This study was aimed to investigate the changes of mitochondrial membrane potential (DeltaPsim) of HL-60 cells induced by cytarabine and the correlation between mitochondrial membrane potential and apoptosis of HL-60 cells. HL-60 cells were stained with Rhodamine 123; change of mitochondrial membrane potential of HL-60 cells was detected by flow cytometry. AO/EB staining and flow cytometry were used to examine the apoptosis of HL-60 cells. The results showed that the levels of HL-60 cell DeltaPsim in experimental groups decreased after cultured for 6 hours. In Ara-C 0.05 mg/ml group, rhodamine 123 fluorescence intensity in mitochondria of HL-60 cells at 6, 12, 24 hours were 117.9+/-7.6, 100.9+/-7.7, 87.6+/-10.7, respectively, there was significant difference between the different culture groups (p<0.05). In Ara-C 0.1 mg/ml group, rhodamine 123 fluorescence intensity in mitochondria of HL-60 cells at 6, 12, 24 hours were 111.9+/-10.1, 86.6+/-9.2, 68.4+/-12.2, respectively, there was significant difference between the different culture groups (p<0.05); rhodamine 123 fluorescence intensity was significantly different between the two groups at 12, 24 hours (p<0.05). In Ara-C 0.05 mg/ml group, the apoptosis rate of HL-60 cells at 6, 12, 24 hours were (41.2+/-3.0)%, (53.7+/-5.1)%, (65.8+/-2.6)% respectively, there was significant difference between the different culture groups (p<0.01); In Ara-C 0.1 mg/ml group, the apoptosis rate of HL-60 cells at 6, 12, 24 hours were (45.7+/-4.1)%, (58.2+/-4.3)%, (70.1+/-2.3)% respectively, there was significant difference between the different culture groups (p<0.01); the apoptosis rates showed no significantly difference between the two groups at same time. The changes of mitochondrial membrane potential and apoptosis rate of HL-60 cells were significantly negatively correlated. In Ara-C 0.05 mg/ml group, r was -0.89, p<0.01, while in Ara-C 0.1 mg/ml group, r was -0.76, p<0.01. It is concluded that the mitochondrial membrane potential on HL-60 cells decrease at HL-60 cells apoptosis induced by Ara-C, therefore the reduction of mitochondrial membrane potential may be one of the important mechanisms at the induced apoptosis.
Antimetabolites, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Cytarabine ; pharmacology ; HL-60 Cells ; Humans ; Membrane Potential, Mitochondrial ; drug effects

Antimetabolites, Antineoplastic ; pharmacology ; therapeutic use ; Drug Resistance, Neoplasm ; Humans ; Methotrexate ; pharmacology ; therapeutic use ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy

This study was aimed to explore the influence of anti-CXCR4 monoclonal antibody 12G5 on killing effect of cytosine arabinoside (Ara C) to HL-60 cell, and to assess its therapeutic value in marrow residual disease. HL-60 cells were cultured and co-cultured with leukemic stromal cells, and SDF-1 activity was inhibited with 10 microg/ml 12G5, then, killing effects of Ara C on HL-60 cells were investigated by MTT and morphology assay. Curves by MTT assay revealed that in the test group of 20 microg/ml Ara C, A(540) values decreased slowly but straightly, however, in control group A(540) values decreased markedly for the first two days, and increased from day 3 or 4. In the test group of 40 microg/ml Ara C, although increasing at constricted range of 7 - 9 days, A(540) values decreased in whole observing period of 12 days, while in control group A(540) values decreased markedly at day 0-3, and increased from day 4. Furthermore, two curves go across each other at day 5, and continue the increasing tendency. Morphology results showed that in both treated groups, the number of HL-60 cell decreased markedly and increased gradually in control group, but just contrary to test group. It is concluded that 12G5 may weaken the killing effect of Ara C on HL60 cell in earlier period, but reinforce the total killing effect and delay the occurrence of drug resistance simultaneously. Thus 12G5 has the therapeutic potential on marrow residual disease.
Antibodies, Monoclonal ; pharmacology ; Antimetabolites, Antineoplastic ; pharmacology ; Cell Line, Tumor ; Cell Survival ; drug effects ; Cytarabine ; pharmacology ; Dose-Response Relationship, Drug ; Drug Synergism ; HL-60 Cells ; Humans ; Receptors, CXCR4 ; immunology

To investigate the effects of rhG-CSF on apoptosis of HL-60 cell induced by cytarabine (Ara-C), HL-60 cell were cultured with Ara-C, with or without rhG-CSF, and the changes of HL-60 cells were detected by morphology, including Wright's and acridine orange (AO) and ethidium bromide (EB) staining, and MTT assay. The results showed that comparing with Ara-C group, the apoptosis level of rhG-CSF + Ara-C group was enhanced (P < 0.01), and the OD value of MTT assay declined (P < 0.01). It is concluded that rhG-CSF can induce the apoptosis of HL-60 cells and enhance the apoptosis induced by Ara-C.
Antimetabolites, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Cell Survival ; drug effects ; Cytarabine ; pharmacology ; Drug Synergism ; Granulocyte Colony-Stimulating Factor ; pharmacology ; HL-60 Cells ; Humans ; Recombinant Proteins

OBJECTIVE: To investigate the apoptosis of human breast cell line MCF-7 cells induced by gemcitabine and radiation. METHODS: The MTT method was applied to study the growth inhibition of MCF-7 treated with gemcitabine, radiation, gemcitabine and radiation. The apoptosis index (AI) was analyzed by flow cytometry. The morphology of the MCF-7 cells apoptosis was observed by transmission electron microscopy. RESULTS: When MCF-7 cells were treated with gemcitabine at different concentrations for 24 h, the cell growth inhibition rate was increased in a concentration-dependent manner. The apoptotic indexes (AI) of MCF-7 of four groups by flow cytometry revealed. The AI of (R+D) group was significantly different from those of the radiation group and the gemcitabine group (P<0.05). Condensed chromation, nuclear fragmentation and apoptotic body of MCF-7 cells were found by transmission electron microscope. CONCLUSION The apoptosis of human breast cancer cell line, MCF-7 cells, could be induced by gemcitabine. Gemcitabine can significantly enhance the radiation-induced apoptosis of MCF-7 cells.
Antimetabolites, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; radiation effects ; Breast Neoplasms ; pathology ; Deoxycytidine ; analogs & derivatives ; pharmacology ; Female ; Flow Cytometry ; Humans ; Radiation ; Radiation-Sensitizing Agents ; pharmacology ; Tumor Cells, Cultured

OBJECTIVETo investigate the apoptosis-inhancing effect of the combination of arsenic trioxide (As2O3 ) and buthionine sulfoximine (BSO) on multidrug-resistant human leukemic K562/ADM cells, to compare the effect of As2O3 alone and As2O3 combined with BSO and As2O3 alone, and to determine the effect of intracellular GSH content on this treatment.

METHODSAs2O3 was used in a dose of 0.5 micromol/L, 2.0 micromol/L and 5.0 micromol/L, respectively, and BSO was used in a dose of 100 micromol/L in the culture of multidrug-resistant human leukenic K562/ADM cells. The cell proliferation activity was assessed with MTT assay. The cell apoptosis was detected by flow cytometry using Annexin-V and propidium iodide (PI) staining. Intracellular GSH content was measured using glutathione assay kit by spectrophotometry.

RESULTSAfter the GSH contents were reduced by the combination of arsenic in clinic dose (0.5, 2 micromol/L) and BSO (100 micromol/L), respectively, the K562/ADM cell proliferation activity was obviously inhibited and the cell apoptosis-inducing effect was advanced in 24 hours. In 48 and 72 hours, the effect of the combination group (clinic dose arsenic group) was significantly stronger than that of clinic dose arsenic alone group and the high dose arsenic alone group.

CONCLUSIONThe cell apoptosis-inducing effect of arsenic in combination of BSO on multidrug resistant human leukemia K562/ADM cells is significantly enhanced in comparison with that of arsenic alone. The reduction of intracellular glutathione content is closely correlated with this apoptosis-enhancing effect.

Antimetabolites, Antineoplastic ; pharmacology ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Buthionine Sulfoximine ; pharmacology ; Cell Proliferation ; drug effects ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; Drug Synergism ; Glutathione ; metabolism ; Humans ; K562 Cells ; Oxides ; pharmacology

OBJECTIVETo establish gemcitabine-resistant pancreatic cancer cell strain and study the role of thioredoxin reductase (TrxR) in drug-resistant process.

METHODSGemcitabine-resistant pancreatic cancer cell strain SW1990/GZ was induced by increasing drug dosage intermittently, then the changes of its biological features and the activity of TrxR were examined.

RESULTSStable drug-resistant SW1990/GZ cell strain was established by culturing with gemcitabine for 9 months. The morphology and growth characteristics of the cell strain changed remarkably. The cells shrunk and became rounder; its endoplasm expanded; granular substances increased; and the doubling-time was prolonged. Resistance of the cell line to gemcitabine, fluorouracil, adriamycin, and mitomycin significantly increased. The TrxR activity of the drug-resistant cells was increased markedly.

CONCLUSIONSW1990/GZ has certain multidrug resistance to some chemotherapy drugs, and TrxR plays a role in the drug-resistant process.

Antimetabolites, Antineoplastic ; pharmacology ; Cell Line, Tumor ; Deoxycytidine ; analogs & derivatives ; pharmacology ; Drug Resistance, Neoplasm ; drug effects ; Humans ; Pancreatic Neoplasms ; enzymology ; pathology ; Thioredoxin-Disulfide Reductase ; drug effects ; metabolism

BACKGROUNDSingle nucleotide polymorphisms (SNPs) in the deoxycytidine kinase (dCK) gene are associated with chemosensitivity to nucleoside analogs. 2',2'-Difluoro 2'-deoxycytidine (gemcitabine) is a first-line nucleoside analog drug in the treatment of pancreatic cancer. However, the association between SNPs in the dCK gene and chemosensitivity to gemcitabine has not been fully established. Therefore, the present study aimed to investigate the relationship between SNPs in the dCK gene and chemosensitivity to gemcitabine in human pancreatic cancer cell lines.

METHODSSeven SNPs in the dCK gene were sequenced in six human pancreatic cancer cell lines. The chemosensitivity of these six cell lines to gemcitabine were evaluated in vitro with a Cell Counting Kit-8 (CCK-8) test. Inhibition rates were used to express the chemosensitivity of pancreatic cancer cell lines to gemcitabine.

RESULTSThe genotype of the A9846G SNP in the dCK gene was determined in six human pancreatic cancer cell lines. The cell lines BxPC-3 and T3M4 carried the A9846G SNP genotype AG, whereas cell lines AsPC-1, Mia PaCa2, SW1990 and SU86.86 carried the GG genotype. Cell lines with the AG genotype (BxPC-3 and T3M4) were more sensitive to gemcitabine compared with cell lines with the GG genotype (AsPC-1, Mia PaCa2, SW1990 and SU86.86) and significantly different inhibition rates were observed between cell lines carrying the AG and GG genotypes (P < 0.01).

CONCLUSIONSVariants in the A9846G SNP of the dCK gene were associated with sensitivity to gemcitabine in pancreatic cancer cell lines. The dCK A9846G SNP may act as a genetic marker to predict chemotherapy efficacy of gemcitabine in pancreatic cancer.

Antimetabolites, Antineoplastic ; pharmacology ; Cell Line, Tumor ; Cell Survival ; drug effects ; Deoxycytidine ; analogs & derivatives ; pharmacology ; Deoxycytidine Kinase ; genetics ; Genotype ; Humans ; Pancreatic Neoplasms ; enzymology ; genetics ; Polymorphism, Single Nucleotide ; genetics

OBJECTIVETo assess the feasibility of MTT colorimetric assay for testing the in vitro chemosensitivity of breast cancer cells to 5 fluorouracil (5-Fu).

METHODSThe chemosensitivity of human breast carcinoma cell lines MCF-7 and MDA-MB-435S to 5-Fu at different concentrations was evaluated with MTT assay.

RESULTS5-FU treatment resulted in dose-dependent growth inhibition of the breast cancer cells with both low and high metastatic capacities.

CONCLUSIONSMTT assay may help select appropriate chemotherapeutic agents and provides evidence for individualized chemotherapy for breast cancer.

Antimetabolites, Antineoplastic ; pharmacology ; Breast Neoplasms ; pathology ; Cell Line, Tumor ; Colorimetry ; methods ; Coloring Agents ; Drug Screening Assays, Antitumor ; Female ; Fluorouracil ; pharmacology ; Humans ; Tetrazolium Salts

OBJECTIVETo investigate the reversal effects of different concentrations of DNA methylation inhibitor, 5-aza-2-deoxycytidine, on the hypermethylation of maternally expressed gene 3 (MEG3) gene promoter, and then the inhibitory effect of restoration of MEG3 expression on the proliferation of ovarian cancer cells.

METHODSHuman ovarian cancer OVCAR3 cells were treated with various concentration of 5-aza-2-deoxycytidine (0, 1, 5, 10, 20 µmol/L, respectively) for 6 days. Then the methylation status of MEG3 promoter was detected by methylation specific PCR (MSP). The alteration of MEG3 gene expression was detected by RT-PCR. Cell proliferation was determined by MTT assay and EdU incorporation assay.

RESULTSAfter treated with 5-aza-2-deoxycytidine, the methylation status of MEG3 in the 0, 1, 5, 10, 20 µmol/L 5-aza-2-deoxycytidine groups were 1.00 ± 0.00, 0.79 ± 0.00, 0.67 ± 0.00, 0.65 ± 0.03 and 0.61 ± 0.01 folds, respectively (P < 0.05 for all). The relative expressions of MEG3 mRNA in the 0, 1, 5, 10, 20 µmol/L 5-aza-2-deoxycytidine groups were 1.00 ± 0.00, 2.04 ± 0.16, 2.44 ± 0.17, 3.19 ± 0.34 and 5.34 ± 0.39, respectively (P < 0.05 for all). In contrast to the negative control, the inhibition rates of the OVCAR3 cell growth were increased significantly when treated with 1, 5, 10, 20 µmol/L 5-aza-2-deoxycytidine in 2, 4 and 6 days. There were (40.78 ± 0.80)%, (35.65 ± 0.33)%, (31.81 ± 0.66)%, (27.33 ± 1.27)% and (17.75 ± 1.85)% of EdU-positive cells in the 0, 1, 5, 10 and 20 µmol/L 5-aza-2-deoxycytidine groups (P < 0.01 for all).

CONCLUSIONSMaternally expressed gene 3 promoter hypermethylation is reversed by 5-aza-2-deoxycytidine in ovarian cancer cells. The downregulation of MEG3 gene might be resulted from the methylation, and the re-expression of MEG3 partly contribute to the growth inhibition of epithelial ovarian cancer cells.

Antimetabolites, Antineoplastic ; pharmacology ; Azacitidine ; analogs & derivatives ; pharmacology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; DNA Methylation ; Female ; Humans ; Neoplasms, Glandular and Epithelial ; Ovarian Neoplasms ; Promoter Regions, Genetic ; RNA, Messenger