1.Anti-proliferation of human cervical cancer HeLa cell line by fascaplysin through apoptosis induction.
Xiao-Ling LU ; Yan-Ling ZHENG ; Hai-Min CHEN ; Xiao-Jun YAN ; Feng WANG ; Wei-Feng XU
Acta Pharmaceutica Sinica 2009;44(9):980-986
This study is to investigate the effect of fascaplysin on human cervical cancer cells (HeLa) in order to provide insights into the mechanisms of growth suppression involved in fascaplysin-mediated apoptosis. Cytotoxic activity of fascaplysin on HeLa cells was determined using MTT assay, cell cycle analysis, and apoptosis (Annexin V-FITC and PI double staining) studies. The role of the molecules in cell cycle regulation and apoptosis was analyzed by Western blotting and flow cytometry. Fascaplysin markedly inhibited HeLa cells proliferation in a dose-dependent manner, however, did not provoke G1 phase arrest in HeLa cells with downregulation of CDK4, cyclin D1 and CDK4-specific Ser795 pRb phosphorylation. Furthermore, fascaplysin induced significantly apoptosis evidenced by sub-G1 peak and Annexin V-FITC and PI double staining. The molecular mechanism of fascaplysin-induced apoptosis was characterized with the activation of caspase-3, -8, and -9, truncation of Bid, release of cytochrome c into cytosol, and down-regulation of Bcl-2 level. Fascaplysin exhibits anti-proliferation effect towards human cervical cancer HeLa cells through induction of apoptosis via extrinsic death pathway and mitochondrial pathway, but not arresting cell cycle progression at G1 phase. All together, these data sustain our contention that fascaplysin has anticancer properties and merits further investigation as a potential therapeutic agent.
Apoptosis
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drug effects
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Cell Cycle Checkpoints
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drug effects
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Cell Proliferation
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drug effects
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HeLa Cells
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Humans
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Indoles
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pharmacology
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Mitochondria
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metabolism
2.Study on the effect of vibsane-type diterpenoids of Viburnum odoratissimum on human HepG2 cell growth and its underlying mechanism.
Hai-Fang ZHANG ; Lin WANG ; Jie LIU ; Wen-Bin ZHOU ; Liu-Zhen ZHANG ; Ya-Jun SHAN ; Zu-Yin YU ; Ping LIU ; Hong-Wei TANG ; Yu-Wen CONG
Chinese Journal of Applied Physiology 2014;30(4):343-347
OBJECTIVETo study the antiproliferation effect on HepG2 cells and its underlying mechanism of the active chemical composition of the Viburnum Odoratissimum.
METHODS3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay and trypan blue dye exclusion assay were used to assess the effect of vibsane-type diterpenoids on the proliferation of various tumor cells. Alterations in cell cycle and apoptosis were determined by flowcytometry. The enzymatic activity of caspase-3/7 was measured by Apo-ONE homogeneous Caspase-3/7 Assay kit.
RESULTSCompound 1 #, a vibsane-type diterpenoid, was found to significantly inhibit the growth of HepG2 cells by anticancer proliferation activity screening. It was demonstrated that the modified groups on side chain coupled to C11 site affected the cell growth-inhibition activity of compounds by structure-activity analysis. In addition, HepG2 cell line was most sensitive to compound 1 #, which induced growth arrest of HepG2 cells in a dose- and time-dependent manner. Study on the mechanisms underlying these effects indicated that compound 1 # induced significant G0/G1 phase arrest of HepG2 cells in a time- and concentration-dependent manner. Meanwhile, It was found that higher concentrations of compound (5-10 micromol/L) caused evident increase in the unmber of apoptotic cells and dose-dependent activation of caspase-3/7.
CONCLUSIONVibsane-type diterpenoids could significantly inhibit the growth of HCC HepG2 cells. Induction of cell cycle arrest and apoptosis may play important roles in their anticancer effects.
Apoptosis ; drug effects ; Cell Cycle Checkpoints ; drug effects ; Cell Proliferation ; drug effects ; Diterpenes ; pharmacology ; Hep G2 Cells ; Humans ; Viburnum ; chemistry
3.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
4.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
5.Cycle arrest of prostate carcinoma DU-145 cells induced by pseudolaric acid B.
Xia MAI ; Zhong-Wei XU ; Xiao-Yi CHEN ; Bo CAO ; Rui-Cheng XU
China Journal of Chinese Materia Medica 2012;37(22):3467-3471
OBJECTIVETo study the effect of pseudolaric acid B (PLAB) on cell proliferation and cycle of human prostate carcinoma DU-145 cells. method: Its inhibitory effect on the cell growth was measured by MTT method. Characteristics of cell death were determined by Hoechest 33342 staining. The cell cycle was detected by flow cytometry. The expressions of cyclin B1, cyclin D1 and CDK1 were detected by Real time-PCR and Western blot, respectively.
RESULTPLAB notably inhibited DU-145 cell growth in a dose- and time dependent manner (P < 0.05). Its IC50 values of PLAB for DU-145 cells for 24, 48 and 72 h were 4.53, 2.39 and 2.08 micromol x L(-1), respectively. Having been treated with 5 micromol x L(-1) PLAB for 24 h, the cells showed such apoptosis characteristics as nuclei chromatin condensation and apoptotic body. With the increase in PLAB concentration, the proportion of G2/M phase cells strikingly increased in a dose- and time dependent manner (P < 0.05), meanwhile cyclin B1 and CDK1 showed over-expressions (P < 0.05), and the cyclin D1 showed under-expression (P < 0.05).
CONCLUSIONPLAB can inhibit the growth of DU-145 cells and induce the cell cycle G2/M arrest, accompanied with the over-expression of cyclin B1 and CDK1, which may be related with its regulation cycle-associated protein degradation.
Apoptosis ; drug effects ; Cell Cycle Checkpoints ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Diterpenes ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Male ; Prostatic Neoplasms ; drug therapy ; physiopathology
6.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
7.Effect of Artesunate on Proliferation, Cell Cycle and Apoptosis of SKM-1 Cells and Its Underlying Mechanisms.
Shu-Kai QIAO ; Ying WANG ; Zhi-Yun NIU ; Jin-Man TAN ; Jun-Li WANG ;
Journal of Experimental Hematology 2016;24(1):131-137
OBJECTIVETo investigate the effects of artesunate (ART) on proliferation, cell cycle and apoptosis of SKM-1 cells in vitro and to explore the underlying mechanisms.
METHODSAfter SKM-1 cells were treated with different concentrations of ART, the cell proliferation was determined by CCK-8 method. Apoptosis and distribution of cell cycle were detected by flow cytometry. Both DCFH-DA fluorescent probe and Fluo-3-Am fluorescent probe were used to detect the changes of intracellular reactive oxygen species (ROS) and calcium ion concentration. Western blot was used to measure the protein levels of BCL-2, BAX, BAD, P-BAD, survivin and XIAP.
RESULTSART obviously inhibited the growth of SKM-1 cells in time and dose-dependent manners (r = -0.841; r = 0.-786). The antioxidant trolox-pretreatment significantly decreased the growth inhibition effect of ART on SKM-1 cells. Caspase inhibitor Ac-DEVD-CHO partially reduced the growth inhibition effect of ART on SKM-1 cells. After treatment with ART for 24 hours, the apoptosis of SKM-1 cells was found, the cell cycle of SKM-1 was arrested in G0/G1 phase, ART could elevate the levels of calciumion and reactive orygen. ART could significantly down-regulate the protein expression levels of P-BAD and survivin in SKM-1 cells, and showed a highly negative correlation with ART dose (r = -0.909; r = -0.849). On the contrary, ART had no significant effect on expression levels of BAD and XIAP in SKM-1 cells, and after ART treatment, although BCL-2 protein expression was not significantly different when compared with control group, but the BCL-2/BAX ratio significantly decreased and highly negatively correlated with ART dose (r = -0.866).
CONCLUSIONThe ART significantly suppresses the cell proliferation, induces the apoptosis and promoted cell cycle arrest at G0/G1 phase in SKM-1 cells. The mechanisms of ART anti-MDS is associated with the increase of intracellular calciumion concentration and ROS levels. In addition, the pro-apoptotic activity of ART may be involved in the regulation of BCL-2 /BAX ratio and the expressions of P-bad and survivin.
Apoptosis ; drug effects ; Artemisinins ; pharmacology ; Calcium ; metabolism ; Cell Cycle ; drug effects ; Cell Cycle Checkpoints ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; drug effects ; Down-Regulation ; Humans ; Inhibitor of Apoptosis Proteins ; metabolism ; Oligopeptides ; pharmacology ; Reactive Oxygen Species ; metabolism
8.Cytotoxic Activities of Total Saponins from Plena Clematis on Human Tumor Cell Lines In Vitro.
Fu-Rong ZHU ; Yong-Ning LI ; Shu-Lan HE ; Qian-Shun CHEN ; Xun-Yu XU
Chinese journal of integrative medicine 2018;24(10):763-767
OBJECTIVE:
To investigate the anti-proliferative effects of saponins prepared from Plena Clematis (PC) cultured in Fujian Province, China on 4 human tumor cell lines and its possible anti-tumor mechanism.
METHODS:
The growth inhibition assays of saponins on human esophageal squamous carcinoma cell line (EC9706), human hepatoma cell line (HepG-2), human oral cancer cell line (KB) and human gastric cancer cell line (BGC-823) were evaluated in vitro by thiazolyl blue (MTT) method. The inhibitory effects on EC9706 treated with different concentrations of saponins (15.62, 31.25, 62.50, 125, 250 and 500 μg/mL) were performed in vitro by MTT method. The morphology and nuclear staining with acridine orange/ethidium bromide of EC9706 cells treated with saponins were illustrated under an inverted phase fluorescence microscope. The apoptotic effects of saponins were further evaluated by annexin-V/propidium iodide dual staining experiment to examine the occurrence of phosphatidylserine externalization onto the cell surface by a flflow cytometer.
RESULTS:
MTT assay showed that the saponins could inhibit the proliferation of 4 tumor cell lines. Among them, the maximum inhibition rate of 73.1% was detected in EC9706 cells at the saponins concentration of 250 μg/mL for 24 h. Further investigation indicated that the saponins induced EC9706 cells apoposis. The EC9706 cells presented apoptotic characteristics when treated with saponins, including that the morphologies of EC9706 cells were appeared round-shaped with higher refraction, and the cell nuclear stained orange with EB after 250 μg/mL saponins exposure. The flow cytometry analysis results showed that the induction of cell cycle arrest in apoptotic system may participate in the anti-proliferative activity of saponins on EC9706 cells.
CONCLUSION
The saponins from PC exhibited significant cytotoxicity against human EC9706, KB, BGC-823, and HepG-2 cells and might be beneficial to development of ethnic pharmaceutical plant for potential anti-tumor drugs.
Antineoplastic Agents, Phytogenic
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pharmacology
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Cell Cycle Checkpoints
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drug effects
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Cell Line, Tumor
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Cell Proliferation
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drug effects
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Clematis
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chemistry
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Humans
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Saponins
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pharmacology
9.Effects of Panax notoginseng saponins on proliferation, apoptosis and cell cycle of K562 cells in vitro and the mechanisms.
Yuyun LI ; Weiwei ZHAI ; Xiangrong YANG ; Juan DING ; Lixin KAN
Journal of Southern Medical University 2015;35(8):1103-1109
OBJECTIVETo investigate the effects of Panax notoginseng saponins (PNS) on the proliferation, apoptosis and cell cycle of K562 cells and explore the molecular mechanisms underlying these effects.
METHODSPNS-induced growth inhibition of K562 cells was detected by MTT assay; the cell apoptosis was evaluated by AO/EB staining and Annexin V-FITC/ PI staining; flow cytometry was used to detect cell cycle changes in the treated cells. The mRNA expressions of the molecules in mTOR signaling pathway were examined by RT-PCR, and the cellular expressions of cleaved caspeas-3, cyclin D1 and major proteins in mTOR signaling pathway were detected using Western blotting.
RESULTSMTT assay showed that treatment with 100-800 µg/mL PNS significantly inhibited the proliferation, promoted the cell apoptosis, and caused cell cycle arrest in G0/G1 phase in K562 cells. Western blotting revealed increased protein expression of cleaved caspase-3 and decreased expression of cyclin D1 in PNS-treated cells, in which the proteins expressions of mTOR, p-mTOR, p-p70S6K and p-4E-BP 1 and the mRNA expression of mTOR were all decreased.
CONCLUSIONPNS can inhibit the proliferation, induce apoptosis and cause cell cycle arrest in K562 cells possibly by up-regulating cleaved caspase 3 and down-regulating cyclin D1 and mTOR signaling pathway.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Cycle ; drug effects ; Cell Cycle Checkpoints ; Cell Proliferation ; drug effects ; Cyclin D1 ; metabolism ; Humans ; K562 Cells ; drug effects ; Panax notoginseng ; chemistry ; Saponins ; chemistry ; Signal Transduction ; TOR Serine-Threonine Kinases ; metabolism ; Up-Regulation
10.Effect of gemcitabine in enhancing the radiosensitivity of HepG2 hepatoma cells and the possible mechanism.
Zhi-hai LING ; Quan-quan SUN ; Yao-wei ZHANG ; Jian GUAN ; Yi DING ; Long-hua CHEN
Journal of Southern Medical University 2011;31(12):1993-1996
OBJECTIVETo evaluate the effect of gemcitabine in enhancing the radiosensitivity of hepatoma cell line HepG2 and explore its mechanisms.
METHODSClonogenic survival assay is employed to calculate the ratios of L-Q model radiation biology parameters and radiosensitization after different doses of irradiation. Flow cytometry was used to detect the changes in HepG2 cell cycle and apoptosis rate after gemcitabine treatment and radiation exposure.
RESULTSThe survival fraction at 2 Gy of HepG2 cells treated with gemcitabine was significantly lower, and the value of alpha was significantly higher than those of untreated cells. GEM treatment increased the percentage of radiation-induced G0/G1 phase cells and cell apoptosis.
CONCLUSIONGemcitabine can significantly enhance the radiosensitivity of HepG2 cells by enhancing radiation-induced cell cycle arrest in G0/G1 phase and cell apoptosis.
Apoptosis ; drug effects ; Cell Cycle Checkpoints ; drug effects ; Deoxycytidine ; analogs & derivatives ; pharmacology ; Hep G2 Cells ; Humans ; Radiation Tolerance ; drug effects ; Radiation-Sensitizing Agents ; pharmacology