OBJECTIVETo investigate the effect of tyrosine kinase inhibitor ZD6474 (Vandetanib) on the proliferative inhibition of K562 cells and its derived imatinib-resistant K562/G cells and its mechanism.

METHODSImatinib-resistant K562/G cells were obtained by culturing cells in gradually increasing concentrations of imatinib. The changed factors related to drug-resistance were tested by Western blot. ZD6474 and imatinib affected K562/G and parental K562 cells proliferation were analyzed by WST assay. Flow cytometry was used to analyze cell cycle. Direct inhibition of Src activity by ZD6474 was measured by a colorimetric ELISA assay with recombinant human Src kinase.

RESULTS10 µmol/L imatinib failed to inhibit K562/G cells proliferation or induce cell cycle arrest. Compared with that in parental K562 cells, there were marked high levels of p-Src and Src protein in K562/G cells. The expression of Bcl-2 and p-STAT3 also increased in K562/G cells. After 48 hours incubation, the IC(50) values of ZD6474 in K562 and K562/G cells were 1.61 µmol/L and 3.18 µmol/L, respectively. ZD6474 treatment caused accumulation of cells in the G(0)/G(1) fraction and cell apoptosis in K562 and K562/G cells. ZD6474 decreased the expression of p-Src and Src at post-transcriptional level. Moreover, ZD6474 increased the ratio of Bax/Bcl-2 and decreased the expression of p-STAT3 at the same concentration for inducing apoptosis.

CONCLUSIONSZD6474 is effective in inhibiting the proliferation of imatinib-resistant K562/G cells and parental K562 cells, and induces their apoptasis by significant inhibition of Src kinase activity. Our study provides a reliable experimental basis for chronic myeloid leukemia treatment with ZD6474.

Apoptosis ; drug effects ; Benzamides ; pharmacology ; Humans ; Imatinib Mesylate ; K562 Cells ; Piperazines ; pharmacology ; Pyrimidines ; pharmacology

OBJECTIVETo investigate the growth inhibitory effect of Imatinib derivative TEB-415 on various multiple myeloma (MM) cell lines, such as U226, H929, RPMI8226, MM1R and MM1S.

METHODSTEB-415, a derivative of Imatinib was synthesized by modifying the chemical structure of Imatinib. MM cell lines (U226, H929, RPMI8226, MM1R and MM1S) were treated with TEB-415, Imatini and Bortezomib of various concentrations. Cells were grown for 72 hours and the growth rate was measured by CCK-8 method, cell morphology was observed and the IC50 was calculated.

RESULTSTEB-415 could inhibit H929 and RPMI8226 growth significantly. When the concentration of TEB-415 was <0.1 nmol/L, >50% H929 cells died. The IC50 of Imatinib was 0.123 mol/L while the IC50 of Bortezomib was 0.03 nmol/L. In RPMI8226 cell line, when the concentration of TEB-415 was 11.9 mol/L, more than 50% of cells died. In contrast, when RPMI8266 were treated with Imatinib of the concentration of 12.8 mol/L, cells grew normally.

CONCLUSIONIn comparison to Imatinib, TEB-415, a derivative of Imatinib, can kill H929 MM cells much effectively, its effecacy is only inferior to Bortezomib. RPMI8226, an MM cell line is insensitive to Imatinib, but still sensitive to TEB-415 and its growth can be inhibited by TEB-415.

Apoptosis ; Bortezomib ; Cell Line, Tumor ; drug effects ; Humans ; Imatinib Mesylate ; analogs & derivatives ; pharmacology ; Multiple Myeloma ; pathology

OBJECTIVE: To investigate the drug resistant related FOXO3/Bcl-6 signaling pathway in K562/G cell line and its related microRNA(miRNA) mechanisms. METHODS: The drug resistance potency of imatinib on K562/G was detected by MTT assay. The expression of FOXO3 and Bcl-6 proteins in K562 and K562/G cells was detected by Western blot. Real-time PCR (RT-PCR) was used to detect the expression of FOXO3 and Bcl-6 mRNA. The miRNA expression profiling in K562 and K562/G cells was analyzed by microarray technique, and the miRNA targeted to FOXO/Bcl-6 signaling pathway was identified. RESULTS: The expression of FOXO3 and Bcl-6 protein was significantly increased in K562/G cells as compared with that in K562 cells (P<0.01), the expression level of Bcl-6 mRNA showed no increase in K562/G cells. However, FOXO3 mRNA was up-regulated in K562/G cells (P<0.05). MiRNA microarray results showed that 109 miRNAs were expressed differentially in K562 and K562/G cells. The expression of 81 miRNAs were up-regulated while 28 miRNAs were down-regulated. Through reverse prediction by bioinformatics, miR-6718-5p, miR-5195-5p, miR-4711-3p, miR-4763-5p, miR-4664-5p and miR-3176 were related to FOXO/Bcl-6 signaling pathway. CONCLUSION The FOXO3/Bcl-6 signaling pathway contributes to imatinib resistance in K562/G cell line, and the miRNA expression profiles showed significant differences between K562/G and K562 cells.
Forkhead Box Protein O3/genetics* ; Humans ; Imatinib Mesylate/pharmacology* ; K562 Cells ; MicroRNAs/genetics* ; RNA, Messenger ; Signal Transduction

OBJECTIVE: To investigate the regulation of chronic myelogenous leukemia （CML） imatinib resistant genes, in order to improve the therapeutic effect of CML imatinib resistant patients. METHODS: The human CML cell line K562 and imatinib-resistant K562 cells (K562/G01) were collected, and transcriptome of the cells were achieved by RNA-seq. The sequencing data were analyzed by using standard procedures. RESULTS: Compared with K562 cells, 464 genes were significantly changed in K562/G01 cells, including 163 up-regulated and 301 down-regulated genes. The GO function annotation analysis and KEGG pathway analysis results showed that the differentially expressed genes were mainly involved in biological processes such as oxidative phosphorylation, localization to protein organelle, ribonucleoprotein complex biogenesis and so on. Gene Set Enrichment Analysis (GSEA) plots showed that 5 gene-sets were up-regulated in K562/G01 significantly, including the pathway of TGF-beta, mTOR and CML. CONCLUSION CML imatinib resistance is associated with oxidative phosphorylation, during which the pathway of TGF-beta and mTOR are significantly up-regulated.
Drug Resistance, Neoplasm ; Gene Expression Profiling ; Humans ; Imatinib Mesylate/pharmacology* ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics*

STI571 is a new antileukemia agent targeting BCR-ABL tyrosine kinase and successfully used in treatment of leukemia patients. Despite strong efficacy of the tyrosine kinase inhibitor STI571, resistance has been observed in a significant proportion of leukemia patients. This review focuses on the diverse mechanisms of ST1571 resistance and its preventive measures.
Animals ; Antineoplastic Agents ; pharmacology ; Benzamides ; Drug Resistance, Neoplasm ; Humans ; Imatinib Mesylate ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; drug therapy ; Piperazines ; pharmacology ; Pyrimidines ; pharmacology

Adult ; Aged ; Benzamides ; pharmacology ; Drug Resistance, Neoplasm ; genetics ; Humans ; Imatinib Mesylate ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Middle Aged ; Piperazines ; pharmacology ; Point Mutation ; Pyrimidines ; pharmacology

OBJECTIVETo investigate the effect of imatinib on rat C6 glioma cell apoptosis and cell cycle.

METHODSMTT assay was used to determine the OD value of C6 glioma cells following treatment with imatinib at different concentrations (0.156, 10 and 15 micromo/L) for 24, 48 and 72 h. The cell apoptosis was assayed by Hochest/PI staining and the cell cycle changes were analyzed by flow cytometry.

RESULTSImatinib treatment resulted in increased number of apoptotic cells in a time- and dose-dependent manner. A 72-h treatment of the cells with imatinib at 10 and 15 micromo/L caused increased cell percentage in G(0)/G(1) phase to (68.53-/+0.83)% and (70.41-/+0.62)%, (P<0.01), decreased the percentage of G(2) phase cells to (14.48-/+0.12)% and (13.84-/+2.86)% (P<0.01), and decreased the percentage of S phase cells to (16.98-/+0.72)% and (15.78-/+2.28)%, respectively (P<0.01).

CONCLUSIONImatinib can induce apoptosis and affect the distribution of the cell cycle of C6 cells in vitro.

Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Benzamides ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Glioma ; pathology ; Imatinib Mesylate ; Piperazines ; pharmacology ; Pyrimidines ; pharmacology ; Rats

The present study is to elucidate the mechanisms underlying Gleevec-induced apoptosis of chronic myeloid leukemia (CML) K562 cells in vitro. The apoptotic cell death and cell cycle distribution after Gleevec treatment and the effect of PDCD4 siRNA on Gleevec-induced apoptosis of K562 cells were analyzed by flow cytometry. The effect of Gleevec on p-Crkl, caspase-3, PARP and PDCD4 protein levels, and the knockdown efficacy of PDCD4 siRNA were detected by Western blotting. The results showed that Gleevec dramatically suppressed the phosphorylation level of Crkl in a dose-dependent manner and induced significant apoptosis and G0/G1 cell cycle arrest of K562 cells in time- and dose-dependent manners. In addition, Gleevec activated caspase-3 and its downstream substrates PARP, and the caspase pan inhibitor Z-VAD-FMK (50 micromol x L(-1)) markedly reduced Gleevec-induced apoptosis from 47.97% +/- 10.56% to 31.05% +/- 9.206% (P < 0.05). Moreover, Gleevec significantly increased the protein expression of programmed cell death 4 (PDCD4). PDCD4 knockdown by siRNA reduced Gleevec-induced apoptosis from 46.97% +/- 14.32% to 42.8% +/- 11.43%. In summary, Gleevec induced apoptosis in K562 cells via caspase-3 activation.
Amino Acid Chloromethyl Ketones ; Apoptosis ; drug effects ; Benzamides ; pharmacology ; Caspase 3 ; metabolism ; Cell Cycle ; drug effects ; Humans ; Imatinib Mesylate ; K562 Cells ; Phosphorylation ; Piperazines ; pharmacology ; Pyrimidines ; pharmacology

OBJECTIVE: To explore the effect of hnRNPK/Beclin1 signaling on the drug resistance of imatinib in Ph⁺ leukemia. METHODS: Expression level of hnRNPK was verified in the imatinib resistant and sensitive Ph⁺ leukemia cell lines by using Western blot. hnRNPK expression was down-regulated by using RNAi. Expression level of LC3I/II and Beclin1 were detected by Western blot and the sensitivity of imatinib was analyzed by CCK-8 assay before and after modulation of hnRNPK expression. RESULTS: hnRNPK showed overexpressed in imatinib resistant leukemia cell line. After the expression level of hnRNPK was down-regulated by RNAi, the sensitivity of drug resistance lines to imatinib restored, while the expression level of LC3I/II and Beclin1 were consistant with the modulation of hnRNPK expression. CONCLUSION hnRNP K/Beclin1 signaling may be involved in the development of imatinib resistance in Ph⁺ leukemia through the regulation of autophagy.
Antineoplastic Agents/pharmacology* ; Beclin-1 ; Cell Line, Tumor ; Drug Resistance ; Drug Resistance, Neoplasm ; Heterogeneous-Nuclear Ribonucleoprotein K ; Humans ; Imatinib Mesylate/pharmacology* ; Leukemia

OBJECTIVETo explore the effect of overexpression of SH2-containing tyrosine phosphatase 1 (SHP-1) on sensitivity of chronic myelogenous 1eukemia (CML) K562 cell line to imatinib and its related mechamism.

METHODSK562 cells were infected with the lentiviral plasmids containing the specified retroviral vector (pEX-SHP-1-puro-Lv105) or the mock vector (pEX-EGFP-puro-Lv105). The expression of SHP-1 in K562 cells treated with 0.2 µmol/L imatinib (IM) for 72 h was determined by Western blot. After transfection the CCK-8 assay was used to determine the proliferation of the tramfected K562 cells (K562(SHP-1) and K562(EGFP) cells) at 72 h after exposure to different doses of IM, the half inhibitary concentration (IC50) was calculated. The mechanisms of the overexpression effects of SHP-1 and IM on the proliferation in K562 cells was investigated, the BCR-ABL1 activity and the level of tyrosine phosphorylation of CrkL (pCrkL) was measured by flow cytometry; the Western blot was used to detect the expression and activity of these molecules controlling cell growth, including MAPK, AKT, STAT5 and JAK2.

RESULTSAfter exposure of K562 cells to 0.08 µmol/L IM for 72 h, there was no significant change of SHP-1 expression in K562 cells. After exposure to 0.2 µmol/L of IM for 72 h, the inhibitory rate of K562(SHP-1) group was higher than that of K562(EGFP) group (P < 0.05), indicating that overexpression of SHP-1 in K562 cells could enhance the proliferation inhtibition effect of IM on K562 cells. The IC50 of IM in K562(SHP-1) cells was the lower as compared with that of K562(EGFP) cells (P < 0.05) after exposure to different concentrations of IM for 72 h. The slope of K562(SHP-1) cells was the largest ranging 0.02 - 0.16 µmol/L of IM. Overexpression of SHP-1 and IM could inhibit the activity BCR-ABL1, MAPK, AKT, STAT5 and JAK2 signaling pathways in the K562 cell line and displayed a synergistic effect.

CONCLUSIONSHP-1 inhibits BCR-ABL1, MAPK, AKT, STAT5 and JAK2 signaling pathways in K562 cells, the overexpression of SHP-1 can enhance the sensitivity of K562 cells to IM.

Cell Proliferation ; Drug Resistance, Neoplasm ; Genetic Vectors ; Humans ; Imatinib Mesylate ; pharmacology ; K562 Cells ; drug effects ; Phosphorylation ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 ; genetics ; metabolism ; Signal Transduction ; Transfection