regulated the expression of Bcl-2 and cyclin B1 at both mRNA and protein level in a concentration-dependent manner. It was concluded that PTPase pathway might be implicated in the regulation of cell proliferation, cell cycle and apoptosis, and PTPase specific inhibitor Na(3)VO(4) could induce Raji cell growth inhibition, G(2)/M arrest and apoptosis via down-regulation of Bcl-2 and cyclin B1, and reduction of mitochondrial transmembrane potential.
Apoptosis ; drug effects ; Cell Division ; drug effects ; Cyclin B ; analysis ; Cyclin B1 ; Enzyme Inhibitors ; pharmacology ; Humans ; Leukocyte Common Antigens ; analysis ; Membrane Potentials ; drug effects ; Mitochondria ; drug effects ; physiology ; Protein Tyrosine Phosphatases ; antagonists & inhibitors ; Vanadates ; pharmacology

OBJECTIVETo assess the ability of tetrandrine (Tet) to enhance the sensitivity to irradiation and its mechanism in cell lines of human breast cancer p53-mutant MCF-7/ADR, p53-wild-type MCF-7 and human colon carcinoma p53-mutant HT-29 as well as in C26 colorectal carcinoma-bearing BALB/c mice.

METHODSMCF-7/ADR, HT-29 and MCF-7 cells were exposed to irradiation in the absence or presence of tetrandrine. The effect of Tet on the cytotoxicity of X-irradiation in these three cells was determined and the effect of tetrandrine on cell cycle arrest induced by irradiation in its absence or presence was studied by flow cytometry. Moreover, mitotic index measurement determined mitosis of cells to enter mitosis. Western blotting was employed to detect cyclin B 1 and Cdc2 proteins in extracts from irradiated or non-irradiated cells of MCF-7/ADR, HT-29 and MCF-7 treated with tetrandrine at various concentrations. Tumor growth delay assay was conducted to determine the radio-sensitization of tetrandrine in vivo.

RESULTSClonogenic assay showed that tetrandrine markedly enhanced the lethal effect of X-rays on p53-mutant MCF-7/ADR and HT-29 cells and the sensitization enhancement ratio (SER) of tetrandrine was 1.51 and 1.63, but its SER was only 1.1 in p53-wt MCF-7 cells. Irradiated p53-mutant MCF-7/ADR and HT-29 cells were only arrested in G2/M phase while MCF-7 cells were arrested in G1 and G2/M phases. Radiation-induced G2 phase arrests were abrogated by tetrandrine in a concentration-dependent manner in MCF-7/ADR and HT-29 cells, whereas redistribution within MCF-7 cell cycle changed slightly. The proportion of cells in M phase increased from 1.3% to 14.7% in MCF-7/ADR cells, and from 1.5% to 13.2% in HT-29 cells, but 2.4% to 7.1% in MCF-7 cells. Furthermore, the levels of cyclin B 1 and Cdc2 expression decreased after X-irradiation in MCF-7/ADR and HT-29 cells, and the mitotic index was also lower. Tet could reverse the decrease and induce the irradiated cells to enter mitosis (M phase). Endosomatic experiment showed that tetrandrine caused tumor growth delay in irradiated mice.

CONCLUSIONTetrandrine boosts the cell killing activity of irradiation both in vitro and in vivo. Tetrandrine is a potent abrogator for G2 checkpoint control and can sensitize the cells to radiation.

Animals ; Benzylisoquinolines ; pharmacology ; CDC2-CDC28 Kinases ; metabolism ; Cell Line, Tumor ; Cyclin B ; metabolism ; Cyclin B1 ; Drug Screening Assays, Antitumor ; G2 Phase ; drug effects ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Radiation Tolerance

OBJECTIVETo investigate the radiosensitizing effect and its mechanism of 3-MA in human hypopharynx cancer cells.

METHODS5 mmol/L of 3-MA combined with 2 Gy or 4 Gy of X-ray was utilized to deal with Fadu cells, and the cell livability (cloning efficiency) and DNA lesion severity (tail moment) of each groups was examined by clonogenic survival assay and comet assay, then differences were compared between groups by independent-sample T test. Fadu cells were then treated with different dose of 3-MA (1, 2, 5, 10 mmol/L), the alteration of cell cycle distribution was detected by flow cytometer, and differences among groups were analyzed through one-way analysis of variance. The expression of p62 and cyclinB1 in each group was examined by western blot.

RESULTSThe livability and DNA lesion severity of cells treated with 3-MA alone showed no notable variation. Compared with non-3-MA groups, the cloning efficiency of cells treated with 3-MA decreased much more after irradiated with 2 Gy or 4 Gy of X-ray (t = 13.41 or 13.98, P < 0.001), and the cells showed a more serious DNA lesion (t = 7.07 or 6.91, P < 0.001). The G2/M percentages of cells in the control group and groups treated with 1, 2, 5, 10 mmol/L of 3-MA were 17.10 ± 1.20, 23.30 ± 2.3, 39.90 ± 3.12, 58.47 ± 1.65, 76.13 ± 3.51 and differences among groups were statistically significant (F = 278.4, P < 0.05). The expression of p62 in cells treated with 3-MA showed a dose-dependent increase, while cyclinB1 showed a dose-dependent decrease.

CONCLUSIONSThe autophagy inhibitor 3-MA could enhance radiosensitivity of human hypopharynx cancer cells by inducing G2/M arrest and enhancing irradiation-induced DNA damage.

Adaptor Proteins, Signal Transducing ; metabolism ; Adenine ; analogs & derivatives ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cyclin B1 ; metabolism ; DNA Damage ; drug effects ; Humans ; Radiation Tolerance ; drug effects ; Radiation-Sensitizing Agents ; pharmacology ; Sequestosome-1 Protein

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

AIMTo determine the anti-angiogenic activity of emodin.

METHODSChick embryo assay and cultured endothelial cells were used.

RESULTSEmodin at doses of 150 and 300 microg/egg caused 37.6% and 63.2% inhibition of angiogenesis, respectively. Emodin was shown to inhibit the proliferation of primary cultured bovine aortic endothelial cells in the absence or presence of basic-fibroblast growth factor (bFGF) or the presence of vascular endothelial growth factor (VEGF) in a dose-dependent manner. The IC50 values by MTT assay were 5.56, 8.40 or 6.91 mg x L(-1), respectively. Emodin at concentrations from 5.4 to 21.6 mg x L(-1) induced apoptosis of endothelial cells for 37.6% to 72.6%. Emodin caused endothelial cell cycle arrest at G2/M phase. After emodin treatment, there was a down-regulation of Cyclin B1, P34cdc2, and Bcl-2 protein expression while the Bax protein expression was unaffected.

CONCLUSIONEmodin shows anti-angiogenic activity and might be useful for the development of novel anti-cancer therapy.

Animals ; Aorta ; cytology ; Apoptosis ; drug effects ; CDC2 Protein Kinase ; metabolism ; Cattle ; Cell Cycle ; drug effects ; Cell Division ; drug effects ; Cells, Cultured ; Chick Embryo ; Cyclin B ; metabolism ; Cyclin B1 ; Emodin ; pharmacology ; Endothelial Cells ; cytology ; metabolism ; Fibroblast Growth Factor 2 ; pharmacology ; Neovascularization, Physiologic ; drug effects ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Vascular Endothelial Growth Factor A ; pharmacology

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

In the present study, a newly synthesized benzofuran lignan 4-formyl-2-(4-hydroxy-3methoxyphenyl)-5-(2-methoxycarbonyethyl)-7-methoxy-benzo [b] furan (ERJT-12) was tested for its antiproliferative activity on human tumor cells. The related mechanisms were also investigated. In vitro growth inhibitory effects of ERJT-12 on various cancer cell lines were determined by MTT assay. Cell cycle distribution and apoptosis were detected by flow cytometry. The integrity of DNA was assessed by agarose gel electrophoresis. Activation of Caspase-3/7 and Caspase-6 was measured by colorimetric assay. The expressions of cell cycle proteins cell divide cycle 25c (Cdc25c), cyclin dependent kinase 1 (CDK1), CyclinB1 and apoptosis-related proteins Bax and Bcl-2 were detected by Western blotting. MTT assay showed that ERJT-12 inhibited the proliferation of several cancer cell lines including multidrug resistant cells. MCF-7 cells were markedly arrested at gap2/mitosis (G2/M) phase after treatment with ERJT-12 and progressed into apoptosis. The increased activities of Caspase-3/7 and Caspase-6 in MCF-7 cells were observed. The expression of CyclinB1 was down-regulated. The activities of Cdc25c and CDK1 protein were suppressed and Bcl-2 protein was phosphorylated. ERJT-12 displays potent antiproliferative activity towards cancer cells through suppressing cell cycle proteins, arresting cell cycle at G2/M phase and inducing apoptosis. It might be a novel candidate for cancer therapy.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Benzofurans ; pharmacology ; CDC2 Protein Kinase ; metabolism ; Caspase 3 ; metabolism ; Caspase 6 ; metabolism ; Caspase 7 ; metabolism ; Cell Cycle Proteins ; metabolism ; Cell Division ; drug effects ; Cell Line, Tumor ; Cyclin B ; metabolism ; Cyclin B1 ; G2 Phase ; drug effects ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; metabolism ; cdc25 Phosphatases ; metabolism