1.Ophiopogonin D inhibits cell proliferation, causes cell cycle arrest at G2/M, and induces apoptosis in human breast carcinoma MCF-7 cells.
Qing-qing ZANG ; Lu ZHANG ; Ning GAO ; Cheng HUANG
Journal of Integrative Medicine 2016;14(1):51-59
OBJECTIVETo investigate the effects of ophiopogonin D on human breast cancer MCF-7 cells.
METHODSCell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation experiments. Cell cycle was measured with cell cycle flow cytometry and a living cell assay. Apoptosis and terminal deoxynucleoitidyl transferase-mediated dUTP nick end labeling assays were performed to detect the apoptosis of MCF-7 cells induced by ophiopogonin D. Finally, Western blotting was used to explore the mechanism.
RESULTSExposure of cells to ophiopogonin D resulted in marked decreases in viable cells and colony formation with a dose-dependent manner. Treatment of these cells with ophiopogonin D also resulted in cell cycle arrest at the G(2)/M phase, and increased apoptosis. Mechanistically, ophiopogonin D-induced G(2)/M cell cycle arrest was associated with down-regulation of cyclin B1. Furthermore, activation of caspase-8 and caspase-9 was involved in ophiopogonin D-induced apoptosis.
CONCLUSIONThe data suggested that ophiopogonin D inhibits MCF-7 cell growth via the induction of cell cycle arrest at the G(2)/M phase.
Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; G2 Phase Cell Cycle Checkpoints ; drug effects ; Humans ; M Phase Cell Cycle Checkpoints ; drug effects ; MCF-7 Cells ; Saponins ; pharmacology ; Spirostans ; pharmacology
2.Oleanolic acid induces G₂/M phase arrest and apoptosis in human hepatocellular carcinoma Bel-7402 cells.
Ling LIU ; Jian-long ZHAO ; Jian-gang WANG
China Journal of Chinese Materia Medica 2015;40(24):4897-4902
This study was to examine the mechanism of oleanolic acid (OA) induces G2/M phase arrest and apoptosis in human hepatocellular carcinoma Bel-7402 cells. MTT and trypan blue exclusion test assay were adopted to detect the proliferate status of cells treated with OA. We assayed the cell cycle by flow cytometry using PI staining. Apoptosis was determined by Annexin V-FITC staining and PI labeling. The expressions of cycle related proteins and apoptotic related proteins were determined by Western blot analysis. OA strongly inhibited human hepatoma cells proliferation. When Bel-7402 cells were pretreated with OA for 24 h, OA induced apoptosis and G₂/M phase cell cycle arrest in a concentration-dependent manner. Analysis of the cell cycle regulatory proteins demonstrated that OA decreased the protein levels of cyclin B1, but increased the protein levels of p-Cdk1 (Tyr15) and p-Cdc25C (Ser 216). Moreover, OA modulated the phosphorylation of protein kinases Chk1 and p2l. Western blotting assay also showed significant decrease of Bcl-2 protein expression and increase of Bax protein expression, the cytosol Cyt c level, cleaved-caspase-9 and cleaved-caspase-3 activity. These data suggest that OA produces anti-tumor effect via induction of G₂/M cell cycle arrest and apoptosis.
Apoptosis
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drug effects
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Carcinoma, Hepatocellular
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drug therapy
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pathology
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Cell Line, Tumor
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G2 Phase Cell Cycle Checkpoints
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drug effects
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Humans
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Liver Neoplasms
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drug therapy
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pathology
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M Phase Cell Cycle Checkpoints
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drug effects
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Oleanolic Acid
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pharmacology
3.The ethanol extract isolated from Weiqi Decoction induces G₂/M arrest and apoptosis in AGS cells.
Hai-lian SHI ; Bao TAN ; Guang JI ; Lan LU ; Jian-qun XIE
Chinese journal of integrative medicine 2014;20(6):430-437
OBJECTIVETo evaluate the effects of the ethanol extract isolated from Weiqi Decoction (WQD-EE) on AGS cell proliferation and apoptosis.
METHODSBy using high-performance liquid chromatography with ultraviolet detectors (HPLC-UV) assay and MTT method, the main compounds in WQD-EE and cell viability were detected. And cell cycle distributions were determined by flow cytometry with propidium iodine (PI) staining while apoptosis was detected by flow cytometry with annexin V/PI double staining. Finally, caspase-3 activities were measured by colorimetric method and protein expression was determined by Western blotting.
RESULTSHPLC analysis showed that naringin (35.92 μg/mg), nobiletin (21.98 μg/mg), neohesperidin (17.98 μg/mg) and tangeretin (0.756 μg/mg) may be the main compounds in WQD-EE. WQD-EE not only inhibited AGS and MCF 7 cell proliferation in a dose-dependent manner, but also blocked cell cycle progression at G2/M stage as well as inducing cell apoptosis at concentrations triggering significant inhibition of proliferation and cell cycle arrest in AGS cells. While at 0.5 mg/mL, WQD-EE significantly increased caspase-3 activity by 2.75 and 7.47 times at 24 h and 48 h, respectively. Moreover, WQD-EE in one hand reduced protein expressions of p53 and cyclin B1, and in other hand enhanced protein expressions of cytochrome c and Bax. Protein levels of Bcl-2, Fas L and Fas were not significantly affected by WQD-EE.
CONCLUSIONSWQD-EE inhibits AGS cell proliferation through G2/M arrest due to down-regulation of cyclin B1 protein expression, and promotes apoptosis by caspase-3 and mitochondria-dependent pathways, but not by p53-dependent pathway.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; pharmacology ; Ethanol ; chemistry ; G2 Phase Cell Cycle Checkpoints ; drug effects ; Humans ; M Phase Cell Cycle Checkpoints ; drug effects ; Neoplasm Proteins ; metabolism ; Plant Extracts ; isolation & purification
4.Knockdown of Bcl-xL Enhances Growth-Inhibiting and Apoptosis-Inducing Effects of Resveratrol and Clofarabine in Malignant Mesothelioma H-2452 Cells.
Yoon Jin LEE ; In Sung HWANG ; Yong Jin LEE ; Chang Ho LEE ; Sung Ho KIM ; Hae Saeon NAM ; Young Jin CHOI ; Sang Han LEE
Journal of Korean Medical Science 2014;29(11):1464-1472
Mcl-1 and Bcl-xL, key anti-apoptotic proteins of the Bcl-2 family, have attracted attention as important molecules in the cell survival and drug resistance. In this study, we investigated whether inhibition of Bcl-xL influences cell growth and apoptosis against simultaneous treatment of resveratrol and clofarabine in the human malignant mesothelioma H-2452 cells. Resveratrol and clofarabine decreased Mcl-1 protein levels but had little effect on Bcl-xL levels. In the presence of two compounds, any detectable change in the Mcl-1 mRNA levels was not observed in RT-PCR analysis, whereas pretreatment with the proteasome inhibitor MG132 led to its accumulation to levels far above basal levels. The knockdown of Bcl-xL inhibited cell proliferation with cell accumulation at G2/M phase and the appearance of sub-G0/G1 peak in DNA flow cytometric assay. The suppression of cell growth was accompanied by an increase in the caspase-3/7 activity with the resultant cleavages of procaspase-3 and its substrate poly (ADP-ribose) polymerase, and increased percentage of apoptotic propensities in annexin V binding assay. Collectively, our data represent that the efficacy of resveratrol and clofarabine for apoptosis induction was substantially enhanced by Bcl-xL-lowering strategy in which the simultaneous targeting of Mcl-1 and Bcl-xL could be a more effective strategy for treating malignant mesothelioma.
Adenine Nucleotides/*pharmacology
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Antimetabolites, Antineoplastic/*pharmacology
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Apoptosis/*drug effects
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Arabinonucleosides/*pharmacology
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Caspase 3/metabolism
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Caspase 7/metabolism
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Cell Line, Tumor
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Cell Proliferation/drug effects
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G2 Phase Cell Cycle Checkpoints/drug effects
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Gene Knockdown Techniques
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Humans
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Leupeptins/pharmacology
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Lung Neoplasms/metabolism/pathology
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M Phase Cell Cycle Checkpoints/drug effects
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Mesothelioma/metabolism/pathology
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Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors/genetics/metabolism
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RNA Interference
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RNA, Messenger/metabolism
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RNA, Small Interfering/metabolism
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Stilbenes/*pharmacology
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bcl-X Protein/antagonists & inhibitors/*genetics/*metabolism