1.Modification of Chinese hamster ovary cells.
Da-Zhi LAI ; Lian-Quan QI ; Chang-Ming YU ; Hai-Tao WANG ; Wei CHEN
Chinese Journal of Biotechnology 2002;18(4):415-419
Chinese hamster ovary cells (CHO) are preferable to prokaryotic, yeast or insect cells as hosts for biopharmaceutical production due to the products are more similar to their natural conformation. However, CHO cells confront tremendous difficulties when cultured in large scale such as mal-adaptation to serum-free medium, apoptosis and over-growth without limitation. So in addition to optimizing CHO system in respect of medium, environment and expression vector, modification of CHO cells themselves has drawn more and more attention. Here the main progress in CHO-modification is reviewed.
Animals
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Apoptosis
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genetics
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CHO Cells
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drug effects
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metabolism
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Cell Cycle
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drug effects
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Cell Cycle Proteins
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drug effects
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Cell Division
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drug effects
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Cricetinae
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Genetic Vectors
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genetics
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Transfection
2.Andrographolide inhibits hepatoma cells growth and affects the expression of cell cycle related proteins.
Kai-Kai SHEN ; Tian-Yu LIU ; Chong XU ; Li-Li JI ; Zheng-Tao WANG
Acta Pharmaceutica Sinica 2009;44(9):973-979
The present study is aimed to investigate the toxic effects of andrographolide (Andro) on hepatoma cells and elucidate its preliminary mechanisms. After cells were treated with different concentrations of Andro (0-50 micromol x L(-1)) for 24 h, cell viability was evaluated with 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay. Furthermore, after hepatoma cells (Hep3B and HepG2) were treated with different concentrations of Andro (0-30 micromol x L(-1)) for 14 d, the number of colony formation was accounted under microscope. Cell cycle related proteins such as Cdc-2, phosphorylated-Cdc-2, Cyclin B and Cyclin D1 were detected with Western blotting assay and the cell cycle was analyzed by flow cytometry using propidium iodide staining. MTT results showed that Andro induced growth inhibition of hepatoma cells in a concentration-dependent manner but had no significant effects on human normal liver L-02 cells. Andro dramatically decreased the colony formation of hepatoma cells in the concentration-dependent manner. Moreover, Andro induced a decrease of Hep3B cells at the G0-G1 phase and a concomitant accumulation of cells at G2-M phase. At the molecular level, Western blotting results showed that Andro decreased the expression of Cdc-2, phosphorylated-Cdc-2, Cyclin D1 and Cyclin B proteins in a time-dependent manner, which are all cell cycle related proteins. Taken together, the results demonstrated that Andro specifically inhibited the growth of hepatoma cells and cellular cell cycle related proteins were possibly involved in this process.
Carcinoma, Hepatocellular
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metabolism
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Cell Cycle Proteins
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metabolism
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Cell Line, Tumor
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Cell Survival
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drug effects
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Diterpenes
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pharmacology
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Humans
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Liver Neoplasms
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metabolism
3.Norcantharidin inhibits DNA replication initiation protein Cdc6 in cancer cells.
Jin-long LI ; Yu-chen CAI ; Zhi-ming HU ; Ji-min GAO
Journal of Southern Medical University 2010;30(8):1851-1853
OBJECTIVETo explore the inhibitory effect of norcantharidin (NCTD) on the expression of DNA replication initiation protein Cdc6 in cancer cells.
METHODSMTT assay was performed to detect the inhibitory effect on different cancer cell lines, including HeLa, HepG2, Jurkat and Ramos cells. The effect of NCTD on Cdc6 protein level was detected by Western blotting, and BrdU incorporation assay was used to evaluate the DNA replication of the cells.
RESULTSNCTD significantly inhibited the proliferation of the cells and caused degradation of Cdc6 protein to result in the inhibition of the DNA replication of the cells shown by BrdU incorporation assay.
CONCLUSIONNCTD can induce the degradation of Cdc6 in cancer cells to produce an anti-cancer effect.
Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; Cell Cycle Proteins ; metabolism ; Cell Line, Tumor ; DNA Replication ; drug effects ; Humans ; Nuclear Proteins ; metabolism
4.Deguelin regulates cell cycle and nuclear pore complex protein Nup98 and Nup88 in U937 cells in vitro.
Yan CHEN ; Hong-Li LIU ; Guo-Hui CUI ; Qiu-Ling WU ; Jing HE ; Wei-Hua CHEN
Chinese Journal of Hematology 2007;28(2):115-118
OBJECTIVETo investigate antitumor activity and molecular mechanism of deguelin to the human U937 leukaemia cells and to explore the mechanisms regulating cell cycle and nucleoporin 98 (Nup98) and nucleoporin 88 (Nup88) in vitro.
METHODSThe effects of deguelin on the growth of U937 cells were studied by MTT assay, and the cell cycle of U937 cells by a propidium iodide method. The localization of the nuclear pore complex protein Nup98 and Nup88 was checked by immunofluorescence and immunoelectron microscopy. The expressions of Nup98 and Nup88 in U937 cells were checked by flow cytometry (FCM) and Western blot respectively.
RESULTSThe proliferation of U937 cells was significantly inhibited in a time-dose dependent manner in deguelin-treated group with a 24 h IC50 value of 21.61 nmol/L and 36 h IC50 value of 17.07 nmol/L. U937 cells treated with deguelin showed reduction in the percentages of cells in G0/G1, whereas accumulation of cells in S and G2/M phase. The ratio of G1/G0 phase cells were 73.01%, 71.15%, 68.42%, 52.45%, 43.99% and 22.82%, and that of S phase cells were 17.18%, 16.30%, 18.09%, 27.56%, 31.21% and 46.85%, and that of G2/M phase cells were 9.75%, 12.31%, 13.09%, 18.99%, 24.83% and 27.79% at deguelin concentrations of 0, 5, 10, 20, 40, 80 nmol/L respectively. Nup88 and Nup98 were found on both the nuclear and cytoplasmic side of the U937 cells. The expression of Nup98 was up-regulated and Nup88 down-regulated in deguelin treated U937 cells.
CONCLUSIONDeguelin is able to inhibit the proliferation of U937 cells by regulating the cell cycle. The antitumor activity of deguelin was related to up-regulating the expression of Nup98 and down-regulating Nup88 protein.
Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Humans ; Nuclear Pore Complex Proteins ; metabolism ; Rotenone ; analogs & derivatives ; pharmacology ; U937 Cells
5.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
6.Erythropoietin increases transferrin receptor expression and the impact of erythropoietin on K562 leukemic cell cycle.
Mi ZHOU ; Qing-kui LIAO ; Feng-yi LI ; Ju GAO ; Ren-yi FU ; Chun-hua LUO ; Qiang LI ; Cang-song JIA
Chinese Journal of Pediatrics 2003;41(7):528-530
OBJECTIVEFunctionally, erythropoietin (EPO) can promote the proliferation and growth of erythroid progenitor cells, and it is widely used in the treatment of anemia in chronic diseases caused by tumor and inflammation. However, it is unclear whether EPO has any effect on tumor cell iron metabolism and tumor cell proliferation. The purpose of this study was to explore the effects of recombinant human EPO (rhEPO) on the expression of transferrin receptor (TfR, CD(71) antigen) of leukemic cell K562 and its relation to cell cycle.
METHODSIn vitro culture of K562 cell was performed with additions of various concentrations of rhEPO and Fe. Treatments were terminated at 24 h and 72 h, respectively. Then each group of cells was incubated with FITC-IgG antibody to CD(71) or PI, a kind of DNA dye. And TfR expression and DNA synthesis status were analyzed by flow-cytometry.
RESULTS(1) The expression of TfR by K562 cells increased significantly when incubated for 72 h with different concentrations of rhEPO. The measurement values of 5 U/ml, 10 U/ml and 20 U/ml groups were 12.2 +/- 1.40, 10.7 +/- 0.99 and 11.1 +/- 0.90, respectively. They were markedly increased when compared with that of control group (6.27 +/- 0.11, P < 0.05). (2) When incubated with rhEPO (5 u/ml) alone or combined with FeCl(3) (100 micro mol/L), the percentages of cells in S phase were 51.1% and 59.6%, respectively. They significantly increased when compared with that of control group (42.9%, P < 0.05).
CONCLUSIONSIron is very important for the proliferation of both normal cells and leukemic cells. It is essential to the activity of ribonucleotide reductase (RR). The authors hypothesized that rhEPO would increase the expression of TfR and intracellular iron content of leukemic cells, which would enhance the DNA synthesis and cell proliferation. Therefore, the clinical application of rhEPO to promote erythropoiesis of cancer patients should be cautious.
Cell Cycle ; drug effects ; Erythropoietin ; pharmacology ; Flow Cytometry ; Humans ; K562 Cells ; Receptors, Transferrin ; metabolism ; Recombinant Proteins
7.Effects of safflower injection on HEL leukemia cell proliferation and apoptosis and relevant molecular mechanisms.
Pei-Feng DUAN ; Xiu-Hong JIA ; Jian-Yong WANG
Chinese Journal of Contemporary Pediatrics 2015;17(9):989-993
OBJECTIVETo study the effect of safflower injection on the proliferation and apoptosis of human leukemia cell line HEL and the relevant molecular mechanisms.
METHODSHEL cells were treated with different concentrations of safflower injection. HEL cells without safflower injection treatment were used as the control group. MTT method was used to detect the inhibitory rate of the HEL cells at 24, 48 and 72 hours after various concentrations of safflower injection treatment (10, 20, 30, 40 and 50 mg/mL). The cell cycle and apoptosis were detected using flow cytometry and the HOXB3-mRNA expression was measured by RT-PCR at 48 hours after safflower injection treatment (10, 20 and 30 mg/mL).
RESULTSCompared with the control group, various concentrations of safflower injection inhibited HEL cell proliferation in a dose-dependent manner (P<0.05). At 48 hours after various concentrations of safflower injection treatment, the number of treated cells in the G2/M phase increased, but that in the S phase decreased, and the apoptosis rate was significantly higher than that in the control group, with a dose-dependent manner (P<0.05). The expression of HOXB3-mRNA in safflower injection-treated cells decreased in a dose-dependent manner compared with the control group (P<0.05).
CONCLUSIONSSafflower injection can inhibit proliferation and induce apoptosis of HEL cells in vitro, and its underlying mechanisms may involve down-regulation of the HOXB3-mRNA expression.
Apoptosis ; drug effects ; Carthamus tinctorius ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Homeodomain Proteins ; genetics ; Humans ; Injections ; Leukemia ; drug therapy ; metabolism ; pathology
9.Effect of SNS-032 on biological activity of hematopoietic stem cells in mice.
Rui-Zhe QI ; Qing JI ; Li-Yan ZHANG ; Yu ZHANG ; Wei-Ping YUAN ; Tao CHENG ; Ying-Dai GAO ; Jing XU
Journal of Experimental Hematology 2013;21(3):741-745
This study was aimed to investigate the effect of SNS-032 (C17 H24 N4O2S2) on cell cycle, apoptosis, differentiation and self-renewal of hematopoietic stem cells (HSC) in mice. The self-renewal capability of bone marrow cells was measured by cobblestone forming cell test. The expressions of self-renewal regulation genes, cell cycle-related genes, apoptosis-related genes were measured by real-time PCR. The cell cycle status and apoptosis of HSC and HPC were detected by flow cytometry. The results showed that there was no significant difference of the frequency of HSC between SNS-032 and control group. The expressions of CDK1, CDK2, CDK7 and p27 decreased in HSC (P < 0.05) while the expressions of CDK4, CDK6, p21, p18, p19, Bcl-2, Bax, Puma, p53, Bim1, Sall4 and Notch1 showed no difference between SNS-032 group and control group (P > 0.05). The fraction of viable HSC in each phase of cell cycle remained unchanged after the treatment of SNS-032 (P > 0.05). Furthermore, there was no statistical difference in the apoptotic fractions between control and drug-treated groups (P > 0.05). It is concluded that SNS-032 induce apoptosis of cancer cells. Interestingly, SNS-032 has no significant inhibitory effect on self-renewal and differentiation of normal HSC, as well as no obvious effect inducing apoptosis of normal HSC and HPC.
Animals
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Apoptosis
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drug effects
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Apoptosis Regulatory Proteins
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metabolism
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Cell Cycle
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drug effects
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Cell Cycle Proteins
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metabolism
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Cells, Cultured
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Hematopoietic Stem Cells
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cytology
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drug effects
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Mice
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Mice, Inbred C57BL
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Oxazoles
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pharmacology
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Thiazoles
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pharmacology
10.Apoptosis-inducing effect of quercetin and kaempferol on human HL-60 cells and its mechanism.
Hui-Juan REN ; Hong-Jun HAO ; Yong-Jin SHI ; Xue-Min MENG ; Yan-Qiu HAN
Journal of Experimental Hematology 2010;18(3):629-633
The purpose of this study was to explore the anti-leukemia effect of quercetin and kaempferol and its mechanism. The HL-60 cells were used as the leukemia models. The inhibitory effects of quercetin and kaempferol on growth of HL-60 cells was assessed by MTT assay. The effect of quercetin and kaempferol on cell cycle in HL-60 cells was detected by flow cytometry. The cytotoxic effect of these 2 drugs was analysed by single cell electrophoresis assay. Western blot analysis was used to study the apoptotic mechanism of HL-60 cells. The results showed that the quercetin and kaempferol had a significant anti-leukemia effect in vitro. The proliferation of HL-60 cells was significantly inhibited in dose-and time-dependent manners after treating with quercetin (r = 0.77) and kaempferol (r = 0.76) respectively, and the cytotoxicity of quercetin was superior to that of kaempferol. The quercetin and kaempferol induced G(2)/M arrest and apoptosis of HL-60 cells. The quercetin and kaempferol could down-regulate the survivin expression. It is concluded that the quercetin and kaempferol have significant anti-leukemia effect in vitro. Furthermore the apoptosis-inducing effect of quercetin is stronger than that of kaempferol, both of which induce apoptosis of HL-60 cells through depressing cell growth, arresting cell cycle and inhibiting expression of survivin.
Apoptosis
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drug effects
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Cell Cycle
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drug effects
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Cell Proliferation
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drug effects
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Gene Expression Regulation, Leukemic
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HL-60 Cells
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Humans
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Inhibitor of Apoptosis Proteins
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metabolism
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Kaempferols
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pharmacology
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Quercetin
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pharmacology