Inhibitory effect of 4-chlorobenzoyl berbamine on imatinib-resistant K562 cells in vitro and in vivo / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the inhibitory effect of 4-chlorobenzoyl berbamine (BBD9) on imatinib-resistant cell line K562 (K562/IR) in vitro and in vivo and explore the mechanisms.</p><p><b>METHODS</b>The IC50 of BBD9 and berbamine (BBM) was determined by MTT assay. The expressions of p210(Bcr-Abl), IKKa, cytoplasmic and nuclear NF-κBp65 were determined using Western blotting in K562/IR cells following a 48-h exposure to 0.5 µg/ml BBD9 or 8 µg/ml BBM. Flow cytometry was used to analyze the cell viability, apoptosis and necrosis; Western blotting was employed to determine the expressions of PARP, caspase-3, caspase-9 and LC3II in K562/IR cells exposed to different concentrations of BBD9 for 48 h. In nude mouse models bearing K562/IR cell xenograft, the tumor weight, tumor regression, and body weight changes of the mice were measured after treatments with 15 mg/kg and 30 mg/kg BBD9 and 100 mg/kg imatinib.</p><p><b>RESULTS</b>The IC50 of BBD9 and BBM was 0.73 µg/ml and 5.43 µg/ml, respectively. In K562/IR cell cultures, the expressions of p210(Bcr-Abl), IKKa and nuclear NF-κB p65 were all decreased following BBD9 and BBM treatments, but BBD9 produced more potent effect; cytoplasmic NF-κB p65 showed no obvious changes after the treatments. The cell apoptosis and necrosis increased with the concentrations of BBD9, which also dose-dependently increased the levels of cleaved caspase-3, csapase-9, PARP, and LC3II expression. In the tumor-bearing mouse model, BBD9 showed stronger effects than imatinib in reducing the tumor weight, promoting tumor regression, and increasing the body weight.</p><p><b>CONCLUSION</b>BBD9 can effectively inhibit the growth of K562/IR cells in vitro and in vivo by activating cell apoptosis, necrosis and autophage pathways, down-regulating expressions of p210(Bcr-Abl) and IKKa and suppressing the cytoplasm-to- nucleus translocation of NF-κBp65.</p>

Berbamine induces apoptosis of multiple myeloma RPMI 8226 cells by activating GADD45/JNK pathway / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effect of berbamine (BBM) on multiple myeloma (MM) cell line RPMI 8226 and its mechanism.</p><p><b>METHODS</b>MTT bioassay was used to examine the effect of berbamine on cell growth and IC(50) was calculated. Apoptosis was observed by flow cytometry (FCM) and DNA gelose electrophoresis. p53, p21, GADD45 mRNA were measured by RT-PCR. The alterations in p53, J NK, p-JNK and c-Jun proteins were detected by Western blot method.</p><p><b>RESULT</b>The growth of RPMI 8226 cells was suppressed in a dose-dependent manner after treatment with BBM(P<0.05), and its IC(50) value was 3.83 microg/ml at 48 h. Both DNA ladder and FCM results showed that BBM induced apoptosis of RPMI 8226 cells with concomitant increase of activated p53, p21 and GADD45gamma mRNA. After treatment with BBM at 8 microg/ml for 24 h, the percentage of apoptotic cells increased from 1.07% to 24.84%. p-JNK and c-Jun proteins were activated.</p><p><b>CONCLUSION</b>BBM can inhibit the growth of RPMI 8226 cells, which is associated with activation of GADD45/JNK signaling pathway and induction of cell apoptosis.</p>

Berbamine induces apoptosis in human hepatoma cell line SMMC7721 by loss in mitochondrial transmembrane potential and caspase activation / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To investigate the effect of berbamine on human hepatoma cell line SMMC7721.</p><p><b>METHODS</b>The effects of 24 h and 48 h incubation with different concentrations (0 to approximately 64 microg/ml) of the berbamine on SMMC7721 cells were evaluated using 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay. Hoechst 33258 staining was conducted to distinguish the apoptotic cell, and the appearance of sub-G1 stage was determined by PI (propidium iodide) staining, the percentage of apoptotic cell was determined by flow cytometry following annexin V/PI staining. Flow cytometry was performed to analyze the cell cycle distribution and the mitochondrial membrane potential (psi(m)); the expression of activated caspase3 and caspase9 was analyzed by Western-blot.</p><p><b>RESULTS</b>The proliferation of SMMC7721 was decreased after treatment with berbamine in a dose- and time-dependent manner. Berbamine could induce apoptosis in SMMC7721 cells and could cause cell cycle arrest in G0/G1 phase, to induce loss of mitochondrial membrane potential (psi(m)) and activate caspase3 and caspase9. Berbamine-induced apoptosis could be blocked by the broad caspase inhibitor z-VAD-fmk.</p><p><b>CONCLUSION</b>Berbamine exerts antiproliferative effects on human hepatocellular carcinoma SMMC7721 cells. The anticancer activity of berbamine could be attributed partly to its inhibition of cell proliferation and induction of apoptosis in cancer cells through loss in mitochondrial transmembrane potential and caspase activation.</p>

Berbamine selectively induces apoptosis of human acute promyelocytic leukemia cells via survivin-mediated pathway / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Currently, resistance and relapse are still major problems in acute promyelocytic leukemia (APL) cases. Thus, new agents that override the resistance are crucial to the development of curative therapies for APL. In this study, we investigated the effects of berbamine on the proliferation of APL cell line NB4 and its possible mechanisms.</p><p><b>METHODS</b>NB4 cells were treated with berbamine at different concentrations (0-64 microg/ml) for 72 hours. MTT assay was used to determine proliferation inhibition of NB4 cells. Cell apoptosis was evaluated by both flow cytometry (FCM) and morphological examination. PML/RAR-alpha and survivin mRNAs were measured by nested-RT-PCR and RT-PCR, respectively. Activated-caspase 3 was determined by FCM.</p><p><b>RESULTS</b>Berbamine greatly inhibited the proliferation of NB4 cells in dose- and time-dependent manners, and its IC50 value was 3.86 microg/ml at 48 hours. Both morphological observations and FCM results showed that berbamine induced apoptosis of NB4 cells with concomitant increase of activated caspase-3 and decrease of survivin mRNA. After treatment with berbamine at 8 microg/ml for 48 hours, the percentage of apoptotic cells increased from 2.83% to 58.44% (P<0.01), and the percentage of cells with activated-caspase 3 elevated from 2.06% to 70.89% (P<0.01), whereas, level of survivin mRNA was reduced to 38.24% of control (P<0.01). However, no significant change was observed in PML/RAR-alpha mRNA.</p><p><b>CONCLUSIONS</b>Berbamine induces caspase-3-dependent apoptosis of leukemia NB4 cells via survivin-mediated pathway, suggesting that berbamine may be a novel potential agent against APL with a mechanism distinct from that of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO).</p>

Effects of berbamine on growth of leukemia cell line NB4 and its mechanism / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To determine effects of berbamine on the growth of leukemia cell line NB4 and explore its possible mechanisms.</p><p><b>METHODS</b>The growth of NB4 cells was examined with MTT assay. Morphological analysis and DNA agarose electrophoresis were used to detect apoptosis in NB4 cells, and the apoptosis rate was measured by flow cytometry. The PML/RAR alpha mRNA was determined by nested-PCR, and the Survivin mRNA was tested by RT-PCR. The expression of caspase 3 protein in NB4 cells was evaluated by flow cytometry.</p><p><b>RESULT</b>The growth of NB4 cells was inhibited significantly after treated with berbamine at different concentrations for different time points, the IC(50)value was 3.860 microg/ml at 48 hours. Morphology analysis showed the characteristics of apoptosis, and the DNA agarose electrophoresis showed the typical DNA ladder. The apoptosis rate increased from 2.83% to 58.44% after treated with berbamine at 12 microg/ml for 48 hours. The expression of PML/RAR alpha mRNA presented no significant changes, however, Survivin mRNA was decreased dramatically. The protein expression of Caspase 3 increased significantly from 2.06% to 70.89% after treated with berberine at a concentration of 12 mug/ml for 48 hours.</p><p><b>CONCLUSION</b>Berbamine could inhibit the growth of leukemia cell line NB4. The induction of cell apoptosis may be one of the mechanisms for suppressing the growth of leukemia cell line NB4. Inhibition of Survivin mRNA and upregulation of Caspase 3 protein might be also involved in cell apoptosis.</p>

Molecular cloning of a novel retroviral NP9 gene of human endogenous retrovirus and its viral protein expression / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the relationship between retroviruses and autoimmune diseases, to clone the novel retroviral NP9 gene from human endogenous retrovirus (HERV), and to construct its expression vector.</p><p><b>METHODS</b>The viral NP9 gene was amplified and cloned by RT-PCR and T-A clone techniques, and its sequence was determined with Perkin-Elmer 377 DNA Sequencer. The amplified viral NP9 gene was subcloned into the prokaryotic express vector pQE30. The recombinant plasmids were identified by restriction endonuclease digestion and sequencing. The recombinant pQE30-NP9 protein was expressed in M15 host cells under the IPTG induction and showed with SDS-PAGE,and the corresponding NP9 viral protein was identified with Western blot analysis.</p><p><b>RESULT</b>A specific band of 250 bp was amplified using RT-PCR from total RNA of peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) and confirmed as the NP9 gene via T-A clone and DNA sequencing analyses. SDS-PAGE profile showed a clear protein band with a relative molecular weight 9 kD in the IPTG-induced samples, which was confirmed as viral NP9 protein by Western blot analysis.</p><p><b>CONCLUSION</b>The NP9 gene has been successfully isolated and cloned from PBMCs of SLE patients and the corresponding NP9 viral protein expressed in prokaryotic expression vector.</p>

Cytotoxicity and apoptosis of human osteosarcoma U2OS cells induced by recombinant soluble AZURIN / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effects of recombinant AZURIN protein of P. aeruginosa on growth and apoptosis of U2OS cells.</p><p><b>METHODS</b>The AZURIN gene was amplified from the genome of P.aeruginosa by PCR, and cloned into prokaryotic expression vector pQE30. The soluble AZURIN protein was expressed in E. coli cells M15, then purified and refolded. After treatment of AZURIN, the cell cycle, proliferation and apoptosis were determined by morphological observation, MTT assay, flow cytometry(FCM) and DNA fragmentation assay. Mitochondrial membrane potential(DeltaPsim) was measured by FCM.</p><p><b>RESULTS</b>The purity of recombinant protein AZURIN reached to 99.1%. Proliferation of U2OS cells were significantly inhibited 12 h after AZURIN (100-200 mg/L) treatment. Apoptosis peak and DNA ladder were observed. Mitochondrial membrane potential decreased gradually from 12 h to 72 h after AZURIN treatment.</p><p><b>CONCLUSION</b>The recombinant AZURIN inhibit the growth of the human osteosarcoma U2OS cells and inducs apoptosis in vitroìwhich may be associated with the decrease of mitochondrial membrane potential.</p>

Expression of a novel human retroviral NP9 gene and potential roles of its protein in systemic lupus erythematosus patients / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the expression of a novel retroviral (NP9) gene transcripts and the possible role of its protein in systemic lupus erythematosus (SLE) patients.</p><p><b>METHODS</b>The retroviral NP9 gene in SLE patients was isolated and cloned using RT-PCR and TA cloning techniques, and it was analyzed by sequencing. The expression of the NP9 genes in 40 patients with SLE and 48 normal controls using RT-PCR was detected. NCBI BLAST and DNASIS 3.1 software were used to analyze the features of protein of NP9 gene.</p><p><b>RESULTS</b>The positive ratio (77.5%) of the mRNA expression of the retroviral NP9 gene in SLE patients is significantly higher than that (8.3%) in normal subjects (P<0.01). The recombinant NP9 protein comprises 74 AA with pI 9.59. Amino acid sequence analysis indicates that the retroviral NP9 protein shares higher homologies with several human proteins with important biological functions.</p><p><b>CONCLUSION</b>SLE patients possess specific novel retroviral NP9 transcripts. The expression of the retroviral NP9 gene may involve in the genesis or development of SLE.</p>

Study on effect of berbamine on multidrug resistance leukemia K562/Adr cells / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the effect and mechanism of berbamine on the apoptosis of multidrug resistant leukemia K562/Adr cells and in reversing the drug resistance.</p><p><b>METHODS</b>IC50 value of K562/Adr cell was determined with MTT method, cell apoptosis rate was analyzed by flow cytometry with Annexin V FITC-PI assay, with the peak and cell cycle detected by PI staining. At the same time, flow cytometry was also used in determining Caspase-3, P-GP protein expression and drug accumulating capacity in cells, and RT-PCR method was used to analyze the gene expression of mdr-1.</p><p><b>RESULTS</b>Berbamine could inhibit human leukemia K562/Adr cell growth in dose-dependent manner, it could also induce cell apoptosis, increase the protein expression of Caspase-3 and the drug excretion capacity of cells, reduce the mRNA and protein expression levels of mdr-1 gene.</p><p><b>CONCLUSION</b>Berbamine could activate Caspase-3 to induce human leukemia K562/Adr cell apoptosis, and by reducing mdr-1 gene expression to reverse its multidrug resistance.</p>

Overexpression of S3 ribosomal protein gene is involved in drug resistance in K562/DOX cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of overexpression of S3 ribosomal protein (S3rp) gene on the resistance of leukemia cell to antitumor drugs.</p><p><b>METHODS</b>Both sense and antisense cDNA recombinants of S3rp gene were constructed with pcDNA3.1 expression vector. Subsequently, the sense S3rp cDNA recombinant was transfected into K562 cells while the antisense one into K562/DOX cells (a multidrug resistant cell line). In addition, empty pcDNA3.1 vector was transfected into the corresponding cells as negative controls. The chemosensitivity of cells was evaluated by MTT assay.</p><p><b>RESULTS</b>Sense S3rp cDNA transfected K562 cells were 5.8 times more resistant to doxorubicin than control cells did, whereas antisense S3rp cDNA transfected K562/DOX cells were 3.2 times less resistant to doxorubicin than control cells did.</p><p><b>CONCLUSION</b>Overexpression of S3rp gene plays an important role in the development of drug resistance in K562/DOX cells.</p>

Study of functional L1 retrotransposon in human type 2 diabetes susceptibility loci / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the susceptibility gene of type 2 diabetes mellitus (T2DM) through a novel strategy.</p><p><b>METHODS</b>Firstly, the common feature of the putative susceptibility genes in the reported susceptibility loci was searched by using NCBI BLAST, and a functional L1 retrotransposon in the loci was found. Secondly, the mRNA expression level of the functional L1 retrotransposon in 25 Han T2DM patients and 22 normal controls was investigated by reverse transcription-polymerase chain reaction, and statistical analysis was implemented in statistical package SPSS10.0. Thirdly, L1 retrotransponson genome mutation screening was performed via sequencing.</p><p><b>RESULTS</b>Screening the human genome for the retrotransposon genome via alignment with the L1 genome using NCBI BLAST showed the functional L1 retrotransposons distribute on most chromosomes except for chromosomes 19, 21 and Y on which rare type 2 diabetes susceptibility loci were reported to reside, and their distribution sites are consistent with the locations of the reported candidate type 2 diabetes susceptibility loci. The mRNA expression level of the functional L1 retrotransposon in the T2DM patients was significantly lower than that in normal subjects (P<0.001). Nonsense mutations including deletion and/or point mutations were observed in all of the 6 T2DM patients tested, but no mutation was observed in all of the 4 normal controls tested.</p><p><b>CONCLUSION</b>The functional L1 retrotransposon may be a candidate susceptibility gene of type 2 diabetes or a key regulator of the susceptibility genes, and it may be an ideal candidate biomarker for screening type 2 diabetes.</p>

Baicalein selectively induce apoptosis in human leukemia K562 cells / 药学学报

<p><b>AIM</b>To study the antitumor effect of baicalein on human leukemia K562 cell and its mechanism.</p><p><b>METHODS</b>The IC50 value and cytotoxity of K562 cell were detected by MTT method. The apoptotic cell was analyzed by FCM using Annexin V FITC--PI staining method. Sub-G1 peak was also measured by FCM. Protein expressions of Bcl-2, Fas, Caspase 3 were evaluated with FCM.</p><p><b>RESULTS</b>Baicalein was shown to significantly inhibit the proliferation of K562 cell in a dose-dependent manner and selectively induce apoptosis of human leukemia K562 cells. Flow cytometric analysis showed that baicalein arrested K562 cells in the S phase. In addition, protein expression of Fas, Caspase 3 of K562 cells increased after exposure to baicalein, but Bcl-2 was unchanged.</p><p><b>CONCLUSION</b>Baicalein can selectively induce apoptosis of human leukemia K562 cell dose and time dependently through up-regulation of caspase-3 and fas gene expression level.</p>

Identification of multidrug resistance related genes in leukemia by suppression subtractive hybridization / 中华血液学杂志

<p><b>OBJECTIVE</b>To clone and screen genes related to multidrug resistance (MDR) in leukemia.</p><p><b>METHODS</b>Suppression subtractive hybridization (SSH) was performed to profile differentially expressed genes between a MDR leukemia cell line (K562/DOX, as tester) and its parent cell line (K562, as driver). Reverse Northern dot blot was carried out to further screen the subtracted cDNA library. The overexpressed cDNA fragments in K562/DOX cells were sequenced and compared with known genes in Genbank. RT-PCR and Northern blot were employed to confirm the differential expression of some identified genes.</p><p><b>RESULTS</b>Eleven genes were identified being overexpressed in K562/DOX, including S3 ribosomal protein (S3rp) gene, NADH dehydrogenase subunit 2 (ND2) gene and My023 gene, which have not been reported to be related to MDR in cancer.</p><p><b>CONCLUSION</b>Several genes, which might be involved in MDR were identified, indicating novel mechanisms of MDR in leukemia.</p>