Antineoplastic Agents ; pharmacology ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Division ; drug effects ; Doxorubicin ; pharmacology ; Humans ; Liver Neoplasms ; metabolism ; pathology ; NF-kappa B ; antagonists & inhibitors ; Transfection ; Tumor Cells, Cultured

BACKGROUNDMultidrug resistance to chemotherapeutic agents is an important clinical problem during the treatment of leukemia. The resistance process is multifactorial. To realize the total factors involved in multidrug resistance, we analyzed the differentially expressed proteins of K562 and K562/ADM cells and we investigated one of the up-regulated proteins (CRKL) using siRNA to determine its role in K562/ADM cells.

METHODSAltered protein expressions between K562/S (K562 ADM-sensitive cell line) and K562/ADM (K562 multidrug resistant cell line induced by adriamycin) were identified by 2D-DIGE coupled with mass spectrometry. Meanwhile, we confirmed the differential expression of CRKL and Stathmin in both K562 and K562/ADM cells by Western blot analysis. Furthermore, we used RNA interference to silence the CRKL gene expression.

RESULTSAmong the 9 differentially expressed proteins, 3 were up-regulated in K562/ADM cells, while 6 were down-regulated in the K562/ADM cells compared with its parent cell line. The expression of CRKL was up-regulated significantly in K562/ADM cells, and it can be decreased by recombinant lentivirus. Moreover, the multidrug resistance of K562/ADM cells was efficiently reversed by silence of CRKL gene expression.

CONCLUSIONSThe data provided the differentially expressed proteins in K562 and its resistant cell line and highlights the power of 2D-DIGE for the discovery of resistance markers in cancer. We found CRKL may be a new protein involved in the multidrug resistance of leukaemia cells.

Adaptor Proteins, Signal Transducing ; analysis ; antagonists & inhibitors ; genetics ; Amino Acid Sequence ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; Humans ; K562 Cells ; chemistry ; drug effects ; Molecular Sequence Data ; Neoplasm Proteins ; analysis ; Nuclear Proteins ; analysis ; antagonists & inhibitors ; genetics ; Proteomics ; Stathmin ; analysis

Apoptosis ; drug effects ; Cell Cycle ; Cell Cycle Proteins ; antagonists & inhibitors ; genetics ; physiology ; Doxorubicin ; metabolism ; pharmacology ; Drug Resistance, Neoplasm ; Humans ; K562 Cells ; cytology ; drug effects ; Protein-Serine-Threonine Kinases ; antagonists & inhibitors ; genetics ; physiology ; Proto-Oncogene Proteins ; antagonists & inhibitors ; genetics ; physiology ; RNA Interference ; RNA, Small Interfering ; pharmacology

To investigate the neutralizing effect of N-acetylcysteine (NAC) and selenium (Se) aganist doxorubicin (DOX) toxicity in rats, NAC (140 mg/kg, p.o.) and Se (0.5 mg/kg, p.o.) were administered for 2 days before DOX injection and then 3 times a week. Cell viability and the level of lipid peroxidation were examined in cultured-rat astrocytes. Severe morphologic changes in the kidney of DOX group; thickening of Bowmans capsule, presence of multifocal tubular casts were observed, but not in the other treated groups. Vacuoles in some hepatic cells and focal aggregation of stellate macrophages were also detected in DOX group, but not in the other treated groups. However, the severe inhibition of spermatogenesis was found in all treated groups. The cell viability of DOX (10 mg/ml) treated group and NAC (5 mM) or Se (0.001 mg/ml) combinedtreated group was 52.5+/-2.0 % , 85.3+/-4.5 % and 75.5+/-1.6 %, respectively. In MDA (malondialdehyde) assay, the level of lipid peroxidation on DOX (10 mg/ml), NAC (5 mM) and Se (0.001 mg/ml) was 0.77+/-0.06, 0.35+/-0.06 and 0.54+/-0.11 nmol/mg protein, respectively. Thus, it is known that NAC and Se have protective effects in kidney and liver but not in the testes. Morphological change was not detected in brain and heart in all groups for experiment period. From this in vitro study, it is known that NAC and Se protect well the astrocytes against DOX induced-cell damage.
Acetylcysteine/*pharmacology ; Animals ; Astrocytes/*cytology/drug effects/pathology ; Cell Survival/drug effects ; Cells, Cultured ; Doxorubicin/antagonists & inhibitors/*toxicity ; Liver/cytology/*drug effects/pathology ; Male ; Rats ; Rats, Sprague-Dawley ; Spermatocytes/cytology/drug effects/pathology

The aim of this study was to evaluate the effect of dimethyl amiloride (DMA), a specific inhibitor of Na(+)/H(+) exchanger-1 (NHE-1), on intracellular pH value (pHi), proliferation and apoptosis of HL-60/ADM cells in vitro. After treatment with DMA at different doses, pHi of HL-60 and HL-60/ADM cell lines were determined by using pH-sensitive fluorescence dye BECEF-AM; the rate of growth inhibition of cells was detected with MTT assay; cell cycle was detected by flow cytometric DNA analysis; cell apoptosis was observed with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The results showed that pHi in HL-60/ADM cells was higher than that in HL-60 cells. After treatment with DMA at different doses, pHi decreased, the rate of growth inhibition and the rate of apoptotic cells in HL-60/ADM cells were all higher than those in HL-60 cells. Meanwhile, after treatment with DMA during 100 micromol/L to 150 micromol/L, the increase amplitude of G(0)/G(1) phase cells and the decrease amplitude of S + G(2)/M cells in HL-60/ADM cells were higher than those in HL-60 cells. It is concluded that by causing intracellular acidification, the NHE-1-specific inhibitor DMA inhibits proliferation of HL-60/ADM cells and induces apoptosis of HL-60/ADM cells, and the degree of this growth inhibition of HL-60/ADM cells is higher than that of HL-60 cells.
Amiloride ; analogs & derivatives ; pharmacology ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; HL-60 Cells ; Humans ; Hydrogen-Ion Concentration ; Sodium-Hydrogen Exchangers ; antagonists & inhibitors

BACKGROUNDThis study aimed at investigating the change and significance of nuclear factor-kappaB (NF-kappaB) in cardiomyopathy induced by adriamycin (ADR) in rats.

METHODSSixty male Wistar rats were randomly divided into three groups: control, ADR and ADR + pyrrolidine dithiocarbamate (PDTC) groups. After 30-day experiment, myocardial histopathological observation was performed. Location and distribution of NF-kappaB p50 was examined by immunohistochemical assay. Expression of NF-kappaB p50 protein was examined by immunobolt assay. Electrophoretic Mobility Shift Assay examined activity of NF-kappaB; Myocardium p53 gene expression was examined by RT-PCR analysis.

RESULTSThe myocardial lesions of rats were less pronounced in ADR + PDTC group than in ADR group. Compared with control group, there were many myocardium nucleuses, which expressed NF-kappaB p50 and distribute under epicardium. Expression of NF-kappaB p50 protein in nucleus increased significantly in ADR group. The NF-kappaB binding activity increased significantly in ADR group. Myocardium expressions of p53 mRNA increased in ADR group.

CONCLUSIONSThe NF-kappaB binding activity increased significantly in cardiomyopathy induced by ADR in rats. Moreover, NF-kappaB plays an important role in causing degeneration of myocardial tissue and regulating expression of related-apoptosis genes.

Animals ; Cardiomyopathies ; chemically induced ; metabolism ; pathology ; Doxorubicin ; toxicity ; Electrophoretic Mobility Shift Assay ; Genes, p53 ; Male ; NF-kappa B ; antagonists & inhibitors ; metabolism ; NF-kappa B p50 Subunit ; Protein Precursors ; Rats ; Rats, Wistar

OBJECTIVETo investigate the effect of a phosphorothioate antisense oligodeoxynucleotide "ASOND" combined with cis-Diamminedichloroplatinum (DDP), 5-fluorouracil (5-FU) and adriamycin (ADM) respectively on inhibiting the proliferation of HepG2 cells.

METHODSA phosphorothioate antisense oligodeoxynucleotide (5'-ACTCACTCAGG CCTCAGACT-3') targeted to human telomerase reverse transcriptase (hTERT) mRNA, which named cantide, was synthesized. ASODN was transfected into HepG2 by lipofectin. And cell growth activity was evaluated by MTT assay. SAS software and Jin Zhengjun Method were used to evaluate the interaction of ASODN and these chemotherapeutic drugs.

RESULTSCombination treatments with 0.1micromol/L ASODN reduced the IC50 of DDP, 5-FU and ADM from 1.07, 4.15 and 0.29microg/ml to 0.25, 1.52 and 0.12microg/ml respectively. The inhibitory ability of combination treatments on HepG2 cells was higher than that of these drugs alone (F=66.92, 25.96, 8.56, P<0.001). And synergism (Q>or=1.15) was observed at the lower concentration of DDP (

CONCLUSIONASODN may enhance therapeutic effectiveness of chemotherapeutic drugs in human hepatocellular carcinoma cells.

Antineoplastic Agents ; administration & dosage ; Cell Line, Tumor ; Cisplatin ; administration & dosage ; DNA-Binding Proteins ; Doxorubicin ; administration & dosage ; Drug Synergism ; Fluorouracil ; administration & dosage ; Humans ; Liver Neoplasms ; drug therapy ; Oligodeoxyribonucleotides, Antisense ; administration & dosage ; Telomerase ; antagonists & inhibitors ; genetics

To study the effects of tyrosine-kinase inhibitor STI571 on the adhesion of RPMI8226 cells to fibronectin (FN), cell adhesion mediated adriamycin-resistance and the Rac1 mRNA expression, the adhesion of RPMI8226 cells to fibronectin and drug resistance mediated by cell adhesion were determined by means of crystal violet staining and MTT assays respectively, Rac1 mRNA levels in RPMI8226 cells were examined by semi-quantitative RT-PCR. The results showed that STI571 could inhibit the adhesion of RPMI8226 cells to fibronectin. When RPMI8226 cells had been adhered to FN or BSA-coated wells for 1, 6 and 12 hours, the adhesion rates were (43.71 +/- 2.18)%, (55.63 +/- 1.56)%, and (63.42 +/- 2.46)% respectively. After treatment with STI571 20 micromol/L, the adhesion rates decreased to (15.12 +/- 1.04)%, (17.58 +/- 1.32)% and (17.24 +/- 1.59)% respectively (P < 0.05). The experiment revealed that growth of RPMI8226 cells adhered to FN-coated plates had a significant advantage over growth on BSA-coated plates when exposed to adriamycin (Adr) for 1 hour followed by a 24-hour culture period, and the mean IC(50) value for FN-adhered cells was (1.46 +/- 0.04) micromol/L while mean IC(50) value for BSA control was (0.78 +/- 0.03) micromol/L (P < 0.05). Following treatment with 20 micromol/L STI571, the mean IC50 values for FN and BSA adhered cells were (0.81 +/- 0.05) micromol/L, (0.74 +/- 0.02) micromol/L respectively, there was no significant difference between them (P > 0.05). RT-PCR demonstrated that the relative Rac1 mRNA level (Rac1/GAPDH) in RPMI8226 cells was downregulated following being treated with 20 micromol/L STI571. It is concluded that STI571 can inhibit the adhesion of RPMI8226 cells to fibronectin, reverse cell adhesion mediated adriamycin-resistance, and downregulate Rac1 mRNA level.
Benzamides ; Cell Adhesion ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; Fibronectins ; metabolism ; Humans ; Imatinib Mesylate ; Multiple Myeloma ; metabolism ; pathology ; Piperazines ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; Pyrimidines ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Tumor Cells, Cultured ; rac1 GTP-Binding Protein ; biosynthesis ; genetics

OBJECTIVETo investigate whether neuroblastoma cells LA-N-6 express Foxp3 and whether the expression of Foxp3 is sensitive to chemotherapy by cyclophosvnamide (CTX)and pirarubicin (THP).

METHODSExpression of Foxp3 on LA-N-6 cells was examined by flow cytometry analysis. The dose-effects of chemotherapy drugs including CTX and THP on LA-N-6 cells were investigated by MTT assay. The effects of CTX and THP on Foxp3 expression were examined by flow cytometry and real-time PCR assays.

RESULTSFlow cytometry analysis showed that LA-N-6 cells expressed Foxp3 at a high level. At sub-optimal concentration, chemotherapy drugs CTX and THP significantly down-regulated expression of Foxp3 on LA-N-6 cells at protein level (P<0.05). CTX also decreased the expression of Foxp3 at mRNA level (P<0.05). CONCLSUSIONS: Neuroblastoma cells LA-N-6 express Foxp3 at a high level, which can be suppressed by chemotherapy drugs CTX and THP. These data suggest that chemotherapy might suppress the growth and metastasis of tumor cells partially through inhibiting the expression of Foxp3.

Antineoplastic Agents ; pharmacology ; Cell Line, Tumor ; Cyclophosphamide ; pharmacology ; Doxorubicin ; analogs & derivatives ; pharmacology ; Flow Cytometry ; Forkhead Transcription Factors ; analysis ; antagonists & inhibitors ; genetics ; Humans ; Neuroblastoma ; drug therapy ; immunology ; pathology ; Reverse Transcriptase Polymerase Chain Reaction

Tropomyosin-related kinase A (TrkA) plays an important role in cell survival, differentiation, and apoptosis in various neuronal and nonneuronal cell types. Here we show that TrkA overexpression by the Tet-On system mimics NGF-mediated activation pathways in the absence of nerve growth factor (NGF) stimulation in U2OS cells. In addition, p53 upregulation upon DNA damage was inhibited by TrkA, and p21 was upregulated by TrkA in a p53-independent manner. TrkA overexpression caused cell death by interrupting cell cycle progression, and TrkA-induced cell death was diminished in the presence of its specific inhibitor GW441756. Interestingly, TrkA-mediated cell death was strongly related to gammaH2AX production and poly (ADP-ribose) polymerase cleavage in the absence of DNA damage inducer. In this study, we also reveal thatgammagammaH2AX production by TrkA is blocked by TrkA kinase inhibitors K-252a and GW441756, and it is also significantly inhibited by JNK inhibitor SP600125. Moreover, reduction of cell viability by TrkA was strongly suppressed by SP600125 treatment, suggesting a critical role of JNK in TrkA-induced cell death. We also found that gammaH2AX and TrkA were colocalized in cytosol in the absence of DNA damage, and the nuclear localization of gammaH2AX induced by DNA damage was partly altered to cytosol by TrkA overexpression. Our results suggest that the abnormal cytosolic accumulation of gammaH2AX is implicated in TrkA-induced cell death in the absence of DNA damage.
Anthracenes/pharmacology ; Apoptosis/drug effects/*genetics ; Carbazoles/pharmacology ; Cell Cycle/drug effects/genetics ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21/*biosynthesis/genetics ; Cytosol/drug effects/enzymology/ultrastructure ; DNA Damage/drug effects/genetics ; Doxorubicin/pharmacology ; Histones/*metabolism ; Humans ; Indole Alkaloids/pharmacology ; MAP Kinase Kinase 4/antagonists & inhibitors ; Nerve Growth Factor/antagonists & inhibitors/metabolism ; Phosphorylation/drug effects ; Protein Binding ; *Protein Transport/drug effects/genetics ; Receptor, trkA/antagonists & inhibitors/*genetics/metabolism ; Signal Transduction ; Transfection