OBJECTIVETo explore the effect of decitabine on Dickkopf-1 (DKK1) gene expression level and its downstream Wnt signaling pathway in acute myeloid leukemia (AML) cell line HL-60.

METHODSFlow cytometry and DNA ladder analysis were performed to detect apoptosis in HL-60 cell treated with different concentration of decitabine. Methylation-specific polymerase chain reaction (MS-PCR) was used to examine the methylation status of DKK1 gene. The expressions of mRNA and protein were determined by qRT-PCR and Western blot, respectively.

RESULTSFlow cytometric detection showed that after treating HL-60 cell line with decitabine of different concentrations for 48 h, the early apoptosis of HL-60 cells increased significantly as compared with control group (P < 0.05). DNA ladder analysis showed that the DNA ladder and demethylation of DKK1 gene appeared. RT-PCR and Western blot showed that the expressions of mRNA and protein increased. The protein expressions of β-catenin and C-MYC decreased.

CONCLUSIONThe decitabine can promote the apoptosis of HL-60 cells throngh demethylation of DDK1 gene and inhibition of Wnt signalling pathway.

Apoptosis ; Azacitidine ; analogs & derivatives ; pharmacology ; DNA Methylation ; Gene Expression Regulation, Bacterial ; Genes, myc ; HL-60 Cells ; drug effects ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Leukemia, Myeloid, Acute ; pathology ; RNA, Messenger ; Wnt Signaling Pathway ; beta Catenin ; metabolism

OBJECTIVETo study the molecular mechanism of cisplatin to enhance the ability of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in reversing multidrug resistance in vincristine-resistant human gastric cancer SGC7901/VCR cells.

METHODSMTT assay was used to measure the 50% inhibiting concentration (IC₅₀) and cell survival in SGC7901 and SGC7901/VCR cells after different treatments. SGC7901/VCR cells were treated with different concentrations of DDP, different concentrations of TRAIL alone or in combination, and then the mRNA and protein levels of several genes were determined by RT-PCR, RT-qPCR and Western-blot analysis. After targeted silencing with specific siRNA and transfection of recombinant plasmid c-myc into the SGC7901/VCR cells, the mRNA and protein levels of DR4, DR5 and c-myc were determined by RT-PCR and Western-blot analysis.

RESULTSAfter combined treatment with TRAIL and DDP of the SGC7901/VCR cells, the IC₅₀ of VCR, DDP, ADM, and 5-Fu treatment was significantly decreased compared with the control group or TRAIL-treated group (P < 0.05). After treatment with 0, 10, 50 ng/ml TRAIL in combination with 0.4 µg/ml DDP, the SGC7901/VCR cells showed significantly higher activation of caspase 3, down-regulation of DNA-PKcs/Akt/GSK-3β signaling pathway, and higher inhibition of MDR1(P-gp) and MRP1 than those treated with TRAIL alone (P < 0.01 for all). The mRNA and protein levels of DR4, DR5, c-myc were significantly decreased after silencing c-myc with specific siRNA in the SGC7901/VCR cells (P < 0.01 for all), and were significantly increased after transfection of recombinant plasmid c-myc into the SGC7901/VCR cells (P < 0.01 foe all). After the treatment with 10 ng/ml TRAIL, 0.25 µg/ml DDP + 10 ng/ml TRAIL and 0.5 µg/ml DDP + 10 ng/ml TRAIL, the relative expression level of c-myc protein in the SGC7901/VCR cells was 0.314 ± 0.012, 0.735 ± 0.026, and 0.876 ± 0.028, respectively, and the relative expression of cytochrome C was 0.339 ± 0.036, 0.593 ± 0.020 and 0.735 ± 0.031, respectively, and the relative expression levels of DR4, DR5, active-caspase 3 and active-caspase 9 in the SGC7901/VCR cells were also increased along with increasing DDP concentrations.

CONCLUSIONSThe activation of DNA-PKcs/Akt/GSK-3β signaling pathway and high expression of MDR1 and MRP1 play an important role in the multi-drug resistance properties of SGC7901/VCR cells. After combining with TRAIL, DDP can enhance the expression of DR4 and DR5 through up-regulating c-myc and enhancing the activation of caspase 3 and caspase 9 by facilitating mitochondrial release of cytochrome C. It may be an important molecular mechanism of DDP-induced sensitization of TRAIL to reverse the multidrug resistancein SGC7901/VCR cells.

ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Antineoplastic Agents ; administration & dosage ; pharmacology ; Antineoplastic Combined Chemotherapy Protocols ; administration & dosage ; pharmacology ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Line, Tumor ; Cisplatin ; administration & dosage ; pharmacology ; Down-Regulation ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Fluorouracil ; administration & dosage ; pharmacology ; Formazans ; Genes, myc ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Humans ; Inhibitory Concentration 50 ; Multidrug Resistance-Associated Proteins ; metabolism ; Neoplasm Proteins ; metabolism ; Plasmids ; Proto-Oncogene Proteins c-myc ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; pharmacology ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; metabolism ; Stomach Neoplasms ; drug therapy ; pathology ; TNF-Related Apoptosis-Inducing Ligand ; administration & dosage ; pharmacology ; Tetrazolium Salts ; Transfection ; methods

OBJECTIVETo explore the antitumoral effect of indirubin on androgen-independent prostate cancer PC-3 cells and its possible mechanisms.

METHODSWe measured the inhibitory effect of indirubin on the proliferation of prostate cancer PC-3 cells using MTT assay, detected their cell cycles by flow cytometry, and determined the expressions of the cell cycle regulatory protein cyclin D1 and its related downstream gene c-myc by Western blot.

RESULTSThe viability of the PC-3 cells was significantly decreased by indirubin in a concentration-dependent manner, reduced to 52. 2% and 13. 6% at 5 and 10 µmol/L, respectively. The cell cycle of the PC-3 cells was markedly inhibited by indirubin at 5 µmol/L, with the cells remarkably increased in the G0 and G1 phases and decreased in the S and G2/M phases. Meanwhile, indirubin also inhibited the expressions of cyclin D1 and c-myc in the Wnt signaling pathway.

CONCLUSIONIndirubin can suppress the proliferation of androgen-independent prostate cancer PC-3 cells, which may be associated with its inhibitory effect on the cell cycle and Wnt signaling pathway.

Antibiotics, Antineoplastic ; administration & dosage ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Coloring Agents ; Cyclin D1 ; metabolism ; Dose-Response Relationship, Drug ; Genes, myc ; Humans ; Indoles ; administration & dosage ; pharmacology ; Male ; Prostatic Neoplasms, Castration-Resistant ; drug therapy ; pathology ; Proto-Oncogene Proteins c-myc ; metabolism ; Tetrazolium Salts ; Thiazoles

This study was aimed to observe the effects of emodin on apoptosis and cell cycle related genes in human myeloid leukemia cell line U937 cells. U937 cells were exposed to 60 µmol/L emodin for 24, 48, 72 h. The expressions of C-MYC, h-TERT, PIM-2, Survivin, wild type P53, P21, TGF β-1 and MCL-1 genes before and after treatment with emodin were determined and quantitated by using reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that the expressions of C-MYC, h-TERT, PIM-2, Survivin in treated U937 cells decreased, but the expressions of WTp53, P21 and TGFβ1 increased, while the expression of MCL-1 gene had no obvious change. It is concluded that multiple pathways may be involved in the processes of emodin-induced U937 cell apoptosis.
Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; Cell Cycle ; drug effects ; Cell Proliferation ; Emodin ; pharmacology ; Genes, myc ; Humans ; Reverse Transcriptase Polymerase Chain Reaction ; U937 Cells

OBJECTIVETo study the effect of astragalus injection on U937 leukemia cells proliferation and apoptosis and relevant molecular mechanisms.

METHODSLeukemia cell line U937 cells were treated with different concentrations of astragalus (62.5, 125, 250, 500, 1 000 μg/mL). The U937 cells without astragalus treatment were used as the control group. The ability of cell proliferation was measured by MTT method. Flow cytometry was used to explore cell apoptosis. The cell morphology changes were observed under a fluorescent microscope by dyeing Hoechst33258. mRNA expression of c-myc and p27 in U937 cells which was exposed in 1 000 μg/mL astragalus after 0, 12, 24 and 48 hours was detected by reverse transcription polymerase chain reaction (RT-PCR).

RESULTSVarious concentrations of astragalus injection inhibited U937 cell proliferation effectively compared with the control group (P<0.05). They also induced U937 cells apoptosis and the apoptosis rate reached to (63 ± 4)% in the 1 000 μg/mL astragalus treatment group. mRNA expression level of c-myc was gradually declined and p27 mRNA expression was gradually increased with astragalus treatment time (P<0.01).

CONCLUSIONSAstragalus injection may inhibit proliferation and induce apoptosis of leukemia cell line U937 in vitro. This contributes to down-regulation of c-myc expression and up-regulation of p27 expression.

Apoptosis ; drug effects ; Astragalus Plant ; Cell Proliferation ; drug effects ; Cyclin-Dependent Kinase Inhibitor p27 ; genetics ; Genes, myc ; Humans ; Injections ; U937 Cells

Estrogen is implicated as playing an important role in aging and tumorigenesis of estrogen responsive tissues; however the mechanisms underlying the mitogenic actions of estrogen are not fully understood. Here we report that estrogen deficiency in mice caused by targeted disruption of the aromatase gene results in a significant inhibition of telomerase maintenance of telomeres in mouse ovaries in a tissue-specific manner. The inhibition entails a significant shortening of telomeres and compromised proliferation in the follicular granulosa cell compartment of ovary. Gene expression analysis showed decreased levels of proto-oncogene c-Myc and the telomerase catalytic subunit, telomerase reverse transcriptase (TERT), in response to estrogen deficiency. Estrogen replacement therapy led to increases in TERT gene expression, telomerase activity, telomere length and ovarian tissue growth, thereby reinstating ovary development to normal in four weeks. Our data demonstrate for the first time that telomere maintenance is the primary mechanism mediating the mitogenic effect of estrogen on ovarian granulosa cell proliferation by upregulating the genes of c-Myc and TERT in vivo. Estrogen deficiency or over-activity may cause ovarian tissue aging or tumorigenesis, respectively, through estrogen regulation of telomere remodeling.
46, XX Disorders of Sex Development ; drug therapy ; genetics ; metabolism ; Aging ; genetics ; metabolism ; Animals ; Aromatase ; deficiency ; genetics ; metabolism ; Cell Proliferation ; drug effects ; Estrogen Replacement Therapy ; Estrogens ; deficiency ; pharmacology ; Female ; Gene Expression ; Genes, myc ; genetics ; Granulosa Cells ; drug effects ; metabolism ; pathology ; Gynecomastia ; drug therapy ; genetics ; metabolism ; Humans ; Infertility, Male ; drug therapy ; genetics ; metabolism ; Metabolism, Inborn Errors ; drug therapy ; genetics ; metabolism ; Mice ; Mice, Knockout ; Telomerase ; genetics ; metabolism ; Telomere ; chemistry ; metabolism ; pathology

OBJECTIVETo investigate the effects of ghrelin on the proliferation and differentiation of 3T3-L1 preadipocyte, and study the possible mechanisms.

METHODS3T3-L1 preadipocytes were cultured in vitro. The proliferation potentials of 3T3-L1 preadipocytes that were treated with different concentrations of ghrelin were evaluated by MTT methods. The levels of c-myc and thymidine kinase mRNA were detected using RT-PCR. 3T3-L1 preadipocytes were differentiated into the matured adipocytes with insulin (INS) or ghrelin. The morphological changes of 3T3-L1 adipocytes were observed and the differentiation rate was assayed by oil-red O staining. Total RNA was extracted from adipocytes at various times, and the levels of peroxisome proliferation activated receptor gamma (PPARgamma) and CAAT/enhancer binding protein(C/EBPalpha) mRNA expressions were detected using RT-PCR.

RESULTSGhrelin at concentrations of 10(-7) to 10(-15) mol/L significantly stimulated preadipocyte proliferation (p<0.05). The levels of c-myc and thymidine kinase mRNA significantly increased in 3T3-L1 preadipocytes with 10(-9) mol/L and 10(-11) mol/L ghrelin treatment (p<0.01). The 3T3-L1 preadipocytes treated with 10(-11) mol/L ghrelin had lots of lipid droplets in the cytoplasma, but the differentiation rate was lower than those treated with INS. Ghrelin of 10(-11) mol/L significantly increased the mRNA expression of PPARgamma and C/EBPalpha in the course of 3T3-L1 preadipocyte differentiation, compared with the normal control group (p<0.05). The PPARgamma and C/EBPalpha mRNA expression increased with the prolonged differentiation of preadipocytes induced by ghrelin or INS. There were significant differences in the levels of PPARgamma and C/EBPalpha mRNA expression between the 2nd and 8th days of differentiation(p<0.01).

CONCLUSIONSGhrelin promotes the proliferation and differentiation of 3T3-L1 preadipocytes. The proliferation of 3T3-L1 preadipocytes induced by ghrelin may be associated with increased c-myc levels. Ghrelin may promote differentiation of 3T3-L1 preadipocytes by increasing mRNA expression of PPARgamma and C/EBPalpha, thus enhances the sensitivity of adipocytes to INS.

3T3-L1 Cells ; Adipocytes ; cytology ; drug effects ; Animals ; CCAAT-Enhancer-Binding Protein-alpha ; genetics ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Genes, myc ; Ghrelin ; pharmacology ; Mice ; PPAR gamma ; genetics ; RNA, Messenger ; analysis ; Stem Cells ; cytology ; drug effects ; Thymidine Kinase ; genetics

OBJECTIVETo establish human bronchial epithelial cell lines over expressing oncogene and to investigate its application in detection of carcinogen-induced cell transformation.

METHODSMediated by retrovirus infection, human telomerase catalytic subunit, hTERT was introduced into immortal human bronchial epithelial cells (16HBE) and followed by introduction of the oncogenic allele H-Ras(V12), or c-Myc or empty vector, creating cell lines 16HBETR, 16HBETM and 16HBETV, respectively. Biological characteristics of these cell lines including morphology, proliferation, and chromosomal aberration were examined to access whether they were transformed. Soft agar experiment and nude mice subcutaneous injection were performed using pre-transformed 16HBE cells induced by known carcinogens, nickel sulfate (NiSO4) and 7, 8, -dihydrodiol-9, 10-epoxide benzo[a] pyrene (BPDE).

RESULTSWith detection of telomerase activity and Western blotting, the expression of target proteins was verified. Thus, the transgenic 16HBE cell lines were successfully established. Cells expressing oncogene H-Ras or c-Myc grew 30.3% or 10.4% faster than control cells. However, these cells failed to form colonies in soft agar or form tumor in nude mice. 16HBETR, 16HBETM cells obtained transformed phenotype at 5 wks, 11 wks, respectively after treatment with BPDE, which are 15 wks and 9 wks earlier than control cells 16HBETV (20 wks). Meanwhile, 16HBETR, 16HBETM cells obtained transformed phenotype at 11 wks, 14 wks, respectively after treatment with nickel sulfate, which are 21 wks and 18 wks earlier than control cells (32 wks).

CONCLUSIONWith the advantage of shorter latency, transgenic human cell transformation models could be used in potent carcinogen screening and applied to chemical-carcinogenesis mechanism study.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide ; toxicity ; Animals ; Carcinogenicity Tests ; Cell Line ; Cell Transformation, Neoplastic ; drug effects ; metabolism ; pathology ; Epithelial Cells ; Gene Expression ; Gene Expression Regulation ; Genes, myc ; Genes, ras ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude

OBJECTIVETo study the effects of selenium on DNA damage, apoptosis and c-myc, c-fos, and c-jun expression in rat hepatocytes.

METHODSSodium selenite at the doses of 5, 10, and 20 micromol/kg was given to rats by i.p. and there were 5 male SD rats in each group. Hepatocellular DNA damage was detected by single cell gel electrophoresis (or comet assay). Hepatocellular apoptosis was determined by TUNEL (TdT-mediated dUTP nick end labelling) and flow cytometry. C-myc, c-fos, and c-jun expression in rat hepatocytes were assayed by Northern dot hybridization. C-myc, c-fos, and c-jun protein were detected by immunohistochemical method.

RESULTSAt the doses of 5, 10, and 20 micromol/kg, DNA damage was induced by sodium selenite in rat hepatocytes and the rates of comet cells were 34.40%, 74.80%, and 91.40% respectively. Results also showed an obvious dose-response relationship between the rates of comet cells and the doses of sodium selenite (r=0.9501, P<0.01). Sodium selenite at the doses of 5, 10, and 20 micromol/kg caused c-myc, c-fos, and c-jun overexpression obviously. The positive brown-yellow signal for proteins of c-myc, c-fos, and c-jun was mainly located in the cytoplasm of hepatocytes with immunohistochemical method. TUNEL-positive cells were detected in selenium-treated rat livers. Apoptotic rates (%) of selenium-treated liver cells at the doses of 5, 10, and 20 micromol/kg were (3.72 +/- 1.76), (5.82 +/- 1.42), and (11.76 +/- 1.87) respectively, being much higher than those in the control. Besides an obvious dose-response relationship between apoptotic rates and the doses of sodium selenite (r=0.9897, P<0.01), these results displayed a close relationship between DNA damage rates and apoptotic rates, and the relative coefficient was 0.9021, P<0.01.

CONCLUSIONSelenium at 5-20 micromol/kg can induce DNA damage, apoptosis, and overexpression of c-myc, c-fos, and c-jun in rat hepatocytes.

Animals ; Apoptosis ; drug effects ; Blotting, Northern ; Comet Assay ; DNA Damage ; Dose-Response Relationship, Drug ; Genes, fos ; drug effects ; genetics ; Genes, jun ; drug effects ; genetics ; Genes, myc ; drug effects ; genetics ; Hepatocytes ; drug effects ; pathology ; Male ; Nucleic Acid Hybridization ; Rats ; Rats, Sprague-Dawley ; Selenium ; pharmacology ; Sodium Selenite ; pharmacology

OBJECTIVETo study the effect of Shuangling Fuzheng anti-tumor preparation (SLAP) five groups on proliferation and c-myc gene expression of SGC-7901 cells in vitro.

METHODThe inhibitory effect of single SLAP (40 -640 microg x mL(-1)) and combined therapy with adriamycin (0.4, 4.0 microg x mL(-1) or cisplatin (0.1,1.0 microg x mL(-1) on human gastric carcinoma SGC-7901 cells proliferation were observed by MTT colorimetric analysis method. Technique of flow cytometry in vitro was used to measure the rate of positive sign of SLAP (80 - 320 microg x mL(-1)) on c-myc gene protein of SGC-7901 cells.

RESULTSGC-7901 cells proliferation were inhibited by single SLAP in dose of 40 - 640 microg x mL(-1) 24 h. Its inhibitory rate was increased with increase of dose. The inhibitory rate on SGC-7901 cells could be increased by SLAP in dose of 40 - 640 microg x mL(-1) plus adriamycin in dose of 0.4 and 4.0 microg x mL(-1) or plus cisplatin in dose of 0.1 and 1.0 microg x mL(-1). At the same time, SLAP (80 - 320 microg x mL(-1)) also could inhibite the expression of c-myc gene of SGC -7901 cells.

CONCLUSIONSingle SLAP had inhibiting effect on human gastric carcinoma cells proliferation with a dose-effect relationship and synergic effect while combined with adriamycin or cisplatin. To inhibit the expression of c-myc gene of human gastric carcinoma cells might be one of action mechanisms of SLAP, which inhibited tumor cells proliferation.

Antibiotics, Antineoplastic ; pharmacology ; Antineoplastic Agents ; pharmacology ; Antineoplastic Agents, Phytogenic ; administration & dosage ; isolation & purification ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; Dose-Response Relationship, Drug ; Doxorubicin ; pharmacology ; Drug Combinations ; Drug Synergism ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacology ; Gene Expression Regulation, Neoplastic ; drug effects ; Genes, myc ; Humans ; Plants, Medicinal ; chemistry ; Proto-Oncogene Proteins c-myc ; metabolism ; Stomach Neoplasms ; metabolism ; pathology