Lixian Wu

Down-regulation of p210~(bcr/abl)by curcumin involves disrupting molecular chaperone functions of Hsp90

AbstractAim:To investigate the effects of curcumin (Cur) on p210bcr/abl level in K562 cells, and the relationship between these effects and the molecular chaperone functions of heat shock protein 90 (Hsp90).Methods:Flow cytometry and Western blot were used to examine the abundance of p210bcr/abl, Hsp90, p23, Hsp70, and p60Hop in K562 cells treated with Cur. Reverse transcription polymerase chain reaction (RT-PCR) was used to determine the bcr-abl mRNA level in K562 cells treated with Cur. After co-immunoprecipitation of p210bcr/abl and its molecular chaperones, the immunoprecipitate was then subjected to Western blot analysis with anti-Hsp90, anti-Hsp70, anti-p23, and anti-p60HopmAb.Results:An exposure of K562 cells to Cur produced time-dependent down-regulation of p210bcr/abl, the inhibition rate of p210bcr/abl in K562 cells determined by flow cytometry after treatment with Cur 27.2 umol/Lfor 1 h, 6 h, 12 hand 24 h was 31.2%, 63.7%, 81.3% and 94.5%, respectively. In contrast, Cur had almost no influence on bcr-abl mRNA level. Treatment with Cur for 24 h reduced the association of p210bcr/abl with Hsp90/p23 complex, while increasing the association of p210bcr/abl with Hsp70/p60Hop complex; however, the total protein abundance of Hsp90, p23, and p60Hop inK562 cells had no apparent change, while Hsp70 increased greatly.Conclusion:Down-regulation of p210bcr/abl by Cur involves dissociating the binding of p210bcr/abl with Hsp90/p23 complex. In contrast, the association of p210bcr/abl with Hsp70/p60Hop complex increased.

Trichosanthin down-regulated p210Bcr-Abl and enhanced imatinib-induced growth arrest in chronic myelogenous leukemia cell line K562

PurposeTrichosanthin (TCS), an active component extracted from the root tubers of traditional Chinese medical herb Tian-Hua-Fen of the Cucurbitaceae family, has long been used for medical purpose in China; there is increasing interest in developing TCS as cancer therapeutic agents. The present study was to investigate the growth arrest of K562 cells and its molecular mechanisms, which the drugs induced by TCS and the possible functional interaction of TCS with imatinib (STI571) to K562 cells.MethodsTrypan blue exclusive staining was used to access the cell growth inhibition; western blot was used to evaluate the p210Bcr-Abl, phosphorylated tyrosine kinase (PTK), and some signaling molecules involving in cell proliferation and apoptosis in K562 cells.ResultsTCS and imatinib inhibited K562 cells at a time- and dose-dependent manners, respectively; TCS down-regulated p210Bcr-Abl at a time- and dose-dependent manners; TCS synergistically enhanced imatinib-induced K562 cell growth arrest and down-regulation of p210Bcr-Abl, PTK activities, procaspase-3, Hsp90,NF-κB and PKC.ConclusionThe results suggest that TCS not only by itself involves but also synergizes activities of imatinib to induce K562 cell growth arrest, down-regulation of p210Bcr-Abl and its downstream signals and to stimulate the effect of the tyrosine kinase inhibition.

Dual inhibition of Bcr-Abl and Hsp90 by C086 potently inhibits the proliferation of imatinib-resistant CML cells

Purpose: Although tyrosine kinase inhibitors (TKI) such as imatinib provide an effective treatment against Bcr-Abl kinase activity in the mature cells of patients with chronic myelogenous leukemia (CML), TKIs probably cannot eradicate the leukemia stem cell (LSC) population. Therefore, alternative therapies are required to target both mature CML cells with wild-type (WT) or mutant Bcr-Abl and LSCs. To investigate the effect of C086, a derivative of curcumin, on imatinib-resistant cells, we explored its underlying mechanisms of Bcr-Abl kinase and heat shock protein 90 (Hsp90) function inhibition. Experimental Design: Biochemical assays were used to test ABL kinase activity; fluorescence measurements using recombinant NHsp90, Hsp90 ATPase assay, immunoprecipitation, and immunoblotting were applied to examine Hsp90 function. Colony-forming unit, long-term culture-initiating cells (LTC-IC), and flow cytometry were used to test CML progenitor and stem cells. Results: Biochemical assays with purified recombinant Abl kinase confirmed that C086 can directly inhibit the kinase activity of Abl, including WT and the Q252H, Y253F, and T315I mutations. Furthermore, we identified C086 as a novel Hsp90 inhibitor with the capacity to disrupt the Hsp90 chaperone function in CML cells. Consequently, it inhibited the growth of both imatinib-sensitive and -resistant CML cells. Interestingly, C086 has the capacity to inhibit LTC-ICs and to induce apoptosis in both CD34+CD38+ and CD34+CD38− cells in vitro. Moreover, C086 could decrease the number of CD45+, CD45+CD34+CD38+, and CD45+CD34+CD38− cells in CML NOD-SCID mice. Conclusions: Dual suppression of Abl kinase activity and Hsp90 chaperone function by C086 provides a new therapeutic strategy for treating Bcr-Abl–induced leukemia resistant to TKIs. Clin Cancer Res; 21(4); 833–43. ©2014 AACR.

Curcumin derivative C817 inhibits proliferation of imatinib-resistant chronic myeloid leukemia cells with wild-type or mutant Bcr-Abl in vitro

Aim:To find new kinase inhibitors that overcome the imatinib resistance in treatment of chronic myeloid leukemia (CML), we synthesized C817, a novel derivative of curcumin, and tested its activities against wild-type (WT) and imatinib-resistant mutant Abl kinases, as well as in imatinib-sensitive and resistant CML cells in vitro.Methods:32D cells harboring WT or mutant Abl kinases (nucleotide binding P-loop mutants Q252H, Y253F, and imatinib contact residue mutant T315I), as well as K562/G01 cells (with whole Bcr-Abl gene amplication) were tested. Kinase activity was measured using Kinase-Glo Luminescent Kinase Assay Platform in recombinant WT and mutant (Q252H, Y253F, and T315I) Abl kinases. Cell proliferation and apoptosis were examined using MTT assay and flow cytometry, respectively. The phosphorylation levels of Bcr-Abl initiated signaling proteins were analyzed using Western blotting. Colony forming units (CFU) growth and long term culture-initiating cells (LTC-ICs) were used to test the effects of C817 on human leukemia progenitor/stem cells.Results:C817 potently inhibited both WT and mutant (Q252H, Y253F, and T315I) Abl kinase activities in a non-ATP competitive manner with the values of IC50 at low nanomole levels. In consistent with above results, C817 suppressed the growth of both imatinib-sensitive and resistant CML cells, including wild-type K562, K562/G01, 32D-T315I, 32D-Q252H, and 32D-Y253F cells with the values of IC50 at low micromole levels. C817 (0.5 or 1 μmol/L) dose-dependently inhibited the phosphorylation of Bcr-Abl and downstream proteins STAT-5 and CrkL in imatinib-resistant K562/G01 cells. Furthermore, C817 significantly suppressed CFU growth and LTC-ICs, implicating that C817 could eradiate human leukemia progenitor/stem cells.Conclusion:C817 is a promising compound for treatment of CML patients with Bcr-Abl kinase domain mutations that confer imatinib resistance.

Curcumin synergistically augments bcr/abl phosphorothioate antisense oligonucleotides to inhibit growth of chronic myelogenous leukemia cells

AbstractAim:To investigate the growth inhibition effect of the combination of bcr/abl phosphorothioate antisense oligonucleotides (PS-ASODN) and curcumin (cur), and the possible mechanisms of cur on the chronic myelogenous leukemia cell line K562.Methods:The K562 cell line was used as a P210bcr/abl-positive cell model in vitro and was exposed to different concentrations of PS-ASODN (0-20 μmol/L), cur (0-20 μmol/L), or a combination of both. Growth inhibition and apoptosis of K562 cells were assessed by MTT assay and AO/EB fluorescent staining, respectively. The expression levels of P210bcr/abl, NF-κB and heat shock protein 90 (Hsp90) were assessed by Western blot.Results:Exposure to cur (5-20 μmol/L) and PS-ASODN (5-20 μmol/L) resulted in a synergistic inhibitory effect on cell growth. Growth inhibition was associated with the inhibition of the proliferation and induction of apoptosis. Western blot analysis showed that the drugs synergistically downregulated the level of P210bcr/abl and NF-κB. Cur downregulated Hsp90, whereas no synergism was observed when cur was combined with PS-ASODN.Conclusion:PS-ASODN and cur exhibited a synergistic inhibitory effect on the cell growth of K562. The synergistic growth inhibition was mediated through different mechanisms that involved the inhibition of P210bcr/abl.

A novel synthetic novobiocin analog, FM-Nov17, induces DNA damage in CML cells through generation of reactive oxygen species

OBJECTIVES To investigate the cytotoxicity of FM-Nov17 against chronic myeloid leukemia (CML) cells, we explored its underlying mechanisms mediating the induction of DNA damage and apoptotic cell death by reactive oxygen species (ROS). METHODS MTT assays were used to measure the proliferation-inhibition ratio of K562 and K562/G01 cells. Flow cytometry (FCM) was used to test the level of extracellular ROS, DNA damage, cell cycle progression and apoptosis. Western blotting was used to verify the amount of protein. RESULTS FM-Nov17 significantly inhibited the proliferation of K562 cells, with an IC50 of 58.28±0.304μM, and K562/G01 cells, with an IC50 of 62.36±0.136μM. FM-Nov17 significantly stimulated the generation of intracellular ROS, followed by the induction of DNA damage and the activation of the ATM-p53-r-H2AX pathway and checkpoint-related signals Chk1/Chk2, which led to increased numbers of cells in the S and G2/M phases of the cell cycle. Furthermore, FM-Nov17 induced apoptotic cell death by decreasing mitochondrial membrane potential and activating caspase-3 and PARP. The above effects were all prevented by the ROS scavenger N-acetylcysteine. CONCLUSIONS FM-Nov17-induces DNA damage and mitochondria-dependent cellular apoptosis in CML cells. The process is mediated by the generation of ROS.

Hsp90 N- and C-terminal double inhibition synergistically suppresses Bcr-Abl-positive human leukemia cells

Heat shock protein 90 (Hsp90) contains amino (N)–terminal domain, carboxyl(C)-terminal domain, and middle domains, which activate Hsp90 chaperone function cooperatively in tumor cells. One terminal occupancy might influence another terminal binding with inhibitor. The Bcr-Abl kinase is one of the Hsp90 clients implicated in the pathogenesis of chronic myeloid leukemia (CML). Present studies demonstrate that double inhibition of the N- and C-terminal termini can disrupt Hsp90 chaperone function synergistically, but not antagonistically, in Bcr-Abl-positive human leukemia cells. Furthermore, both the N-terminal inhibitor 17-AAG and the C-terminal inhibitor cisplatin (CP) have the capacity to suppress progenitor cells; however, only CP is able to inhibit leukemia stem cells (LSCs) significantly, which implies that the combinational treatment is able to suppress human leukemia in different mature states.

A Novobiocin Derivative, XN4, Inhibits the Proliferation of Chronic Myeloid Leukemia Cells by Inducing Oxidative DNA Damage

XN4 might induce DNA damage and apoptotic cell death through reactive oxygen species (ROS). The inhibition of proliferation of K562 and K562/G01 cells was measured by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide). The mRNA levels of NADPH oxidase 1-5 (Nox1-5) genes were evaluated by qRT-PCR. The levels of extracellular reactive oxygen species (ROS), DNA damage, apoptosis, and cell cycle progression were examined by flow cytometry (FCM). Protein levels were analyzed by immunoblotting. XN4 significantly inhibited the proliferation of K562 and K562/G01 cells, with IC50 values of 3.75±0.07 µM and 2.63±0.43 µM, respectively. XN4 significantly increased the levels of Nox4 and Nox5 mRNA, stimulating the generation of intracellular ROS, inducing DNA damage and activating ATM-γ-H2AX signaling, which increased the number of cells in the S and G2/M phase of the cell cycle. Subsequently, XN4 induced apoptotic cell death by activating caspase-3 and PARP. Moreover, the above effects were all reversed by the ROS scavenger N-acetylcysteine (NAC). Additionally, XN4 can induce apoptosis in progenitor/stem cells isolated from CML patients’ bone marrow. In conclusion, XN4-induced DNA damage and cell apoptosis in CML cells is mediated by the generation of ROS.