Yuanzhong Chen

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.

Bone marrow stromal cells protect acute myeloid leukemia cells from anti-CD44 therapy partly through regulating PI3K/Akt-p27(Kip1) axis.

The anti‐CD44 monoclonal antibody (mAb) A3D8 induces differentiation or apoptosis in vitro in various subtypes of acute myeloid leukemia (AML) via p27Kip1 upregulation. Bone marrow (BM) stromal cells play a vital role in the development of chemoresistance in AML cells attached to the stroma. To investigate the effect of BM stroma adhesion induced AML resistance to A3D8, we developed a co‐culture system composed of an AML‐derived cell line (NB4) cultured with either a human BM stroma cell line (HS‐5) or mesenchymal stem cells (MSCs). We found that NB4 cells adhered to HS‐5 cells or MSCs developed resistance against the anti‐proliferative effects of A3D8, and this action is caused by the activation of PI3K/Akt signaling following p27Kip1 down‐regulation and cytoplasmic re‐localization. The stromal co‐culture‐induced resistance can be partially abolished by inhibiting the PI3K/Akt signaling pathway. Such findings were confirmed in two additional AML‐derived cell lines as well as in primary AML cells. Our results suggest that BM stroma can induce A3D8 resistance in part via the PI3K/Akt–p27Kip1 axis, and blocking PI3K/Akt pathway maybe necessary for anti‐CD44 treatment on AML in BM microenvironment.

Mitochondria-dependent apoptosis induced by a novel amphipathic photochemotherapeutic agent ZnPcS2P2 in HL60 cells

AbstractAim:To investigate the mechanism underlying the killing effects of a novel amphipathic photosensitizer, disulfonated diphthalimidomethyl phthalocyanine zinc (ZnPcS2P2), mediated photodynamic therapy (ZnPc-PDT) in human myelogenous leukemia HL60 cells.Methods:After incubation for 5 h with 0.5 μmol/L ZnPcS2P2, the HL60 cells were exposed to a light source of 670 nm wavelength. Thereafter, the cells were detected at different time intervals after PDT. The characteristics of apoptosis were detected by observation of ultrastructure assay, DNA fragmentation assay and terminal deoxynucleotidyl transferase deoxyuridine nick-end labeling method (TUNEL). Mitochondria-dependent apoptosis was determined by the detection of mitochondrial membrane potential (Av|/m), activities of caspase family protease and of caspase-3, cytosol cytochrome c. Proteins Bcl-2 and Bax were detected by immunoblot analysis.Results:Evident characteristics of apoptosis were observed post-ZnPc-PDT with ultrastructure assay, DNA fragmentation assay and TUNEL staining. TUNEL assay showed that apoptotic rates in the cells collected from 6 h, 12 h and 24 h after PDT were 9. 6%, 24.4%, and 33.0%, respectively. HL60 cells underwent mitochondria-dependent apoptosis as a result of cytochrome c release from mitochondria into cytosol accompanied by a reduction of Δψm. The activities of caspase family protease and of caspase-3 were elevated. Furthermore, ZnPc-PDT could remarkably down-regulate the Bcl-2 pro-apoptotic protein and up-regulate the anti-apoptotic Bax protein. Conclusion: ZnPc-PDT could induce mitochondria-dependent apoptosis in HL60 cells.

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.

Efficacy of ZnPcS2P2 photodynamic therapy solely or with tumor vaccines on mouse tumor models

BACKGROUND AND OBJECTIVESnGranulocyte-macrophage colony-stimulating factor (GM-CSF) and B7.1 transduced tumor vaccine cells could induce efficient anti-tumor immune response. It is interesting to study whether they could be an adjuvant to photodynamic therapy (PDT). Recent in vitro study proved that novel photosensitizer ZnPcS2P2 has capability of effective photodynamic killing of leukemic cells. In this preliminary study, we evaluated the photodynamic efficacy of ZnPcS2P2 on two tumor models and the improving anti-tumor efficacy of PDT in combination with GM-CSF gene-transduced vaccine and B7.1 gene-transduced vaccine.nnnMETHODSnNude mice bearing human leukemia xenograft and C57BL/6 mice bearing EL-4 thymic lymphoma were used to evaluate photodynamic efficacy of ZnPcS2P2. The EL-4 thymic lymphoma was used to test the improving anti-tumor efficacy of ZnPcS2P2-PDT in combination with GM-CSF gene-transduced vaccine and B7.1 gene-transduced vaccine. Each vaccine was administered near the tumor bed three times: 2 days before PDT, 0 and 2 days after PDT.nnnRESULTSnZnPcS2P2-PDT could significantly reduce tumor growth and prolong the survival time in both tumor models. Improving anti-tumor efficacy of ZnPcS2P2-PDT was demonstrated when utilizing GM-CSF-transduced vaccine and B7.1-transduced vaccine prior to and after ZnPcS2P2-PDT in lymphoma-bearing mice. Twenty-five percent of lymphoma-bearing mice were completely cured with a combination of PDT and vaccine cells.nnnCONCLUSIONSnZnPcS2P2-PDT may be a beneficial treatment for hemotopoietic malignance. GM-CSF-transduced vaccine and B7.1-transduced vaccine could strengthen ZnPcS2P2-PDT-elicited anti-lymphoma potency.

Apoptosis induced by ZnPcH1-based photodynamic therapy in Jurkat cells and HEL cells

Photodynamic therapy (PDT) can selectively and effectively kill tumor cells, and photosensitization is the key to these anti-tumor effects. In this study, we investigated the killing mechanisms of the photosensitizer ZnPcH1 (a mono-α-substituted zinc(II) phthalocyanine synthesized in China), in the acute lymphoid leukemia cell line Jurkat and the acute erythroleukemia cell line HEL. Results from acridine orange/ethidium bromide fluorescence staining, DNA gel electrophoresis, and Annexin-VFITC/PI double-stained flow cytometry analysis indicated that ZnPcH1-PDT induced apoptosis in Jurkat and HEL cells, with Jurkat cells being more sensitive. Following ZnPcH1-PDT treatment, upregulation of p53 and Bax, downregulation of HSP70, Bcl-2 and Akt, and inhibition of the phosphorylation of Akt and GSK3β were observed. Our results establish a theoretical basis for the application of ZnPcH1-PDT in the treatment of acute leukemia.

Reduction of transforming growth factor-β1 expression in leukemia and its possible role in leukemia development

Abstract The expression of transforming growth factor-β1 (TGF-β1) in leukemic cells and sera from patients with leukemia and its possible role in leukemia development were studied. TGF-β1 levels in culture supernatants from leukemic cells were significantly lower than those from normal bone marrow mononuclear cells. Serum TGF-β1 levels in leukemic patients were significantly lower compared with healthy controls, but returned to normal in patients achieving complete remission, and decreased when patients relapsed. TGF-β1 mRNA expression levels were significantly higher in normal bone marrow mononuclear cells but lower in leukemic cells compared with normal CD34 + cells. After transfection of the TGF-β1 gene to HL-60 cells, cell apoptosis was detected. Moreover, by flow cytometry analysis, cells arrested in G1 phase were 62% for TGF-β1 transfected cells and 44% for controls. Transfection of exogenous TGF-β1 gene inhibited HL60 cells xenograft growth in nude mice, and prolonged survival of tumor-bearing mice compared with the controls. Decreased endogenous TGF-β1 expression in leukemia cells may be involved in leukemia development, Transfection of exogenous TGF-B1 gene to HL60 can inhibit the proliferation of the cells and induce cell apoptosis by down regulating bcl-2, hTERT (human telomerase reverse transcriptase) and c-myc expression.