A stable dark variant separated from photobacterium phosphoreum (A2) was fixed in agar-gel membrane and immobilized onto an exposed end of a fiber-optic linked with bioluminometer. The variant could emit a luminescent signal in the presence of genotoxic agents, such as Mitomycin C (MC). The performance of this whole-cell optical fiber sensor system was examined as a function of several parameters, including gel probe thickness, bacterial cell density, and diameter of the fiber-optic core and working temperature. An optimal response to a model genotoxicant, Mitomycin C, was achieved with agar-bacterial gel membrane: the thickness of gel membrane was about 5 mm; the cell density of bacteria in gel membrane was about 2.0 x 10(7)/ml; the diameter of fiber-optic core was 5.0 mm; the working temperature was 25 degrees C. Under these optimized conditions, the response time was less than 10 h to Mitomycin C, with a lower detection threshold of 0.1 mg/L.
Biosensing Techniques ; Chemiluminescent Measurements ; Fiber Optics ; Luminescent Proteins/*genetics ; Mitomycin/*pharmacology ; Mitomycin/toxicity ; Photobacterium/*genetics ; Transcription, Genetic/drug effects ; Variation (Genetics)

OBJECTIVETo investigate the relationship between aldose reductase like protein (ARL-1) gene overexpressed in HCC cells and drug-resistance of the cell to drugs containing carbonyl group.

METHODSTo establish ARL-1 stable expression positive cell line, eukaryotic expression vectors containing ARL-1 gene cDNA were transfected into Hep cell mediated by lipofect AMINE. The positive monoclones were determined by PCR and RT-PCR, respectively. Then MTT assay was used to study the drug resistance ability of the cells to drugs containing carbonyl after incubating three days with those drugs.

RESULTSAfter ARL-1 gene transfection mediated by lipofect AMINE, one positive monoclonal cell overexpressing ARL-1 gene was selected. Compared with the control cell group, drug resistance ability of the positive cells to ADM and MMC which contain carbonyl group increased 2.3 and 3.17 fold, respectively (t=6.39, P=0.016 in ADM group and t=30.06, P=0.001 in MMC group). In the same time, drug resistance ability to 5-FU which has no carbonyl group had no statistical difference between positive monoclonal cell group and control cell group (t=0.684, P=0.531).

CONCLUSIONSThe Hep ARL-1 positive cell line with stable expression of ARL-1 gene has been established successfully and the up-regulation of ARL-1 gene may plays an important role in drug resistance of the cells to anticancer drugs containing carbonyl group.

Aldehyde Reductase ; genetics ; metabolism ; Antineoplastic Agents ; pharmacology ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; genetics ; physiology ; Fluorouracil ; pharmacology ; Humans ; Inhibitory Concentration 50 ; Mitomycin ; pharmacology ; Transfection ; Tumor Cells, Cultured ; drug effects

OBJECTIVETo evaluate the effect of combination of glutamine (GLN) and mitomycin C (MMC) on the human gastric carcinoma cell line MGC-803 in vitro.

METHODSThe effects of GLN and MMC were measured by MTT assay, and the interaction between the two agents was evaluated by the median-effect principle. Flow cytometry was used for cell cycle analysis.

RESULTSGLN did not significantly stimulate the cell growth in vitro. High-concentration of GLN could inhibit the cell growth. MMC could effectively inhibit the cell growth in a time-dependent manner. The interaction of these two agents showed a weak antagonistic activity (1 < CI < 1.2703). MMC induced remarkable S-phase arrest. Low-dose GLN has limited effect on the S-phase arrest of MMC, while high-dose GLN significantly attenuated the S-phase arrest and lowered the proliferation index of MGC-803 cell.

CONCLUSIONSCombination of GLN and MMC has a a weak and dose-dependent antagonistic activity in the treatment of gastric carcinoma cell line MGC-803. The combination of high-dose MMC and low-dose GLN may achieve better efficacy.

Adenocarcinoma ; pathology ; Antibiotics, Antineoplastic ; pharmacology ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Drug Synergism ; Glutamine ; pharmacology ; Humans ; Mitomycin ; pharmacology ; Stomach Neoplasms ; pathology

The defect or block of apoptosis is an important factor involved in the drug resistance of tumor cells. Blm gene plays a great role in DNA damage and repair. This study was aimed to explore the relationship of blm gene expression with cell cycle and apoptosis after Jurkat DNA damage. The apoptosis rate and change of cell cycle were detected by flow cytometry, the expression level of blm mRNA in Jurkat cells was determined by semi-quantitative RT-PCR. The results indicated that after induction with 0.4 g/L of mitomycin C (MMC) for 24 hours the apoptosis rate of Jurkat cells were (11.42±0.013)%, and (66.08±1.60)% Jurkat cells were arrested in G2/M phase. After induction for 48 hours, the apoptosis rate of Jurkat cells declined from (11.42±0.013)% to (8.08±0.27)%, and cell count of Jurkat cells arrested in G2/M phase decreased from (66.08±1.60)% to (33.96±1.05)%. When induced with 0.4 g/L of MMC for 24 hours, the apoptosis rate of fibroblasts and the percentage of fibroblasts in G2/M, G0-G1 and S phase all showed no significant change until 48 hours. The range of apoptosis rate and the change of cell percentage in three phases were significantly different between Jurkat cells and fibroblasts (p<0.01). Expression level of blm mRNA in Jurkat cells was remarkably higher than that in normal fibroblasts (p<0.01), at 48 hours expression level of blm mRNA was remarkably higher than that at 24 hours. The 2 groups showed clear difference of blm mRNA expression after treated by MMC (p<0.01). It is concluded that the blm gene may play a significant role in repair of DNA damage of Jurkat cells after MMC induction. Abnormal expression of blm is correlated to the drug resistance of leukemia cells.
Apoptosis ; Cell Cycle ; DNA Damage ; drug effects ; DNA Repair ; drug effects ; Humans ; Jurkat Cells ; Mitomycin ; pharmacology ; RecQ Helicases ; genetics

To provide the profiles of metabolism of mitomycin C (MMC) by human liver microsomes in vitro, MMC was incubated with human liver microsomes, then the supernatant component was isolated and detected by HPLC. Types of metabolic enzymes were estimated by the effect of NADPH or dicumarol (DIC) on metabolism of MMC. Standard, reaction, background control (microsomes was inactivated), negative control (no NADPH), and inhibitor group (adding DIC) were assigned, the results were analyzed by Graphpad Prism 4. 0 software. Reaction group compared with background control and negative control groups, 3 NADPH-dependent absorption peaks were additionally isolated by HPLC after MMC were incubated with human liver microsomes. Their retention times were 10. 0, 14. 0, 14. 8 min ( named as Ml, M2, M3) , respectively. Their formation was kept as Sigmoidal dose-response and their Km were 0. 52 (95% CI, 0. 40 - 0.67) mmol x L(-1), 0. 81 (95% CI, 0. 59 - 1. 10) mmol x L(-1), 0. 54 (95% CI, 0. 41 -0. 71) mmol x L(-1) , respectively. The data indicated that the three absorption peaks isolated by HPLC were metabolites of MMC. DIC can inhibit formation of M2, it' s dose-effect fitted to Sigmoidal curve and it' s IC50 was 59. 68 (95% CI, 40. 66 - 87. 61) micromol x L(-1) , which indicated DT-diaphorase could take part in the formation of M2. MMC can be metabolized by human liver microsomes in vitro, and at least three metabolites of MMC could be isolated by HPLC in the experiment, further study showed DT-diaphorase participated in the formation of M2.
Antibiotics, Antineoplastic ; metabolism ; Chromatography, High Pressure Liquid ; methods ; Dicumarol ; pharmacology ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; pharmacology ; Humans ; Microsomes, Liver ; drug effects ; enzymology ; metabolism ; Mitomycin ; metabolism

OBJECTIVETo compare morphological differences of three drug-resistant hepatocellular carcinoma (HCC) cell subclones (Huh-7/ADM, Huh-7/CBP, Huh-7/MMC) and their parental Huh-7 cell line, to analyze differential microRNA (miRNA) expression profiles in these cells and, finally to screen for the abnormal expressed miRNAs in drug-resistant HCC cells.

METHODSCellular morphology was observed by histology and transmission electron microscopy. MiRNA microarray was used to analyze the differential miRNA expression profiles in these cells (Huh-7, Huh-7/ADM, Huh-7/CBP, Huh-7/MMC) followed by real time quantitative PCR validation.

RESULTSThe drug-resistant cells had more intracytoplasmic organelles and were larger in size along with increased cytological pleomorphism than the parental Huh-7 cells. Compared with the parental Huh-7 cells, 32 simultaneously up-regulated and 22 down-regulated miRNAs were found in three drug-resistant cells. Up-regulation of miR-15a, miR-16, miR-27b, miR-30b, miR-146a, miR-146b-5p, miR-181a, miR-181d and miR-194 was verified by RT-qPCR.

CONCLUSIONDrug-resistant HCC cells have abnormal expressed miRNAs, which may be explored to further investigate the association of miRNA expressions with multidrugs resistance in HCC.

Antineoplastic Agents ; pharmacology ; Carboplatin ; pharmacology ; Carcinoma, Hepatocellular ; genetics ; pathology ; ultrastructure ; Cell Line, Tumor ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Gene Expression Profiling ; Humans ; Liver Neoplasms ; genetics ; pathology ; ultrastructure ; MicroRNAs ; genetics ; metabolism ; Mitomycin ; pharmacology ; Oligonucleotide Array Sequence Analysis

AIMTo examine the effect of inducible nitric oxide synthase (iNOS) on tumour cells chemosensitivity to mitomycin C (MMC) analogue 5-aziridinyl-3-hydroxyl-1-methylindole-4,7-dione (629) in vitro, and elucidate the possible role of iNOS in the metabolism of 629.

METHODSHuman sarcoma cells (HT1080) and its iNOS gene transfected clones (iNOS9, iNOS12) were exposed to 629 at concentrations of 1 nmol x L(-1) - 100 micromol x L(-1). 3-[4, 5-Dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT) assay, agarose electrophoresis and flow cytometric analysis were used to determine cell sensitivity, deoxyribonucleic acid (DNA) damage and the change of cell cycle in above process, respectively. All experiments were performed both in air and under hypoxia parallelly.

RESULTS629 was more toxic than MMC, and enhanced cytotoxicity under hypoxia, which resulted in cell necrosis. Sixteen hours after treated with 629, HT1080 cells and related iNOS-transfected clone cells were obviously blocked in G2/M phase.

CONCLUSIONiNOS plays dual roles in 629 metabolism, enhancing or decreasing the cytoxicity of 629 depending on the intracellular oxygen pressure P(O2), which caused higher cytotoxicity to hypoxia cells of 629 with the increasing of iNOS activity.

Antibiotics, Antineoplastic ; pharmacology ; Aziridines ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Survival ; drug effects ; DNA Damage ; Fibrosarcoma ; metabolism ; pathology ; Flow Cytometry ; Humans ; Indoles ; pharmacology ; Mitomycin ; chemistry ; pharmacology ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Transfection

OBJECTIVETo study the combined damage-effects of low-intensity 2,450 MHz microwave (MW) with three chemical mutagens on human lymphocyte DNA.

METHODSDNA damage of lymphocytes exposed to microwave and(or) with chemical mutagens were observed at different incubation time (0 h or 21 h) with comet assay in vitro. Three combination-exposure ways of MW with chemicals were used: MW irradiation before chemical exposures, simultaneously exposed to MW and chemicals and MW irradiation after chemical exposures. The three chemical mutagens were mitomycin C (MMC, DNA crosslinker), bleomycin (BLM, radiometric agent), methyl methanesulfonate (MMS, alkylating agent). The exposure time of MW and chemical mutagens were 2 h and 3 h respectively.

RESULTSThe differences of comet tail length between MW group and control group were not significant when lymphocytes were incubated for 0 h or 21 h (P > 0.05). However, when lymphocytes were incubated for 21 h with 30.00 micro mol/L of MMC, the comet tail lengths of MW + MMC group, MW-MMC group and MMC + MW group were (18.00 +/- 5.96), (21.79 +/- 11.47) and (22.32 +/- 8.10) micro m respectively; while with 3.00 micro mol/L of MMC, the comet tail lengths were (8.99 +/- 3.75), (12.40 +/- 5.35) and (14.00 +/- 5.38) micro m respectively, which were significantly higher than those of corresponding MMC groups [(9.42 +/- 3.34) and (6.50 +/- 2.89) micro m, P < 0.01 or P < 0.05]. The DNA damage of MW plus BLM groups and MW plus MMS groups were not significantly different from the corresponding BLM and MMS groups (P < 0.05).

CONCLUSION2 450 MHz MW (5 mW/cm(2)) did not induce DNA damage directly, but could enhance the DNA damage effects induced by MMC. The synergistic effects of 2 450 MHz MW with BLM and MMS were not obvious.

Bleomycin ; pharmacology ; Comet Assay ; DNA ; drug effects ; genetics ; radiation effects ; DNA Damage ; Humans ; Lymphocytes ; drug effects ; metabolism ; radiation effects ; Methyl Methanesulfonate ; pharmacology ; Microwaves ; adverse effects ; Mitomycin ; pharmacology ; Mutagens ; pharmacology ; Time Factors

OBJECTIVETo investigate survivin mRNA and protein expressions in mitomycin (MMC)-treated hepatoma carcinoma Hepa1-6 cells in vitro.

METHODSHepa1-6 cells were cultured in vitro in the presence of MMC at the concentrations of 1.0, 3.0 and 9.0 microg/ml, respectively, and 1 day and 3 days after the culture, the cell growth inhibition was assessed using MTT assay and the expressions of survivin were detected by RT-PCR and Western blotting.

RESULTSMMC at the concentration of 9.0 microg/ml resulted in significantly greater growth inhibition of the Hepa-6 cells than MMC at 1.0 and 3.0 microg/ml, and at the latter two concentrations, MMC treatment for 3 days did not produce obvious cell growth inhibition. Survivin expressions at both the mRNA and protein levels in Hepa1-6 cells were significantly decreased 1 day after MMC treatment at the 3 concentrations, and after 3-day MMC treatment at 1.0 and 3 microg/ml, survivin expressions increased to exceed the control level, whereas survivin maintained the low expression levels in cells treated with 9 microg/ml MMC for 3 days.

CONCLUSIONSurvivin expression in Hepa1-6 cells increases in response to MMC treatment at low doses, which might be one of the reasons for chemotherapeutic drug resistance.

Carcinoma, Hepatocellular ; metabolism ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Humans ; Inhibitor of Apoptosis Proteins ; Liver Neoplasms ; metabolism ; Microtubule-Associated Proteins ; metabolism ; Mitomycin ; pharmacology ; RNA, Messenger ; metabolism

OBJECTIVETo investigate the effects of overexpression of second mitochondria-derived activator of caspases (Smac) gene on apoptosis of gastric cancer cells.

METHODSUnder the induction of liposome, MKN-45 cells were transfected by Smac gene and incubated with G418 for subclone selection. Reverse transcriptase-polymerase chain reaction and Western blot were used to determine cellular Smac gene expression. After induction of apoptosis by mitomycin (MMC), cell viabilities were analyzed using trypan blue stain. Apoptosis was measured by electronic microscopy, acridine orange-ethidium bromide fluorescent staining and in situ terminally labelled transferase technique (TUNEL). Western blot and colorimetry were used to assess cellular caspase-3 expression and its activity.

RESULTSThe Smac mRNA and protein levels in MKN-45/Smac subclone cells (subclone consistently expressing Smac gene) were significantly higher than those in MKN-45 (P < 0.01). When compared with those in MKN-45, cell viabilities of MKN-45/Smac were reduced by 10.0% to 30.8% (P < 0.01), after treatment with 10 microg/ml MMC for 6 to 24 hours. Some of the MKN-45/Smac cells showed characteristic morphologic changes of apoptosis, their apoptotic rate being increased by 21.2% (P < 0.01). After treatment with MMC, caspase-3 expression and its activity in MKN-45/Smac cells were significantly higher than those in MKN-45 (P < 0.01).

CONCLUSIONSOverexpression of Smac in gastric cancer cell line significantly improves expression and activity levels of caspase-3 after induction by MMC. Such apoptosis-inducing effect establishes a novel strategy for regulating the apoptosis activity of gastric cancer.

Apoptosis ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; Mitochondrial Proteins ; biosynthesis ; genetics ; Mitomycin ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Stomach Neoplasms ; metabolism ; pathology ; Transfection