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.

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>

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.

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.

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>