OBJECTIVETo investigate the effect of monoamine oxidase inhibitor phenelzine on proliferation, apoptosis and histone modulation in acute lymphoblastic leukemia cell line Molt-4 cells.

METHODSThe effect of Phenelzine on cell proliferation was detected by MTT. Apoptotic rate was measured by flow cytometry. The variation of apoptosis associated proteins Caspase-3, Bcl-2 and Bax, cyclin-dependent kinase inhibitor p21, tumor suppressor protein p15, and the expression level of histone methylation of H3K4, H3K9 and histone acetylation of H3, DNMT1 were detected by Western Blot.

RESULTS① Molt-4 cell proliferation rates were (87.68±3.54)%, (67.84±3.24)%, (51.48±3.37)%, (28.72±2.56)% respectively after exposured to phenelzine at 5, 10, 15, 20 μmol/L for 24 h, P<0.05. ② After 10 μmol/L of phenelzine exposure for 24, 48, 72 h, cell proliferation rates were (67.84±3.24)%, (50.24±2.01)%, (40.31±2.25)%, P<0.05. ③ The apoptotic rates were (13.64±2.58)%, (31.24±3.42)%, (56.37±4.26)% after phenelzine treatment at 5, 10, 20 μmol/L for 24 h, which was concentration dependent. ④ Phenelzine could upregulate the expression of Bax, caspase-3, p21, and downregulate Bcl-2 expression. Phenelzine upregulated the methylation level of histone H3K4me1, H3K4me2 and histone acetylated H3, while it didn't change the level of histone H3K4me3, H3K9me1, H3K9me2. ⑤ Phenelzine inhibited DNMT1 expression and promoted p15 expression.

CONCLUSIONSPhenelzine increased the methylation of histone H3K4me1, H3K4me2, acetylation of histone H3 and p21, and decreased the expression of DNMT1 and p15, and ultimately inhibited the proliferation and apoptosis of Molt-4 cells.

Acetylation ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p15 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; Histones ; metabolism ; Humans ; Methylation ; Phenelzine ; pharmacology

Objective:To establish a gas chromatography(GC) fingerprints analysis method of farmed musk,natural musk and artificial musk for effective control and scientific evaluation of the quality of musk. Method:The sample was split in the 250℃ injection port,with 1:1 split ratio,and separated on a Capillary column(325℃,0.25 mm×30 m,0.25 μm). Nitrogen was used as a carrier gas,the flow rate was 1 mL·min^-1. The detector temperature was 280℃. The temperature program was 80℃ for 2 min,increase by 5℃·min^-1 to 160℃, and by 1℃·min^-1 to 200℃ for 20 min,then 5℃·min^-1 to 260℃ for 20 min. GC of the 30 batches of farmed musk,24 batches of natural musk and 8 batches of artificial musk were analyzed by adopting temperature programmer. The similarity evaluation system for chromatographic fingerprint of TCM(2004A)was used to evaluate the similarity of these chromatograms. The fingerprint peaks were identified by using GC-MS combination technology. The ion source temperature was set at 230℃. The quadrupole temperature was set at 150℃. The GC-MS interface temperature was set at 280℃. Electron impact(EI)was used as the ionization source,and the electron energy was set at 70 eV. The scanning scope was m/z 30-550. Result:The GC fingerprints of different kind of musk were established,and the similarities all conformed to regulations. Totally 17 common compounds in farmed musk,10 common compounds in natural musk,and 8 common compounds in artificial musk were determined and compared. Conclusion:This method features a high precision,reproducibility,and stability. The established fingerprints could be used for the overall quality evaluation for musk.

Near infrared spectroscopy(NIRS) was used for rapid quantitative analysis of saponins in Pien Tze Huang troches and powders. The near infrared spectra of Pien Tze Huang were collected,and the contents of notoginsenoside R1,ginsenoside Rg1 and ginsenoside Rb1 in Pien Tze Huang were determined by high performance liquid chromatography(HPLC) as the reference values. Then the near infrared spectra of the samples were associated with the reference values to establish the quantitative analysis models by using partial least squares(PLS) method. Finally,the models were verified by unknown samples. The results showed that root mean square error of cross-validation(RMSECV) of R1,Rg1,Rb1 and the total content was 0.095 1,0.555,0.414,0.960 mg·g-1 for the troches models,0.085 6,0.443,0.405,0.913 mg·g-1 for the powders models. After external validations,root mean square error of prediction(RMSEP) of R1,Rg1,Rb1 and the total content was 0.111,0.274,0.276,0.807 mg·g-1 for the troches models,0. 059 2,0. 322,0. 327,0. 705 mg·g-1 for the powders models. The averages of relative standard deviation between the predicted values and the chemical measured values were all less than 2.0%. According to the results of paired-t tests at the level of α = 0.05,there were no significant differences between the predicted values and the measured values. The established quantitative analysis models can be used to predict the contents of saponins in Pien Tze Huang accurately and the proposed method is simple,fast,non-destructive and environmentally friendly for the rapid detection and quality control of saponins in Pien Tze Huang.
Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; Least-Squares Analysis ; Phytochemicals ; analysis ; Saponins ; analysis ; Spectroscopy, Near-Infrared

To investigate the protective effect of Yindan Pinggan Capsules(YDPG)on acute alcoholic liver inflammatory injury and oxidative stress,sixty male Sprague-Dawley rats were divided into 6 groups randomly:normal group,model group,YDPG-L group,YD-PG-M group,YDPG-H group and Haiwang Jinzun(HWJZ)group.Except for the normal rats,while the remaining rats were administered orally with alcohol(50%,5 g·kg~(-1)).One hour after drinking,the rats in normal or model group were administered orally with PS,while the remaining rats were separately given YDPG(0.15,0.3,0.6 g·kg~(-1))or HWJZ(0.3 g·kg~(-1))for 10 days.After the last intragastric administration and fasting for 8 h,the HE staining was used to observe the pathological changes.The serum was used to determine the content of ALT,AST,ALP,TB,TP,LDH.The expressions of IL-1β,TNF-α,IL-10 and IL-4 in serum were detected by enzyme-linked immuno sorbent assay(ELISA).The content of SOD,MDA,GSH,GSP-Px in liver were determined.The results showed that both YDPGand HWJZ alleviated the liver injury induced by alcohol,decreased the activities of ALT,AST,ALP and LDH,down-regulated the expressions of TNF-αand IL-1β,up-regulated the expressions of IL-4 and IL-10;increased the activities of SOD,GSH,GSH-Px,but decreased the content of MDA.Therefore,the above results indicated that YDPG could protect the liver from alcoholic injury.
Animals ; Capsules ; Liver ; Male ; Oxidative Stress ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha

This study is to determine seven flavonoids( narirutin,naringin,hesperidin,neohesperidin,baicalin,oroxylin A-7-glucoronide and wogonoside) in Qingre Zhike Granules and intermediate products by quantitative analysis of multi-components with a single marker( QAMS). High performance liquid chromatography( HPLC) was performed on Thermo BETASIL C18 chromatographic column( 4.6 mm×250 mm,5 μm) with acetonitrile-0.1% formic acid solution as mobile phase for gradient elution at a flow rate of 0. 8 m L·min^-1. The detection wavelength was set at 280 nm and the column temperature was 30 ℃. Six relative correction factors( RCFs)of narirutin,naringin,hesperidin,baicalin,oroxylin A-7-glucoronide and wogonoside were established in the HPLC method with neohesperidin as the internal standard,and then these RCFs were used to calculate the mass fractions of the six components. Such mass fractions were compared with the mass fraction determined by the external standard method( ESM) at the same time to verify the feasibility and accuracy of QAMS method. Within the linear range,the RCFs of narirutin,naringin,hesperidin,baicalin,oroxylin A-7-glucoronide and wogonoside were 1.150,1.109,1.110,0.658,0.723 and 0.572,respectively,with good repeatability in different experimental conditions. There was no significant difference between the QAMS method and ESM method. The QAMS method with neohesperidin as the internal standard is feasible and accurate for the determination of narirutin,naringin,hesperidin,baicalin,oroxylin A-7-glucoronide and wogonoside,and it can be used for quality control of Qingre Zhike Granules and intermediate products.
Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; Flavonoids ; Quality Control

OBJECTIVETo investigate the effect of silencing LSD1 gene by RNA interference on the proliferation, apoptosis on human lymphocytic leukemia Jurkat cell line and its mechanism.

METHODSThe hairpin- like oligonucleotide sequences targeting LSD1 gene was transfected into Jurkat cells by lipofectamine(TM) 2000. The LSD1 mRNA and protein were detected by RQ- PCR and Western blot. Cell growth was determined by MTT. Cell apoptosis was analyzed by flow cytometry. The expression of Bcl-2, Bax, procaspase- 3, and histone H3K4me, H3K4me2, H3K4me3, Act- H3, H3K9me were detected by Western blot.

RESULTSLSD1 mRNA was markedly suppressed by the shRNA targeting LSD1. LSD1 shRNA suppressed the proliferation and induced cells apoptosis of Jurkat cells. The cell apoptotic rate was (41.34±3.58)%, (3.45±1.54)%, (1.76±0.52)% in LSD1 shRNA, Neg-shRNA and Blank respectively, the difference among them was statistically significant (P<0.05). LSD1 shRNA down- regulated the expressions of Bcl- 2 and procaspase- 3, and up- regulated the expression of Bax. The methylation of H3K4me1, me2 and acetylation of Act- H3 improved without change of the methylation of H3K4me3.

CONCLUSIONSDeplete of LSD1 gene maybe through modifying the methylation of histone H3K4 to promote the cell apoptosis and inhibit cell growth in Jurkat cell line.

Acetylation ; Apoptosis ; Caspase 3 ; metabolism ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; Histone Demethylases ; genetics ; Histones ; metabolism ; Humans ; Jurkat Cells ; Methylation ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA Interference ; RNA, Messenger ; RNA, Small Interfering ; Transfection

This study is to investigate the effect of downregulation histone deacetylases 1 (HDAC1) gene by the technology of RNA interference on the differentiation of HL-60 cells line. The optimal segment targeting HDAC1 gene was designed and transfected into HL-60 cells by Lipofectamine 2000. The HDAC1 mRNA and protein level were detected by RT-PCR and Western blotting. The morphologic change of HL-60 cells was detected by an optical microscope with Wright-Giemsa. Cell differentiation was tested by NBT reduction assay. Expression of CD13, CD33 and CD14 was measured by flow cytometry. The results indicated that HDAC1 mRNA and protein were markedly suppressed by the siRNA targeting HDAC1 in a concentration-dependent manner. HDAC1 siRNA promoted cell differentiation. HL-60 cells became more mature in morphology after transfected to HDAC1 siRNA at a concentration of 30-60 nmol x L(-1) for 24 h. NBT reduction ability of HDAC1 siRNA with 30 nmol x L(-1) was 0.31 +/- 0.09, compared with negative control (0.20 +/- 0.02) (t = -3.1, P < 0.01), and with 60 nmol x L(-1) was 0.25 +/- 0.02 in comparison with negative control (0.21 +/- 0.04) (t = -2.12, P < 0.05). But it has no change in HDAC1 siRNA > or = 120 nmol x L(-1). After transfection with 60 nmol x L(-1) HDAC1 siRNA to HL-60 cells, the expression of CD13 was (96.50 +/- 0.70)% in compared to siRNA-NC (3.39 +/- 0.68) % (t = 164.9, P < 0.000 5), CD33 was (66.73 +/- 0.50) % in compared to siRNA-NC (96.80 +/- 1.70) % (t = 43.4, P < 0.000 5). CD14 was (0.53 +/- 0.00) % by comparison with siRNA-NC (0.49 +/- 0.02) % (t = -0.97, P > 0.1). HDAC1 siRNA promoted cell differentiation in indicated concentration. HDAC1 might be one of the targets of gene therapy for leukemia.
CD13 Antigens ; metabolism ; Cell Differentiation ; Down-Regulation ; HL-60 Cells ; Histone Deacetylase 1 ; genetics ; metabolism ; Humans ; Lipopolysaccharide Receptors ; metabolism ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Sialic Acid Binding Ig-like Lectin 3 ; metabolism ; Transfection

This study was purposed to investigate the effect of phenylhexyl isothiocyanate (PHI) on Wnt/β-catenin signaling pathway, histone acetylation, histone methylation and cell apoptosis in Jurkat cell line. The viability of Jurkat cells after treatment with PHI was tested by MTT. Apoptotic rate of Jurkat cells was measured by flow cytometry. The levels of Wnt/β-catenin related proteins including β-catenin, TCF, c-myc, and cyclinD1, histone acetylated H3 and H4, histone methylated H3K9 and H3K4 were detected by Western blot. The results showed that PHI inhibited the cell growth and induced apoptosis in Jurkat cells in time-and dose-dependent manners. Its IC50 at 48 h was about 20 µmol/L. Expression of histone acetylated H3, H4 and histone methylated H3k4 increased after exposure to PHI for 3 h, while histone methylated H3K9 decreased. Expression of β-catenin was not changed after exposure to PHI for 3 h, but expression of β-catenin, and its cell cycle-related genes such as TCF, c-myc and cyclinD1 decreased after exposure to PHI for 7 h. It is concluded that PHI regulates acetylation and methylation of histone, inhibits Wnt/β-catenin signal pathway, and is able to induce apoptosis and inhibits growth of Jurkat cells.
Acetylation ; Acylation ; Cyclin D1 ; metabolism ; Histones ; metabolism ; Humans ; Isothiocyanates ; pharmacology ; Jurkat Cells ; Methylation ; Proto-Oncogene Proteins c-myc ; metabolism ; TCF Transcription Factors ; metabolism ; Wnt Signaling Pathway ; drug effects ; beta Catenin ; metabolism

OBJECTIVETo investigate the effect of phenylhexyl isothiocyanate (PHI) on the drug-resistance to imatinib in K562/G01 cell line and to elucidate its possible mechanisms.

METHODSMTT assay was employed to access K562/G01 cell growth inhibition after exposure to PHI and/or imatinib at different concentrations. Apoptotic rate of K562/G01 cells was measured by flow cytometry. The levels of P-gp, P210(bcr-abl) and p-P210(bcr-abl) protein were detected by Western blot.

RESULTSPHI inhibited proliferation and induced apoptosis of K562/G01 cells after treated with PHI alone for 24 h. PHI concentration increased from 0 to 40 µmol/L, the inhibitory rate of cell proliferation from 0 to (51.22 ± 1.41)%, and the apoptosis rate from (3.76 ± 1.46)% to (35.35 ± 3.70)%. Combination of 10, 20, 40 µmol/L PHI and various concentrations of imatinib, IC50 s of imatinib were (10.49 ± 1.24), (6.33 ± 1.42), and (0.85 ± 0.17) µmol/L, respectively. When K562/G01 cells treated with 20 µmol/L PHI combined with 10 and 20µmol/L imatinib for 24 hours, apoptosis rate were (43.62 ± 4.23)% and (55.41 ± 4.35)%, respectively, being significantly higher than that with imatinib or PHI alone. PHI concentrations increased from 0 to 40 µmol/L for 7 hours, the P210(bcr-abl)/β-actin decreased from (0.944 ± 0.034) to (0.392 ± 0.025), and the p-P210(bcr-abl)/β-actin decreased from (0.906 ± 0.019) to (0.361 ± 0.021), while the alteration of P-gp was not seen.

CONCLUSIONSPHI inhibits the proliferation and induces apoptosis of K562/G01 cell line. PHI has synergistic effect with imatinib. It partially reverses the drug-resistance to imatinib. The mechanism of reversal of drug resistance in K562/G01 cells might be by inhibiting P210(bcr-abl) and p-P210(bcr-abl).

ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Apoptosis ; drug effects ; Benzamides ; pharmacology ; Cell Proliferation ; drug effects ; Drug Resistance, Neoplasm ; Fusion Proteins, bcr-abl ; metabolism ; Humans ; Imatinib Mesylate ; Isothiocyanates ; pharmacology ; K562 Cells ; Piperazines ; pharmacology ; Pyrimidines ; pharmacology

OBJECTIVETo investigate the effect of short hairpin RNA (shRNA) and XAV939, a specific inhibitor for β-catenin, on growth and apoptosis of mantle cell lymphoma(MCL) Jeko-1 cell line.

METHODSβ-catenin shRNA eukaryotic expression vector was transfected into Jeko-1 cells, the antiproliferative effect of shRNA on Jeko-1 cells was detected by RT-PCR and Western blot. The proliferation inhibitory rate of Jeko-1 cells treated by different doses of XAV939 was assayed by MTT method; the level of apoptosis of Jeko-1 cells was detected by flow cytometry; the expression levels of apoptosis-related protein BCL-2, BAX, CyclinD1, C-MYC and Caspase-3 in Jeko-1 cells were determined by Western blot.

RESULTSThe expression of β-catenin mRNA and growth of Jeko-1 cell line were inhibited by shRNA; after Jeko-1 cells treated with 0,2 and 8 µmol/L XAV939 for 48 hours, the cell proliferation rate decreased, while the cell apoptosis rate increased, the expressions of apoptosis-related protein BCL-2, CyclinD1 and C-MYC were down-regulated, on the contrary, the expression of BAX and caspase-3 were up-regulated.

CONCLUSIONThe specific inhibition of β-catenin can inhibit Jeko-1 cell proliferation and promote the cell apoptosis.

Apoptosis ; Caspase 3 ; Cell Line, Tumor ; Cell Proliferation ; Heterocyclic Compounds, 3-Ring ; Humans ; Lymphoma, Mantle-Cell ; RNA, Messenger ; RNA, Small Interfering ; Transfection ; beta Catenin