Effects of Garcinia Acid Combined with Daunorubicin on Expression of Pregnane X Receptor in Leukemia Cell Line K562/A02 / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the expression of PXR (Pregnane X receptor) in several malignant hematological cell lines, and to investigate the reversal effect of Gambogic acid (GA) on multi-drug resistance (MDR) of K562/A02 cell line and its reversal mechanism.</p><p><b>METHODS</b>Transcription of PXR was detected by real-time PCR in several malignant hematological cell lines. The growth inhibition rate of K562/A02 in different experimental groups was assayed by MTT method, and the expression of PXR protein was measured by Western blot.</p><p><b>RESULTS</b>PXR gene transcription could be detected in several hematological malignancy cell lines, and it was significantly higher in K562/A02 cell line, compared with the other cell lines used in this experiment. Low-dose GA could enhance cell growth inhibition rate, increasing the effect of chemotherapy, which may be associated with down-regulation of PXR expression. PXR gene transcription and protein expression in GA and DNR+GA groups decreased as compared with control group and the DNR group, suggesting that low-dose GA can down-regulate PXR gene transcription and protein expression.</p><p><b>CONCLUSION</b>PXR gene transcription can be detected in several hematological malignancy cell line, which is significantly higher in K562/A02 cell line, as compared with the other cell lines used in this experiment. Low-dose GA can enhance cell growth inhibition rate, increasing the effect of chemotherapy, which may be associated with down-regulation of PXR expression.</p>

Effect of gambogic acid on proliferation of SKM-1 cells and its mechanism / 中国实验血液学杂志

The aim of this study was to explore the effect of gambogic acid (GA) on MDS SKM-1 cell proliferation, apoptosis and their possible mechanism. Cell proliferation was determined by MTT method. The apoptosis percentage and cell cycle regulation of SKM-1 cells were analyzed by flow cytometry. Morphological features were observed by light microscopy. The mRNA expression of bcl-2 and bax were detected by RT-PCR. The results showed that GA could inhibit the proliferation of SKM-1 cells in a dose- and time-dependent manner (IC50 was 0.37 µg/ml at 48 h), increase the apoptotic percentage of SKM-1 cells, and arrest cell cycle at the G0/G1. The expression of bax mRNA was up-regulated while that of bcl-2 mRNA was down-regulated in SKM-1 cells treated with GA for 48 h. It is concluded that GA can induce apoptosis, which may be related to its effect of arresting cells at phase of G0/G1 and down-regulating bcl-2/bax ratio.

Association of polymorphisms of cytosine arabinoside-metabolizing enzyme gene with therapeutic efficacy for acute myeloid leukemia / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The cytosine arabinoside (Ara-C)-based chemotherapy is the major remedial measure for acute myeloid leukemia (AML). Deoxycytidine kinase (DCK) and cytidine deaminase (CDA) are the key enzymes in the metabolism of Ara-C. Many single nucleotide polymorphisms (SNPs) and haplotypes of DCK and CDA, which contribute to susceptibility to Ara-C, have been identified in Africans and Europeans. However, there has been no report about the relation among three SNPs in DCK (rs115543896, rs72552079, and rs111454937) and two SNPs in CDA (rs2072671 and rs60369023), and their clinical response to Ara-C for a Chinese population. In this study, we aimed to investigate whether these five SNPs are associated with the therapeutic outcomes of Ara-C-based chemotherapy regimens in patients with AML.</p><p><b>METHODS</b>A total of 151 Chinese patients with AML were enrolled in our study. SNPs genotyping were performed using the MassARRAY system by means of the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) method.</p><p><b>RESULTS</b>The results illustrated that DCKrs111454937 AA genotype was more frequent in patients with higher platelet count, and A allele frequency was significantly higher in the group £40 years, lower white blood cell (WBC) count patients group and the group with platelet counts > 60'10(9)/L. Meanwhile, both DCKrs72552079 TC (OR = 1.225, 95%CI = 1.225 - 9.851, P = 0.0192) and CDArs60369023 GA (OR = 9.851, 95%CI = 1.31 - 77.93, P = 0.0263) significantly improved Ara-C-based chemotherapy response. While DCKrs11554389 AA (OR = 0.147, 95%CI = 0.027 - 0.801, P = 0.0267) was associated with the decrease of Ara-C-based chemotherapy response.</p><p><b>CONCLUSION</b>It is evident that the DCK and CDA polymorphisms might be the important markers for the AML patients' therapy outcomes in a Chinese population.</p>

Effects of advanced glycosylation end products and tetrandrine on proliferation of K562 and K562/A02 cells / 中国实验血液学杂志

This study was aimed to investigate the effect of advanced glycosylation end products (AGE) on the proliferation of K562 and K562/A02 cells, the effect of tetrandrine (Tet) on proliferation of K562 and K562/A02 cells induced by AGE, and their mechanisms. The effects of AGE on proliferation of K562 and K562/A02 cells and Tet on the proliferation of AGE-induced K562 and K562/A02 cells were assayed by CCK8 kit, the apoptosis rate and the expression of receptor of advanced glycosylation end products (RAGE) in K562 and K562/A02 cells were determined by flow cytometry, the expression of RAGE mRNA was detected by semi-quantitative RT-PCR. The results showed that AGE could promote the proliferation of K562 and K562/A02 cells in a concentration-dependent manner, the cell proliferation was enhanced with time increasing in 0 - 48 h, and was higher than control group after 72 h. AGE up-regulated the RAGE mRNA and protein expressions of K562 and K562/A02 cells in a concentration-dependent manner. Treatment of Tet combined with AGE for 48 h could inhibit the proliferation of K562 and K562/A02 cells promoted by AGE in a concentration-dependent manner, which probably by inducing cell apoptosis, however, there was no obvious effect in the up-regulating expression of RAGE mRNA and protein induced by AGE. It is concluded that AGE can promote the proliferation of K562 and K562/A02 cells, which is probably induced by up-regulating the expression of RAGE mRNA and protein. Tet can inhibit the proliferation of K562 and K562/A02 cells induced by AGE, and the mechanism may be not closely associated with changes of the up-regulating expression of RAGE mRNA and protein induced by AGE.

Reversal effect of gambogic acid on multidrug resistance of K562/A02 cell line / 中国实验血液学杂志

This study was purposed to investigate the reversal effect of gambogic acid (GA) on multidrug resistance of K562/A02 cells and its mechanism. The IC(50) (half maximal inhibitory concentration) of adriamycin (ADM) was evaluated by MTT. Cell apoptosis was detected by flow cytometry. Morphological changes of K562/A02 cells were observed by fluorescent microscopy with DAPI staining. The expressions of Survivin and P-gp were determined by Western blot. The results showed that the IC(50) of ADM on K562 and K562/A02 cell proliferation were (1.42 ± 0.07) µg/ml and (28.42 ± 1.40) µg/ml respectively. GA ≤ 0.0625 µmol/L had no inhibitory effect on proliferation of K562 and K562/A02. 0.0625 µmol/L GA could enhance the sensitivity of K562/A02 cells to ADM (P < 0.05) and the reversal multiples was 1.53. The apoptotic rate was raised after treating with ADM combined with 0.0625 µmol/L GA for 48 h (P < 0.05). Morphological differences were typical and obvious between cells of control and treated groups under fluorescence microscopy using DAPI staining. After treating K562/A02 cells with ADM combined with 0.0625 µmol/L GA for 48 h, the expressions of Survivin and P-gp were down-regulated at protein levels. It is concluded that GA can enhance the sensitivity of K562/A02 cells to ADM, which may be related to increasing cell apoptosis and down-regulating expressions of Survivin and P-gp.

Inducing apoptosis and reversal effect of nilotinib in combination with tetrandrine on multidrug resistance of K562/A02 cell line / 中国实验血液学杂志

This study was aimed to investigate the relevance of nilotinib in combination with tetrandrine (Tet) on reversing multidrug resistance and inducing apoptosis of K562/A02 cell line and its mechanism. Methyl-thiazol tetrazolium (MTT) assay was employed to examine the pharmacological effect of nilotinib or Tet alone on K562/A02 cell line, the IC(50) of daunorubicin (DNR) on K562/A02 cell line treated with nilotinib and Tet was calculated; the flow cytometry (FCM) was employed to detect the apoptosis rate of K562/A02. The expression of bax/survivin mRNA was determined by RT-PCR, and the expression of bax/survivin protein was assayed by Western blot. The results showed that after being treated by 5 nmol/L nilotinib or 1.0 µml/L Tet for 48 hours, IC(50) of DNR to K562/A02 was 5.71 ± 0.72 mg/L or 6.52 ± 0.43 mg/L, respectively, while in their combined treatment, IC(50) decreased to 3.12 ± 0.13 mg/L. Nilotinib or Tet alone could increase DNR-inducing apoptosis rate of K562/A02 cell, while the apoptosis rate of K562/A02 increased remarkably in combination treatment of nilotinib with Tet. After being treated with 5 nmol/L nilotinib or 1.0 µml/L Tet alone for 48 hours, the expressions of bax mRNA and BAX protein was up-regulated, while both effects were more obvious in combination treatment of nilotinib with Tet. Treatment with 5 nmol/L nilotinib or 1.0 µmol/L Tet alone for 48 hours down-regulated the expression of survivin mRNA and its protein, while treatment of nilotinib in combination with Tet had more significant effect on down-regulation of their expression. It is concluded that the K562/A02 cells are resistant to DNR, nilotinib or Tet alone both can partially reverse resistance of K562/A02 cells to DNR, increase the apoptosis rate of K562/A02 cells. Combination of nilotinib with Tet shows obvious synergistic action, mechanism of which may associate with up-regulation of bax mRNA and BAX protein expressions and down-regulation of survivin mRNA and its protein expressions.

Effect of desferrioxamine on K562/A02 cell line and its mechanism / 中国实验血液学杂志

Iron is an essential element for cell growing including tumor cells. This study was purposed to explore the effect of desferrioxamine (DFO) on cell line K562/A02 and its mechanism. K562/A02 cells were cultured with different concentrations of DFO. The inhibitory effects of adriamycin (ADM) used alone or combined with DFO on the proliferation of K562/A02 was evaluated by MTT assay. The apoptosis rate of K562/A02 cells after treatment with 0, 12.5, 25 and 50 µmol/L DFO alone or in combination with 1 mg/L ADM were analyzed by flow cytometry. ADM accumulation in K562/A02 cells after treatment with different concentrations of 0, 12.5, 25 and 50 µmol/L DFO were also analyzed by flow cytometry. The levels of BAX/BCL-2 and MDR1 mRNA were determined by RT-PCR, and then the protein level of P-glycoprotein (P-gp) was detected by Western blot. The results showed that the IC(50) of ADM for K562 and K562/A02 cells were (1.46 ± 0.07) mg/L and (40.98 ± 3.05) mg/L respectively. The resistance of K562/A02 cells to ADM was 28.06 times as that of K562 cells. After treatment of K562/A02 cell with DFO of 12.5, 25 and 50 µmol/L for 48 hours, the resistance of K562/A02 cells to ADM were increased by 24.95, 16.11 and 9.99 times respectively. When K562/A02 cells were incubated with different concentrations of DFO of 12.5, 25, 50 µmol/L for 48 hours, the apoptosis rat were (3.50 ± 0.30)%, (7.27 ± 0.32)% and (12.53 ± 1.21)% respectively. After co-culture with DFO and ADM for 48 hours, apoptosis rate were (6.13 ± 0.29)%, (9.57 ± 0.40)% and (18.97 ± 1.10)% respectively. The above apoptosis rates was much higher than that of control group (p < 0.05) and they were dose-dependent. In comparison between DFO + ADM group and DFO group, there was no significant difference (p > 0.05). Expression rate of BAX/BCL-2 increased. The levels of MDR1 mRNA reduced. Furthermore, expression of P-gp also decreased in K562/A02 cells. It is concluded that iron increase can promote K562/A02 cells growth and inhibit their apoptosis. Otherwise, iron-deprivation can induce K562/A02 cells apoptosis. DFO disturbs the iron metabolism and inhibits DNA synthesis of K562/A02 cells. This action of DFO may enhance the susceptibility of K562/A02 cells to apoptosis induced by chemotherapeutic drugs. The iron-deprivation may play a role in the treatment of leukemia with combination of DFO with other anticancer agents.

Detection of single nucleotide polymorphisms of mthfr and dpyd genes in leukemia cell lines K562 and K562/A02 / 中国实验血液学杂志

This study was purposed to detect single nucleotide polymorphisms (SNP) of 2 pharmacokinetics-related genes in K562 and K562/A02 cell lines. Leukemia cell line K562 and its resistant line K562/A02 were cultured, the genomic DNA was isolated by QIAamp DNA Blood Mini kit, primers were designed, the related DNA fragments were amplified by PCR. The SNP genotyping of mthfr gene rs1801131, rs1801133 and rs2274976 and dpyd gene rs1801159, rs1801160 and rs17376848 was performed by means of matrix assisted laser desorption ionization-time of flight mass spectrometry method (MALDI-TOFMS). The results showed that the genotype of mthfr gene locus 1801131 was AC, rs1801133 was CC, rs2274976 was GG, genotype of dpyd gene locus 1801159 was GG, rs1801160 was GG, rs17376848 was AA in both K562 and K562/A02 cell lines. It is concluded that the above-mentioned loci of mthfr and dpyd genes in K562 and K562/A02 cell lines are not expressed differently.

Gene expression in patients with myelodysplastic syndrome / 中国实验血液学杂志

This study was aimed to investigate the expression of c-FLIPL, c-FLIPS and DLK1 mRNA in the patients with myelodysplastic syndrome (MDS) and its clinical significance. The mRNA expression of c-FLIPL, c-FLIPS and DLK1 in bone marrow mononuclear cells (BMMNC) of 16 patients with MDS and 3 controls were detected by RT-PCR. The results indicated that the expression of DLK1 mRNA was up-regulated in MDS, including RA and RAEB, as compared with controls (p < 0.05). There was no significant difference in expression of DLK1 between RA and RAEB patients (p > 0.05); the expression of c-FLIPL mRNA both in RA and RAEB patients was higher than that in controls (p < 0.05). There was no significant difference in expression of c-FLIPL between RA and RAEB patients (p > 0.05); the expression of c-FLIPS mRNA was not significantly different between MDS patients and controls (p > 0.05), but its expression in RAEB patients was significantly higher as compared with RA patients and controls (p < 0.05). It is concluded that the mRNA expressions of DLK1, c-FLIPL and c-FLIPS in MDS patients are abnormal, some of which may be useful as an important indicator for the evaluation of development in MDS.

Effects of imatinib and 5-bromotetrandrine on the reversal of multidrug resistance of the K562/A02 cell line / 癌症

<p><b>BACKGROUND AND OBJECTIVE</b>Research has shown that 5-bromotetrandrine (BrTet) can effectively reverse multidrug resistance (MDR). Imatinib plays an important role in cell proliferation. This study explored the efficacy of the combination of imatinib and BrTet on reversing MDR of tumor cells and its mechanism.</p><p><b>METHODS</b>Cytoxicity was assessed by MTT assay. Apoptosis of K562/A02 cells was analyzed by flow cytometry. The expressions of mdr1 mRNA and P-glycoprotein (P-gp) were detected using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis.</p><p><b>RESULTS</b>After 48 h of treatment with 0.0625 micromol/L imatinib, 0.5 micromol/L BrTet, or both, the 50% inhibition concentration (IC50) of daunorubicin (DNR) for the K562/A02 cells were 5.69 mg/L, 5.41 mg/L, and 2.19 mg/L, respectively. The gray-scale values of mdr1 mRNA expression in the K562/A02 cells were 0.65+/-0.02, 0.64+/-0.01, and 0.25+/-0.03, respectively. The expression levels of P-gp were 0.74+/-0.02, 0.52+/-0.02, and 0.29+/-0.02, respectively. All decreased significantly in the K562/A02 cells treated with both imatinib and BrTet compared to cells treated with imatinib and BrTet alone (P<0.05). The apoptosis rates of the K562/A02 cells increased without a significant difference after treatment with DNR, imatinib, or BrTet (P>0.05), while increased significantly after treatment with DNR combined with imatinib, BrTet, or both (P<0.05).</p><p><b>CONCLUSIONS</b>The MDR of K562/A02 cells may be partially reversed by imatinib or BrTet, and the mechanism may be related to the downregulation of mdr1 mRNA and P-gp expression and the upregulation of the rate of apoptosis in K562/A02 cells. Imatinib combined with BrTet showed a synergistic effect on K562/A02 cells.</p>

Effect of sodium valproate on human myelodysplastic syndrome cell line SKM-1 and its mechanism / 中国实验血液学杂志

This study was aimed to investigate the effect of sodium valproate(VPA) on human myelodysplastic syndrome cell line SKM-1 and its mechanism. The cell proliferation was determined by MTT assay, cell apoptosis was analyzed by flow cytometry. The expressions of c-flipl, c-flips and dlk1 mRNA were detected by RT-PCR. The results showed that VPA could inhibited the growth of SKM-1 cells in dose- and time-dependent manners. The flow cytometric analysis indicated that VPA could induce cell apoptosis, apoptosis rate increased in dose-dependent manner. The expressions of c-flipl, c-flips and dlk1 mRNA in SKM-1 cell treated with VPA decreased using of VPA. It is concluded that VPA can induce apoptosis and inhibited proliferation of SKM-1 cells. In this process, the decreasing of c-flipl, c-flips and dlk1 mRNA expression may play important roles.

Effects of magnetic nanoparticle of Fe3O4 on apoptosis induced by Gambogic acid in U937 leukemia cells / 中国实验血液学杂志

This study was aimed to explore the potential therapy of Gambogic acid (GA) combined with magnetic nanoparticle of Fe3O4 (Fe3O4-MNP) on leukemia. The proliferation of U937 cells and the cytotoxicity were evaluated by MTT assay. Cell apoptosis was observed and analyzed by microscopy and flow cytometry respectively. The expressions of gene and protein were detected by quantitative real-time polymerase chain reaction and Western blot respectively. The results showed that GA enhanced the cytotoxicity for U937 cells in dose- and time-dependent manners. The Fe3O4-MNP itself had not cytotoxicity, but could enhance the inhibitory effect of GA on proliferation of U937 cells. The apoptotic rate of U937 cells induced by combination of GA with Fe3O4-MNP was higher than that by GA alone. The typical apoptotic features of cells treated with GA and Fe3O4-MNP were observed. The expression levels of caspase-3 and bax after co-treatment of GA and Fe3O4-MNP were higher than that exposed to GA or Fe3O4-MNP alone, but the expressions of bcl-2, NF-kappaB and survivin were down-regulated. It is concluded that Fe3O4-MNP can promote GA-induced apoptosis in U937 cells, and the combination of GA with Fe3O4-MNP may be a safer and less toxic new therapy for leukemia.

Effects of hypoxia-inducible factor inhibitor on expression of HIF-1alpha and VEGF and induction of apoptosis in leukemic cell lines / 中国实验血液学杂志

This study was purposed to investigate the effect of a hypoxia-inducible factor inhibitor (YC-1) on expression of hypoxia-inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) as well as induction of apoptosis in leukemic cell lines. RT-PCR was used to determine the levels of HIF-1alpha mRNA and VEGF mRNA in K562, U937 and Jurkat cells. After treatment of U937 cell with 4 micromol/L YC-1, cell apoptosis was assayed by DAPI staining under fluorescent microscope and flow cytometry with Annexin V-FITC/PI staining; the expression levels of HIF-1alpha mRNA and VEGF mRNA were measured with RT-PCR; the expression levels of HIF-1alpha, VEGF, BAX, BCL-2 and caspase-3 proteins were measured by Western blot. The results showed that HIF-1alpha mRNA and VEGF mRNA were expressed in all three leukemia cell lines. After treatment of U937 cell with 4 micromol/L YC-1 for 0, 8, 16 and 24 hours, the changes of morphologic features of U937 cells could be observed under fluorescent microscope and the apoptotic rates significantly increased in time-dependent manner, they were (4.87 +/- 0.70)%, (27.27 +/- 2.00)%, (51.53 +/- 2.81) and (60.5 +/- 3.20)% respectively, the expression levels of VEGF mRNA reduced, while the expression levels of HIF-1alpha mRNA had no obviously changes.Furthermore, the expression of HIF-1alpha, VEGF and BCL-2 decreased, while the expression of BAX and caspase-3 increased, the ratio of BAX/BCL-2 increased in time-dependent manner (r = 0.973, p < 0.01). It is concluded that HIF-1alpha mRNA and VEGF mRNA are all expressed in in K562, U937 and Jurkat cells, YC-1 has significant effect on down-regulating the protein expression of HIF-1alpha and VEGF, and induces the apoptosis in U937. The mechanism of apoptosis in leukemic cells may involve in up-regulating BAX/BCL-2 ratio and expression of protein caspase-3.

Effect of PDMP, a glucosylceramide synthase inhibitor, on reversion of daunorubicin resistance in human leukemia cell line K562/A02 / 中国实验血液学杂志

This study was purposed to investigate the reversal effect of glucosylceramide synthase (GCS) inhibitor D, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) hydrochloride, on multidrug resistance in K562/A02 cells and its mechanism. The IC(50) (the half maximal inhibitory concentration) of PDMP was measured by MTT method. Cell apoptosis and intracellular daunorubicin (DNR) concentration were detected by flow cytometry. The expression of GCS and mdr1 genes were assayed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot. The results showed that the IC(50) of DNR in K562 and K562/A02 cells were 0.23 +/- 0.02 and 7.15 +/- 0.24 microg/ml respectively. When the concentration of PDMP was equal to or less than 20 micromol/L ( < / = 20 micromol/L), the obviously inhibitory effect on proliferation of K562 and K562/A02 cells was not observed, but both 20 micromol/L and 10 micromol/L PDMP could enhance the sensitivity of K562/A02 cells to DNR (p < 0.01) and the reversal multiples were 2.59 and 1.69 respectively. After treating with 20 micromol/L and 10 micromol/L PDMP for 48 hours, the concentration of DNR in K562/A02 cells increased (p < 0.05) and the apoptotic rate also was elevated (p < 0.01). The expressions of GCS and mdr1 genes were down-regulated at mRNA and protein levels after treating K562/A02 cells with 20 micromol/L PDMP for 48 hours. It is concluded that PDMP can enhance the sensitivity of K562/A02 cells to DNR by increasing cell apoptosis rate and accumulation concentration of DNR in cells, which may be related to down-regulated expressions of GCS and mdr1 genes.

Reversal of multidrug-resistance in human leukemia cell line K562/A02 by tyrosine kinase inhibitors / 中国实验血液学杂志

This study was aimed to investigate the reversal effect of tyrosine kinase inhibitors (TKI) Imatinib and Nilotinib on multidrug-resistant cell line K562/A02. The expression levels of mdr-1 mRNA and bcr-abl mRNA were assayed by RT-PCR. The protein levels of P-glycoprotein (P-gp) and P210 were detected by Western blot. The daunorubicin (DNR) accumulation in K562/A02 cells were analyzed by flow cytometry (FCM). The results showed that the 0.0625 micromol/L Imatinib or 5 nmol/L Nilotinib alone had no cytotoxic effect on the inhibition of K562/A02 cells. When K562/A02 cells were treated with Imatinib or Nilotinib alone for 48 hours, the expressions of mdr-1 mRNA, der/abl mRNA, P-gp and P210 protein were all down-regulated, furthermore the effect of Nilotinib was stronger than that of Imatinib. The detection of fluorescence intensity revealed that the DNR concentration in K562/A02 cells treated with Imatinib or Nilotinib alone for 48 hours were 7.85% and 12.02% of K562 cells respectively. It is concluded that the tyrosine kinase inhibitors show great effect reversing drug resistance of cells, moreover, the effect of Nilotinib is stronger than that of Imatinib.

Construction and identification of Mdr-1-shRNA eukaryotic expression vector / 中国实验血液学杂志

This study was purposed to construct and identify the short hairpin RNA (shRNA) eukaryotic expression vector for targeting gene mdr-1 which may play an important role in K562/A02. Short hairpin RNA (shRNA) aiming at the target sequence was to synthesized, the 3491-3509, 1539-1557and 3103-3121 nucleotide of mdr-1 mRNA were selected as targets. The selected nucleotides were cloned in the plasmid pGCSilencer-U6-neo-GFP respectively, and the resultant recombinant plasmids were named as pGY1-1, pGY1-2 and pGY1-3. The sequences of the recombinant plasmids were identified by DNA sequencing and PCR electrophoresis. The recombinant plasmids were transfected into the cell line K562/A02 by lipofection. After being transfected for 48 hours, the inhibition of mdr-1 mRNA was detected by real time-PCR, and P-gp expression was detected by Western blot. The results showed that the specific oligonucleotide was cloned into the vector successfully, and the expression of mdr-1 mRNA and P-gp in K562/A02 cells was reduced after transfecting the recombinant plasmid, as compared to the control group. It is concluded that the shRNA eukaryotic expression vector has been successfully established which can inhibit the expression of mdr-1 mRNA, setting up the basis to futher explore the effects of mdr-1 on cell line of K562/A02.

Influence of magnetic Fe3O4 nanoparticle on functions of lymphocytes and macrophages in mice / 中国实验血液学杂志

This study was purposed to investigate the effects of magnetic nanoparticle of Fe3O4 (Fe3O4-MNPs) on murine immune system. ICR mice were assigned randomly into four groups which were treated with normal saline, low, middle and high dose of MNP-Fe3O4 respectively. The mice were killed after being exposed by intragastric administration for 2 weeks. The ratios of spleen weight to body weight, lymphocyte transformation rate in spleen suspension and phagocytic index of macrophage in abdominal cavity were detected. The results showed that the ratios of spleen weight to body weight in Fe3O4-MNP groups were not significantly different in comparison with the control (p > 0.05). The lymphocyte transformation rate in spleen suspension in Fe3O4-MNP groups were all higher than that in control group (-0.1775 +/- 0.0246), especially in the middle dose group (0.1833 +/- 0.0593) (p < 0.05), and the phagocytic index of macrophages in abdominal cavity of middle dose group (0.2051 +/- 0.0213) was higher than that of control group and other two Fe3O4-MNP group (low dose 0.1538 +/- 0.0100, high dose 0.1511 +/- 0.0184) (p < 0.05). It is concluded that suitable dose of Fe3O4-MNP can enhance the cellular immune activity and phagocytic function of macrophages of mice.

Study on reversal effect of nilotinib in combination with 5-BrTet on multidrug resistance of K562/A02 cell line / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the reversible effect of nilotinib, BrTet (5-bromotetrandrine) and their combination on multidrug resistance cell line K562/A02 and its mechanism.</p><p><b>METHODS</b>Cell proliferation inhibition was assessed by MTT method and cell apoptosis by flow cytometry (FCM). The expression of mdr1 mRNA was determined by RT-PCR, and the expression of P-gp was assessed by Western blot.</p><p><b>RESULTS</b>After 48 h 5 nmol/L nilotinib or 0.5 µmol/L BrTet treatment, IC(50) of daunorubicin (DNR) to K562/A02 was 4.52 mg/L or 5.41 mg/L respectively; While on combinative treatment, its IC(50) decreased to 2.98 mg/L. Nilotinib or BrTet alone was not able to increase the DNR induced apoptosis rate of K562/A02 cell (P > 0.05), while on combination treatment the apoptosis rate increased remarkably. After 48 h 5 nmol/L nilotinib or 0.5 µmol/L BrTet treatment alone, gray-scale value of mdr1 mRNA was 0.48 ± 0.04 or 0.64 ± 0.01, respectively; while on combinative treatment the value decreased to 0.35 ± 0.04. The P-gp expression level in K562/A02 cells was 0.61 ± 0.05, or 0.52 ± 0.02 when treated with 5 nmol/L nilotinib or 0.5 µmol/L BrTet alone for 48 h, but on combination treatment, the level decreased to 0.44 ± 0.03.</p><p><b>CONCLUSION</b>Nilotinib or BrTet alone can partially reverse drug resistance of K562/A02 cells. The mechanism may be associated with the decrease of mdr1 mRNA and P-gp expression and increase of the apoptosis rate. And there is a synergistic action with these two agants in combination.</p>

Effect of hypoxia inducible factor1-α inhibitor on reversal of multidrug resistance of K562/A02 cell line / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the reversal effect of the hypoxia inducible factor (HIF)-1α inhibitor, YC-1, on multidrug resistance of K562/A02 cells and its mechanism.</p><p><b>METHODS</b>Pre- and post- incubation with adriamycin (ADM) alone or in combination with YC-1 for 48 h, the proliferation capacity of K562/A02 and K562 cells were evaluated by MTT assay. The apoptosis rate of K562/A02 cells after treated with 0, 5, 10 and 20 µmol/L YC-1 alone or in combination with 1 mg/L ADM and intracellular ADM concentration were analyzed by flow cytometry (FCM). The mRNA levels of HIF-1α and mdr1 genes were determined by semi-quantitative RT-PCR. The protein levels of HIF-1α and P-glycoprotein (P-gp) were detected by Western blot.</p><p><b>RESULTS</b>The IC(50) of ADM for K562 and K562/A02 cells were (1.56 ± 0.07) mg/L and (42.98 ± 3.15) mg/L respectively. The resistance of K562/A02 cells to ADM was 27.55- fold higher of that of K562 cells. After treatment with YC-1 (5 µmol/L, 10 µmol/L, 20 µmol/L) for 48h, the resistances of K562/A02 cells to ADM were 24.63-, 16.38- and 10.71- fold increase respectively. After treatment of K562/A02 cell with YC-1 (0 µmol/L, 5 µmol/L, 10 µmol/L, 20 µmol/L) alone or in combination with 1 mg/L ADM for 48 h, the apoptotic rates were (1.9 ± 0.9)%, (4.9 ± 0.9)%, (5.8 ± 1.1)%, and (9.3 ± 1.4)% and (2.3 ± 0.7)%, (8.2 ± 1.2)%, (19.0 ± 1.7)%, and (34.5 ± 2.4)% respectively. The intracellular flucorescence intensity of ADM were 232 ± 33, 1300 ± 219, 1961 ± 240 and 3342 ± 269 in the combined treatment group. With the increase in YC-1 concentration, the levels of mdr1 mRNA reduced, while that of HIF-1α mRNA had no obvious change. Furthermore, the expressions of HIF-1α and P-gp were also decreased in K562/A02 cells.</p><p><b>CONCLUSION</b>YC-1, as a HIF-1 inhibitor, can reverse multidrug resistance of K562/A02 cells through down-regulating HIF-1α and P-gp.</p>

Detection of single nucleotide polymorphisms of Jak2 gene in multiple myeloma by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry / 中国实验血液学杂志

This study was purposed to investigate the practicality of matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) for detection of single nucleotide polymorphisms (SNP) on jak2 gene in multiple myeloma (MM) cells. The DNA fragment containing 2 SNPs of jak2 (C428T and C643T) was amplified using PCR and was purified. The purified product was used as the template for primer extension (PEX). The small products of allele-specific reaction were purified, the SNPs on jak2 gene of 5 patients with MM and 5 healthy persons were detected by MALDI-TOF MS. The results showed that the distribution of genotype C428T and C643T was not different between MM patients and healthy persons, both of which are homozygous T/T. In conclusion, the method based on MALDI-TOF MS and PEX technique for detecting SNP in jak2 gene is rapid, accurate and reliable method, and can be used in clinical practice.