OBJECTIVETo investigate the antitumor efficacy and mechanism of HSP90 inhibitor FW-04-806 against Bcr/Abl(+) leukemia K562 and HL60 cells and their mechanisms of action.

METHODSMTT assay was used to assess the proliferation-inhibiting effect of FW-04-806. Cell cycle was analyzed with propidium iodide by flow cytometry. Cell apoptosis was determined using the FITC mV apoptosis detection kit. Western blot was applied to reveal the protein expression of related proliferative and apoptotic signaling pathways. The changes of mitochondrial membrane potential were detected by flow cytometry. Protein-protein interactions was shown by co-immunoprecipitation. The level of mRNA was assessed by real-time RT-PCR.

RESULTSFW-04-806 obviously inhibited cell proliferation in the HL60, K562 and HL60/Bcr-Abl cell lines, with an IC50 of (30.89 ± 0.12) µmol/L, (9.76 ± 0.19) µmol/L and (8.03 ± 0.26) µmol/L, respectively (P<0.001). Compared with the vehicle group, the two increasing doses of FW-04-806 showed inhibition of tumor growth at a rate of (17.40 ± 0.34)% and (34.33 ± 5.00)%, respectively, in the K562 cell line groups (P=0.003), and (18.90 ± 1.45)% and (35.60 ± 3.55)% (P=0.001) in the HL60/Bcr-Abl cell line groups. FW-04-806 dissociated Hsp90/Cdc37 chaperon/co-chaperon complex, followed by degradation of the Hsp90 proteins through proteasome pathway without affecting mRNA expression. FW-04-806 induced apoptosis and led to G2/M arrest.

CONCLUSIONOur findings indicate that FW-04-806 displays potential antitumor effect by suppressing the proliferation and apoptosis in Bcr/Abl(+) leukemia cells in vivo.

Apoptosis ; drug effects ; Cell Cycle ; Cell Proliferation ; drug effects ; Fusion Proteins, bcr-abl ; HL-60 Cells ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; Humans ; K562 Cells ; Leukemia ; drug therapy ; metabolism ; pathology ; Membrane Potential, Mitochondrial ; Oxazoles ; pharmacology ; RNA, Messenger ; metabolism ; Signal Transduction

No abstract available.
Adult ; Antineoplastic Agents/therapeutic use ; *Chromosome Deletion ; *Chromosomes, Human, Pair 9 ; Cytogenetic Analysis ; DNA Mutational Analysis ; Fusion Proteins, bcr-abl/*antagonists & inhibitors/genetics/metabolism ; Gene Expression Regulation ; Humans ; Incidental Findings ; Klinefelter Syndrome/complications/diagnosis/*genetics ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/complications/diagnosis/*drug therapy/enzymology/genetics ; Male ; Molecular Targeted Therapy ; Protein Kinase Inhibitors/*therapeutic use ; Remission Induction ; Time Factors ; Treatment Outcome

OBJECTIVETo evaluate the molecular response and prognostic factors of patients with Philadelphia chromosome/BCR-ABL-positive acute lymphoblastic leukaemia (Ph⁺ ALL) treated by imatinib with chemotherapy.

METHODSFrom May 2006 to July 2012, 82 adult Ph⁺ ALL patients were enrolled in the study. Forty-eight patients combined imatinib in, and 34 patients after induction therapy. Forty-nine patients underwent allogeneic hematopoietic stem cell transplant (allo-HSCT) after 3 to 5 cycles of consolidation therapy. The molecular response of BCR-ABL mRNA was evaluated by real-time quantitative PCR in every chemotherapy course ending.

RESULTSThe complete remission (CR) rate after the first cycle of induction chemotherapy was 76.8% (63/82), with overall CR rate of 92.7% (76/82). The CR rate in the patients combined imatinib in was higher than of those combined imatinib after the first cycle of induction chemotherapy (93.8% vs 52.9%, P<0.001). 55.3% patients BCR-ABL decreased >1 log after induction therapy. Among 76 CR patients, cumulative incidence of relapse was 27.6%, the probabilities of disease-free survival (DFS) and overall survival (OS) at 3 years were 60.5% and 70.2%, respectively. allo-HSCT was an independent favorable factor for decrease of leukemia relapse (P<0.001). allo-HSCT, imatinib combined in the first cycle of induction therapy and female were independent favorable factors for DFS (P<0.01, 0.05 and 0.01, respectively), BCR-ABL mRNA reduction at least 1 log from baseline after the first induction therapy and allo-HSCT were independent favorable factors for OS (P=0.011 and 0.027, respectively).

CONCLUSIONImatinib combined in the first cycle of induction therapy, BCR-ABL mRNA reduction at least 1 log from baseline after the first induction therapy and allo-HSCT improved outcomes of Ph⁺ ALL patients.

Adolescent ; Adult ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Benzamides ; therapeutic use ; Disease-Free Survival ; Female ; Fusion Proteins, bcr-abl ; antagonists & inhibitors ; Hematopoietic Stem Cell Transplantation ; Humans ; Imatinib Mesylate ; Male ; Middle Aged ; Philadelphia Chromosome ; Piperazines ; therapeutic use ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; diagnosis ; genetics ; therapy ; Prognosis ; Protein Kinase Inhibitors ; therapeutic use ; Pyrimidines ; therapeutic use ; Transplantation, Homologous ; Treatment Outcome ; Young Adult

OBJECTIVETo explore the prognostic significance of Ph-positive and/or BCR-ABL positive acute lymphoblastic leukemia (Ph⁺ ALL).

METHODSA retrospective analysis of 72 patients with Ph⁺ ALL to probe prognostic factors including sex, age, high white cell counts at diagnosis, additional chromosome abnormality, BCR-ABL transcripts type, imatinib based therapy, allo-HSCT and complete remission (CR) after one-course induction on the outcomes of Ph⁺ALL patients.

RESULTSOf 72 patients with median age 40.5 (13-68) years, 38 patients received imatinib plus chemotherapy. With median follow-up of 11 (0.2-96) months, total CR rate in patients receiving imatinib plus chemotherapy was higher than of patients receiving chemotherapy only (97.4% vs 62.3%, P=0.019). High white blood counts at diagnosis or additional chromosome abnormality had no effects on CR rate. 2-year overall survival (OS) and disease free survival (DFS) in imatinib plus chemotherapy group were (28.9±7.4) % and (25±7.4) %, respectively, which were higher than those in chemotherapy group (P<0.001). OS rate in HSCT group was significantly higher than that in non-HSCT group[ (61.1±11.5) % vs (5.6±3.1) %, P<0.001]. Multivariate prognostic analysis for OS showed that imatinib-based therapy [RR=0.413 (95% CI 0.237-0.721), P=0.002], allo-HSCT [RR=0.175 (95% CI 0.075-0.389), P=0.000] and CR after one-course induction [RR=0.429 (95% CI 0.245-0.750), P=0.003] were of importance for survival.

CONCLUSIONallo-HSCT was an optimal choice for Ph⁺ALL patients. Imatinib-based therapy could increase CR rate, maintain CR duration and decrease relapse, resulting in more chance of HSCT. Imatinib improved the outcomes of Ph⁺ALL patients who were not eligible for HSCT.

Adolescent ; Adult ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Benzamides ; therapeutic use ; Disease-Free Survival ; Female ; Fusion Proteins, bcr-abl ; antagonists & inhibitors ; Hematopoietic Stem Cell Transplantation ; Humans ; Imatinib Mesylate ; Male ; Middle Aged ; Philadelphia Chromosome ; Piperazines ; therapeutic use ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; diagnosis ; genetics ; therapy ; Prognosis ; Protein Kinase Inhibitors ; therapeutic use ; Pyrimidines ; therapeutic use ; Retrospective Studies ; Young Adult

OBJECTIVECompare the correlation and the concordance of simultaneous bone marrow cytogenetic and peripheral blood molecular responses in patients with chronic myeloid leukemia (CML) during tyrosine kinase inhibitor (TKI) treatment.

METHODSA total of 1 030 pairs of simultaneous bone marrow and peripheral blood samples from 419 patients with CML during TKI treatment were analyzed with standard G-banding techniques and real-time quantitative reverse transcriptase PCR (Q-PCR).

RESULTSThe Spearman correlation coefficient for the paired analysis of the percentage of Ph- positive metaphases versus Q-PCR (BCR-ABL/ABL) values was 0.655 (n=1 030, P<0.01). There was a significant difference in terms of BCR-ABL values among all three cytogenetic response groups of no partial cytogenetic (no PCyR), partial cytogenetic (PCyR) and complete cytogenetic responses(CCyR) (P<0.01). 93.8% of the patients in CCyR had BCR-ABL≤1% [International Scale (IS)], and 97.5% of those with BCR-ABL≤0.1% (IS) were in CCyR. There was good concordance of 86.2% (888 of 1 030 samples) when BCR-ABL values according to cutoffs of BCR-ABL>10%(IS), ≤10%->1%(IS)and ≤1%(IS) were coupled with cytogenetic responses including no PCyR, PCyR and CCyR. Furthermore, 497 pairs of samples from 279 patients with newly diagnosed CML in chronic phase during the first year on TKI as first-line therapy were analyzed. Concordances between major cytogenetic response and BCR-ABL≤10%(IS) at 3 months, CCyR and BCR-ABL≤1%(IS) at 6 months and 12 months were 89%, 83.5% and 92.1%, respectively.

CONCLUSIONThere were significant correlation and concordance between cytogenetic and molecular responses, and some differences in assessment of early responses between using cytogenetic and molecular analyses in CML patients during TKI treatment.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Bone Marrow Cells ; cytology ; drug effects ; Child ; Cytogenetic Analysis ; Female ; Fusion Proteins, bcr-abl ; antagonists & inhibitors ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; drug therapy ; genetics ; Male ; Middle Aged ; Philadelphia Chromosome ; Protein Kinase Inhibitors ; therapeutic use ; Treatment Outcome ; Young Adult

Studies on chronic myeloid leukemia (CML) have served as a paradigm for cancer research and therapy. These studies involve the identification of the first cancer-associated chromosomal abnormality and the subsequent development of tyrosine kinase inhibitors (TKIs) that inhibit BCR-ABL kinase activity in CML. It becomes clear that leukemia stem cells (LSCs) in CML which are resistant to TKIs, and eradication of LSCs appears to be extremely difficult. Therefore, one of the major issues in current CML biology is to understand the biology of LSCs and to investigate why LSCs are insensitive to TKI monotherapy for developing curative therapeutic strategies. Studies from our group and others have revealed that CML LSCs form a hierarchy similar to that seen in normal hematopoiesis, in which a rare stem cell population with limitless self-renewal potential gives rise to progenies that lack such potential. LSCs also possess biological features that are different from those of normal hematopoietic stem cells (HSCs) and are critical for their malignant characteristics. In this review, we summarize the latest progress in CML field, and attempt to understand the molecular mechanisms of survival regulation of LSCs.
Animals ; DNA-Binding Proteins ; genetics ; metabolism ; Fusion Proteins, bcr-abl ; antagonists & inhibitors ; metabolism ; Humans ; Hypoxia-Inducible Factor 1 ; metabolism ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; drug therapy ; metabolism ; pathology ; Lipid Metabolism ; Neoplastic Stem Cells ; drug effects ; metabolism ; Protein Kinase Inhibitors ; pharmacology ; therapeutic use ; Proto-Oncogene Proteins c-bcl-6 ; src-Family Kinases ; metabolism

The aim of this study was to investigate the effect of suppression of nicotinamide phosphoribosyltransferase (NAMPT) expression on imatinib-sensitivity in chronic myelogenous leukemia (CML) cell line K562 and its mechanisms, NAMPT siRNA was synthesized and transfected into K562 cells. PI/Calcein staining technique was used to determine survival rate of transfected K562 cells at 48th hour after exposure to 1 µmol/L imatinib. MTS method was used to determine the proliferation changes of transfected K562 cell at 48th hour after exposure to different doses of imatinib, then half inhibitory concentration (IC(50)) was calculated. Expression of NAMPT at 3rd-48th hour after exposure to 1 µmol/L imatinib was determined by Western blot. To explore the effect of NAMPT-siRNA and imatinib on the expression of apoptosis-related genes, the microarray data from NCBI GEO Data-Sets was analyzed, then the results were confirmed by Western blot. The luciferase reporter assay was used to determine the effect of NAMPT and imatinib on transcriptional activity of NF-κB transcription factors. The results showed that after exposure to 1 µmol/L imatinib for 3 - 48 h, there was no significant change of NAMPT expression in K562 cells. The expression of NAMPT could be effectively inhibited by the NAMPT-siRNA. After exposure to 1 µmol/L of imatinib for 48 h, the survival rate of NAMPT-siRNA interference group was lower than that of negative control group (P < 0.05), indicating that suppression of NAMPT expression can increase the sensitivity of K562 cells to imatinib and enhance the killing effect of imatinib on K562 cells. The IC(50) of imatinib in NAMPT-siRNA interference group was the lowest compared with that of control group (P < 0.05) after exposure to different concentrations of imatinib for 48 h, the fitted survival curves showed that the slope of NAMPT-siRNA interference group was the largest ranging between 0.01 - 0.1 µmol/L of imatinib. Data mining of expression profiling indicated that the anti-apoptotic factor Bcl-2 decreased in K562 cells treated with either NAMPT-siRNA or imatinib, which was confirmed by Western blot. The inhibitory effect was much more significant when both NAMPT-siRNA and imatinib were used. The results of luciferase reporter assay showed that either NAMPT-siRNA or imatinib decreased transcriptional activity of NF-κB. The decreased effect was much more significant when both NAMPT-siRNA and imatinib were used. It is concluded that survival of K562 cells affected by imatinib may not be due to regulation of expression of NAMPT. When expression of NAMPT decreases, the K562 cells are more sensitive to imatinib, this may be related with the decreased transcriptional activity of NF-κB and its downstream effector Bcl-2.
Benzamides ; Cytokines ; antagonists & inhibitors ; metabolism ; Fusion Proteins, bcr-abl ; metabolism ; Humans ; Imatinib Mesylate ; K562 Cells ; NF-kappa B ; metabolism ; Nicotinamide Phosphoribosyltransferase ; antagonists & inhibitors ; metabolism ; Piperazines ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Pyrimidines ; pharmacology

Imatinib, a breakpoint cluster region (BCR)-Abelson murine leukemia(ABL) tyrosine kinase inhibitor (TKI), has revolutionized the treatment of chronic myelogenous leukemia (CML). However, development of multidrug resistance(MDR) limits the use of imatinib. In the present study, we aimed to investigate the mechanisms of cellular resistance to imatinib in CML. Therefore, we established an imatinib-resistant human CML cell line(K562-imatinib) through a stepwise selection process. While characterizing the phenotype of these cells, we found that K562-imatinib cells were 124.6-fold more resistant to imatinib than parental K562 cells. In addition, these cells were cross-resistant to second- and third-generation BCR-ABL TKIs. Western blot analysis and reverse transcription-polymerase chain reaction(RT-PCR) demonstrated that P-glycoprotein(P-gp) and MDR1 mRNA levels were increased in K562-imatinib cells. In addition, accumulation of [14C]6-mercaptopurine (6-MP) was decreased, whereas the ATP-dependent efflux of [14C]6-MP and [3H]methotrexate transport were increased in K562-imatinib cells. These data suggest that the overexpression of P-gp may play a crucial role in acquired resistance to imatinib in CML K562-imatinib cells.
ATP Binding Cassette Transporter, Sub-Family B ; genetics ; metabolism ; Antineoplastic Agents ; pharmacology ; Benzamides ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Fusion Proteins, bcr-abl ; antagonists & inhibitors ; Gene Expression Regulation, Neoplastic ; Humans ; Imatinib Mesylate ; K562 Cells ; Mercaptopurine ; metabolism ; Methotrexate ; metabolism ; Piperazines ; pharmacology ; Protein Kinase Inhibitors ; pharmacology ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; Pyrimidines ; pharmacology ; RNA, Messenger ; metabolism

OBJECTIVETo 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.

METHODSThe 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.

RESULTSThe 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.

CONCLUSIONBBD9 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.

Animals ; Antineoplastic Agents ; pharmacology ; therapeutic use ; Benzamides ; Benzylisoquinolines ; pharmacology ; therapeutic use ; Drug Resistance, Neoplasm ; Female ; Fusion Proteins, bcr-abl ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; I-kappa B Kinase ; metabolism ; Imatinib Mesylate ; K562 Cells ; Liver Neoplasms, Experimental ; drug therapy ; metabolism ; Mice ; Mice, Nude ; Piperazines ; pharmacology ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; Pyrimidines ; pharmacology ; Transcription Factor RelA ; metabolism ; Xenograft Model Antitumor Assays

In order to investigate the molecular mechanisms of SARI expression regulation in chronic myeloid leukemia (CML), 46 patients with CML and 40 healthy volunteers were recruited in this study. SARI expression in the peripheral blood mononuclear cells (PBMNC) of CML patients and healthy volunteers was assayed by using real-time quantitative PCR. K562 cells were in vitro incubated with the BCR-ABL inhibitor STI571 (imatinib) at 37°C and 5% CO2 for 24 hours, then SARI expression was detected by using real-time quantitative PCR. All experiments were repeated three times. The results showed that as compared with healthy volunteers, the expression of SARI mRNA in PBMNC of CML patients presented a lower level (p < 0.001). After exposure of K562 cells to STI571 (2.5 µmol/L) for 24 hours, the SARI expression was higher than that in K562 cells treated without STI571 (p < 0.001). It is concluded that the suppression of SARI expression is involved in CML pathogenesis, and BCR-ABL mediates the down-regulation of SARI mRNA expression in K562 cells. These findings suggest a new orientation for gene therapy in CML patients.
Basic-Leucine Zipper Transcription Factors ; genetics ; Benzamides ; Fusion Proteins, bcr-abl ; antagonists & inhibitors ; Gene Expression Regulation, Leukemic ; drug effects ; Humans ; Imatinib Mesylate ; K562 Cells ; Piperazines ; pharmacology ; Pyrimidines ; pharmacology ; Tumor Suppressor Proteins ; genetics