Antimetabolites, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; pathology ; Caspase 3 ; Caspase 8 ; Caspases ; metabolism ; Fluorouracil ; pharmacology ; Humans ; Liver Neoplasms ; pathology ; Tumor Cells, Cultured

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

OBJECTIVETo investigate the relationship between the expression of thymidine phosphorylase (TP) and the sensitivity of gastric carcinoma to 5-fluorouracil (5-FU) and its prodrugs.

METHODSGastric carcinoma cell line AGS was transfected with recombinant plasmid pEGFP-N1-TP or control plasmid pEGFP-N1 by lipofectamin 2000. The expression of green fluorescence labeled protein was observed under fluorescence microscope. Positive clones AGS-p and AGS-pTP were selected by G418 treatment. Expression of TP protein and mRNA was detected by immunocytochemistry and RT-PCR, respectively. Drug sensitivity to 5-FU and its prodrugs was assessed by MTT assay.

RESULTSCell clones with the expression of green fluorescent protein (AGS-p) and a clone with TP and green fluorescent fusion protein (AGS-pTP) were established. Immunostaining of TP protein was strongly positive in AGS-pTP and negative in AGS-p and AGS. The expression of TP mRNA was significantly higher in AGS-pTP (0.8090 ± 0.0450) than that in AGS (0.0490 ± 0.0046) and AGS-p (0.0610 ± 0.0069; P < 0.01). The sensitivity to doxifluridine and capecitabine in AGS-pTP was significantly increased, as compared with that in AGS-p. IC50 values of AGS-pTP to doxifluridine and capecitabine were estimated 1.7 folds and 2.2 folds as much as that of AGS-p, respectively. The sensitivity to 5-FU was not different between AGS-pTP and AGS-p.

CONCLUSIONSEnhancement of TP expression improves the sensitivity of gastric carcinoma cells to doxifluridine and capecitabine. But it does not affect the sensitivity to 5-FU.

Antimetabolites, Antineoplastic ; pharmacology ; Capecitabine ; Cell Line, Tumor ; drug effects ; Deoxycytidine ; analogs & derivatives ; pharmacology ; Floxuridine ; pharmacology ; Fluorouracil ; analogs & derivatives ; pharmacology ; Humans ; Plasmids ; RNA, Messenger ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Sensitivity and Specificity ; Stomach Neoplasms ; metabolism ; pathology ; Thymidine Phosphorylase ; biosynthesis ; genetics ; Transfection

OBJECTIVETo evaluate in vitro antitumor effects of chemotherapeutic drugs, and investgate the relationship with expression of hTERT mRNA in human gastric cancer tissues.

METHODSFresh samples of gastric cancer obtained from operation room were prepared to single-cell suspension (3 x 10(5) to 5 x 10(5) cells ml(-1)) and were separately exposed to taxol (TAX), cisplatin (CDDP), 5-fluorouracil (5-Fu), adriamycin (ADM), mitomycin (MMC) for 48 hours. Metabolic activity and inhibitory rate of the cells were determined by trypan blue exclusion and MTT assay. Expression of hTERT mRNA was detected by in situ hybridization (ISH).

RESULTSThe inhibition rate of cancer cells exposed to chemotherapeutic drugs was different, and that of TAX, CDDP, 5-Fu was significantly higher than that of ADM and MMC. The positive rate of hTERT mRNA expression was 90.0% (54/60) and positive cells showed resistance to 5-Fu and ADM.

CONCLUSIONOverexpression of hTERT mRNA may contribute to primary drug-resistance of tumors. Chemosensitivity tests by MTT assay may contribute to prediction of effectness in applying chemotherapeutic drugs and identify drug-resistant cases.

Adenocarcinoma, Mucinous ; metabolism ; pathology ; Adenocarcinoma, Papillary ; metabolism ; pathology ; Adult ; Aged ; Antibiotics, Antineoplastic ; pharmacology ; Antimetabolites, Antineoplastic ; pharmacology ; Antineoplastic Agents ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Carcinoma, Signet Ring Cell ; metabolism ; pathology ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; Female ; Fluorouracil ; pharmacology ; Humans ; Male ; Middle Aged ; Mitomycin ; pharmacology ; Paclitaxel ; pharmacology ; RNA, Messenger ; metabolism ; Stomach Neoplasms ; metabolism ; pathology ; Telomerase ; genetics ; metabolism

OBJECTIVE: To explore the effect of fluorouracil (FU) combined with epigenetic drug FK228 (depsipeptide FR901228) on the proliferation, apoptosis and Fas mRNA level in HepG2 hepatoma cell lines. METHODS: There were 4 groups in this experiment: an untreated group, an FK228 treated group, a FU treated group,and a FU combined with FK228 group.Tetrazolium salt colorimetry (MTT) assay was used to assess the cell inhibition rate. Q value of Kingos formula and multi-factor analysis of variance (ANOVA ) were used to judge the combination treatment effect,and flow cytometry was used to detect the apoptosis rate. Fas mRNA level was analyzed by RT-PCR. RESULTS: FK228 or FU would inhibit the growth of HepG2 cells in a concentration-dependent and time-dependent manner. Cell inhibition rates of HepG2 were significantly enhanced in the FU combined with FK228 group, compared with that in the FU treated group alone (P<0.05). Both Q values were more than 1, the 2 drug combinations showed interaction,and FU combined with FK228 had synergistic effect.Compared with the FK228 treated group and the FU treated group, apoptosis rate of HepG2 cells was significantly increased (P<0.05), and the Fas mRNA level was up-regulated in HepG2 cells in FU combined FK228 group (P<0.05). CONCLUSION Combination of FK228 and FU can enhance the proliferation inhibition and apoptosis induction of FU in hepatoma cell lines, up-regulate the Fas mRNA level, and increase the sensitivity of hepatoma cell lines to FU.
Antibiotics, Antineoplastic ; pharmacology ; Antimetabolites, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Depsipeptides ; pharmacology ; Drug Synergism ; Fluorouracil ; pharmacology ; Hep G2 Cells ; Humans ; RNA, Messenger ; genetics ; metabolism ; fas Receptor ; genetics ; metabolism

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 investigate the expression of LTF mRNA in several nasopharyngeal cancer (NPC) cell lines, and analyze the relationship between the genetic and epigenetic changes and expression of LTF gene.

METHODSThe expression level of LTF was detected in NPC cell lines HNE1, HNE2, HNE3, CNE1, CNE2, 5-8F, 6-10B cells and tissues of 15 cases of chronic nasopharyngitis by RT-PCR. The LTF protein level was analyzed by Western blotting in 6-10B cells. Then LOH, mutation and methylation status of LTF was examined by microsatellites analysis, PCR-SSCP, MSP and bisulfite genomic sequencing, respectively.

RESULTS15 chronic nasopharyngitis tissues showed stable LTF expression, while there were weak expression in 6-10B cells and absent expression in remaining detected NPC cell lines. There was a significantly lower LTF expression in chronic nasopharyngitis tissues (Z = -3.738, P = 0.000). No LTF protein expression was observed in 6-10B cells. LOH analysis demonstrated that allele loss of LTF wasn't found in NPC cell lines. LTF mutation was noted in 14.3% (1/7) of NPC cell lines. DNA sequencing confirmed the mutation point in the promoter region (-305 bp to -50 bp) was at -218 bp (del T) of LTF gene in the HNE1 cell line. Methylation of LTF gene was not found in chronic nasopharyngitis. However, methylation of LTF promoter was detected in all NPC cell lines. LTF mRNA expression was increased in 5-8F and 6-10B cell lines after treatment with 5-aza-2-deoxycytidine.

CONCLUSIONThere is an inactivation of expression of LTF gene in the NPC cell lines. Its molecular mechanism may be related with methylation of promoter region and deletion mutation.

Antimetabolites, Antineoplastic ; pharmacology ; Azacitidine ; analogs & derivatives ; pharmacology ; Cell Line, Tumor ; DNA Methylation ; Epigenesis, Genetic ; Gene Deletion ; Humans ; Lactoferrin ; genetics ; metabolism ; Loss of Heterozygosity ; Nasopharyngeal Neoplasms ; genetics ; metabolism ; pathology ; Nasopharyngitis ; genetics ; metabolism ; Promoter Regions, Genetic ; genetics ; RNA, Messenger ; metabolism

Puromycin aminonucleoside (PAN) -induced nephrosis is a well-described model of human idiopathic nephrotic syndrome, but the mechanism of PAN's effect is not completely understood. To investigate whether proteinuria in the PAN model is associated with an alteration of zonula occludens-1 (ZO-1) expression within the glomeruli, and whether cyclosporin A (CsA) has an effect on proteinuria and ZO-1 expression in this model, eighteen Sprague Dawley (SD) rats were assigned into three groups. Twelve rats received a single intraperitoneal injection of PAN (15 mg/100 g). The other six rats received an equal volume of saline (normal control group; control). CsA solution was administered intraperitoneally once a day for 20 days after the PAN injection (n=6, PAN+CsA). The remaining six rats received PAN, but they didn't receive CsA (n=6, PAN). Compared to control rats (35.1 +/- 5.4 mg/day), the 24-hour urinary protein excretion on day 18 was significantly higher in the PAN rats (1021.9 +/- 128.9 mg/day, p< 0.01), and the CsA treatment partly reversed the increase in proteinuria in the PAN rats (556.4 +/- 102.3 mg/day, p< 0.05). Glomerular ZO-1 protein expressions were significantly increased in the PAN rats as compared to the control group on day 20 (176%, p< 0.01). CsA treatment for 20 days in the PAN rats inhibited the increase in ZO-1 protein expression by 71.1% (p< 0.05). CsA treatment significantly diminished the glomerular ZO-1 expression in the PAN rats as assessed by immunohistochemistry. CsA treatment significantly reduced proteinuria and the diminished glomerular ZO-1 expression in a PAN nephrosis rat model. These findings suggest the potential role of the slit diaphragm associated proteins in the development of the nephrotic syndrome, and CsA decreased the proteinuria probably by a direct action on the expression of these proteins in podocytes. Further investigations are needed to clarify the role of slit diaphragm associated proteins in the development of PAN nephrosis.
Animals ; Antimetabolites, Antineoplastic ; Cyclosporine/*pharmacology ; Immunosuppressive Agents/*pharmacology ; Kidney Glomerulus/*drug effects/metabolism ; Male ; Membrane Proteins/*metabolism ; Nephrosis/chemically induced/*drug therapy/*metabolism ; Phosphoproteins/*metabolism ; Puromycin Aminonucleoside ; Rats ; Rats, Sprague-Dawley ; Research Support, Non-U.S. Gov't

BACKGROUND AND OBJECTIVEmiRNA-200c can not only inhibit the aggressiveness of cancer cells but also increase the sensitivity of cells to antitumor drugs. However, some mechanisms are still unclear. Recent researches revealed that E-cadherin is more than an inhibitor of metastasis, and it also plays important roles in reversing drug resistance. We had previously found that miRNA-200c could not only induce the expression of E-cadherin but also increase the sensitivity of gastric cancer SGC7901/DDP cells to cisplatin (DDP). This study aimed to explore the effects of miRNA-200c on biological characteristics of SGC7901/DDP cells and the roles of E-cadherin in the regulatory pathway of miRNA-200c.

METHODSSGC7901/DDP cells and its parental cell line SGC7901 cells were transfected with miRNA-200c precursor (Pre-200c) and E-cadherin siRNA, respectively. Real-time RT-PCR was used to detect miRNA-200c expression after transfection with Pre-200c in SGC7901/DDP cell line. Drug sensitivities to DDP, 5-fluorouracil (5-FU), paclitaxel, and adriamycin (ADR) after transfection were tested using MTT assay. The proliferation of SGC7901/DDP cells was also detected after transfection. The protein changes of E-cadherin, Bax, and Bcl-2 after transfection were detected by Western blot.

RESULTSThe miRNA-200c expression in SGC7901/DDP cells after transfection of Pre-200c was 7.128 ± 0.159 times of that in negative control (P < 0.05). The IC50 of DDP, 5-FU, paclitaxel, and ADR in Pre-200c-transfected group were significantly lower than that in negative control group (P < 0.05). Compared to the control group, cell proliferation was significantly decreased (P < 0.05). The relative protein expressions of E-cadherin and Bax in Pre-200c-transfected group were significantly higher than those in negative control group (P < 0.05), whereas Bcl-2 was significantly lower than that in control (P < 0.05). Additionally, E-cadherin protein expression was significantly inhibited after transfected with E-cadherin siRNA in SGC7901 cells. The Bax protein expression was significantly down-regulated by E-cadherin siRNA (P < 0.05), whereas the Bcl-2 expression was significantly up-regulated (P < 0.05).

CONCLUSIONmiRNA-200c can indirectly regulate apoptosis through E-cadherin in SGC7901/DDP cells, which may be a possible mechanism of miRNA-200c in reversing drug resistance and inhibiting proliferation.

Antibiotics, Antineoplastic ; pharmacology ; Antimetabolites, Antineoplastic ; pharmacology ; Antineoplastic Agents ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Cadherins ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Cisplatin ; pharmacology ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Fluorouracil ; pharmacology ; Humans ; MicroRNAs ; genetics ; metabolism ; physiology ; Paclitaxel ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA, Small Interfering ; genetics ; Sensitivity and Specificity ; Stomach Neoplasms ; metabolism ; pathology ; Transfection ; bcl-2-Associated X Protein ; metabolism

The study was purposed to explore the effect and mechanisms of decitabine and/or Trichostatin A (TSA) on SKM-1 cells in vitro. The effect of decitabine and/or TSA on proliferation of SKM-1cells was analyzed with trypan blue exclusion; the differentiation of SKM-1 cells was detected by nitro-blue tetrazolium (NBT) reduction and flow cytometry; the apoptosis of cells was measured by Annexin V-FITC; the mRNA expression of Fas, survivin and P15(INK4B) in cells treated with decitabine and/or TSA was evaluated by RT-PCR. The results showed that decitabine and/or TSA were capable of inhibiting SKM-1 cell growth and promoting cell differentiation; they stimulated the expression of CD14 and CD11b and inhibited HLA-DR expression; meanwhile and decitabine or/and TSA could induce cell apoptosis, up-regulate mRNA expression of Fas and P15(INK4B), and down-regulate survivin mRNA expression. It is concluded that decitabine can induce apoptosis/differentiation of SKM-1 cells, whose mechanisms may related to the expression of Fas, survivin and P15(INK4B). Decitabine has the synergistic effect with TSA.
Antimetabolites, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Azacitidine ; analogs & derivatives ; pharmacology ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drug Synergism ; Humans ; Hydroxamic Acids ; pharmacology ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; genetics ; metabolism ; Myelodysplastic Syndromes ; pathology ; fas Receptor ; genetics ; metabolism