Huang Hf

EFFECTS OF TREADMILL EXERCISE ON THE EXPRESSION OF NETRIN-1 AND ITS RECEPTORS IN RAT BRAIN AFTER CEREBRAL ISCHEMIA

Recent evidence suggests that exercise improves functional outcome in animal models of cerebral ischemia. Since netrin-1 and its receptors, deleted in colorectal cancer (DCC) and uncoordinated gene 5B (Unc5B), act as important regulators in neural and vascular activities, we sought to determine whether netrin-1 and DCC and Unc5B are involved in the neuroprotective effects of exercise on rats with induced cerebral ischemia. A total of 108 rats were randomly distributed into three groups: sham-operated group (n = 12), middle cerebral artery occlusion (MCAO) group (n = 48), MCAO+treadmill exercise group (n = 48). Behavioral testing indicated that treadmill exercise could significantly improve neurologic deficits of rats with cerebral ischemia at day 14 and 28 after MCAO (n = 12, P<0.05 and P<0.01), but there was no significant difference at day 4 and 7. Quantitative reverse transcription polymerase chain reaction (qPCR) and Western blot analysis revealed that treadmill exercise enhanced netrin-1 and DCC expression, while it suppressed Unc5B expression in rat peri-ischemic brain area, especially at day 14 and 28 after MCAO (n = 4, P<0.05 or P<0.01). Immunofluorescence analysis showed that in the peri-ischemic area, netrin-1 was expressed in neuronal perikarya, DCC, however, was expressed in neural processes and peri-vascular astrocytes, while Unc5B was expressed mostly in neuronal perikarya and some processes. These results suggest that netrin-1 and its receptors DCC and Unc5B may engage in exercise-induced neural circuit remodeling in the peri-ischemic area, and exercise may promote survival of neurons in this area by regulating netrin-1-Unc5B signaling. Additionally, netrin-1 may also play a role in brain-blood barrier via DCC-immunoreactive peri-vascular astrocytes. In conclusion, we demonstrate that treadmill exercise has beneficial effects that may be attributed, at least in part, to the involvement of netrin-1 and its receptors DCC and Unc5B in the neuronal and vascular activities in brain-ischemic rats.

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.

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

BACKGROUND AND OBJECTIVES Granulocyte-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. METHODS Nude 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. RESULTS ZnPcS2P2-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. CONCLUSIONS ZnPcS2P2-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.

Induction of Multidrug Resistance of Acute Myeloid Leukemia Cells by Cocultured Stromal Cells via Upregulation of the PI3K/Akt Signaling Pathway.

This study aimed to investigate the role of the PI3K/Akt signaling pathway in multidrug resistance of acute myeloid leukemia (AML) cells induced by cocultured stromal cells. Human AML cell lines HL-60 and U937 were adhesion cocultured with human bone marrow stromal cell line HS-5 cells. Such coculturing induced HL-60 and U937 cells resistant to chemotherapeutic drugs including daunorubicin (DNR), homoharringtonine (HHT), and cytosine arabinoside (Ara-C). The coculturing-induced resistance of AML cells to DNR, HHT, and Ara-C can be partially reversed by inhibition of the PI3K/Akt signaling pathway. Clinically, AML patients with a low level of PTEN and a high level of CCND1 had high relapse rates within 1 year, and newly diagnosed AML patients with extramedullary infiltration had a low level of PTEN. This study confirms the involvement of the PI3K/Akt signaling pathway in multidrug resistance in AML cells induced by stroma and suggests that the expression of PTEN and CCND1 may be a prognostic indicator for AML.

Prognostic Significance of CD44v6/v7 in Acute Promyelocytic Leukemia

CD44v, especially splice variants containing exon v6, has been shown to be related closely to development of different tumors. High levels of CD44v6/v7 have been reported to be associated with invasiveness and metastasis of many malignancies. The objective of this study was to detect expression of CD44v6-containing variants in patients with acute promyelocytic leukemia (APL) and evaluate the potential of CD44v6/v7 for risk stratification. Reverse transcription polymerase chain reaction (RT-PCR) followed by PCR product purification, ligation into T vectors and positive clone sequencing were used to detect CD44 v6-containing variant isoforms in 23 APL patients. Real-time quantitative PCR of the CD44v6/v7 gene was performed in patients with APL and in NB4 cells that were treated with all-trans retinoic acid (ATRA) or arsenic trioxide (As2O3). Sequencing results identified four isoforms (CD44v6/v7, CD44v6/v8/v10, CD44v6/v8/v9/v10, and CD44v6/v7/v8/v9/v10) in bone marrow mononuclear cells of 23 patients with APL. The level of CD44v6/v7 in high-risk cases was significantly higher than those with low-risk. Higher levels of CD44v6/v7 were found in three patients with central nervous system relapse than in other patients inthe same risk group. Furthermore, in contrast to ATRA, only As2O3 could significantly down-regulate CD44v6/v7 expression in NB4 cells. Our data suggest that CD44v6/v7 expression may be a prognostic indicator for APL.

Purging efficacy of ZnPcH1‐based photodynamic therapy on chronic myeloid leukemia bone marrow

Introduction:  Ex vivo purging is an emerging technique for successful transplantation of autologous hematopoietic stem cells in the treatment of leukemia. Among them, photodynamic therapy (PDT) might be a promising modality. The objective of this study was to evaluate the purging efficacy of ex vivo PDT on chronic myeloid leukemia (CML).

PI3K/Akt inhibitor LY294002 potentiates homoharringtonine antimyeloma activity in myeloma cells adhered to stromal cells and in SCID mouse xenograft

Homoharringtonine (HHT) is a known anti-leukemia drug that inhibits multiple myeloma (MM) cells both in vitro and in vivo. Our prior study demonstrated that the potency of HHT in MM cells was compromised significantly when myeloma cells were co-cultured with BM stromal cells. This study aimed to investigate whether PI3K/Akt inhibitor LY294002 could potentiate the antimyeloma activity of HHT against MM cells adhered to BM stromal cells and in vivo xenograft models. A co-culture system composed of MM cells and human stromal cells was employed to mimic MM cells in bone marrow niche. The inhibitory and pro-apoptotic effect of HHT and LY294002 was determined by CCK-8 assay or flow cytometry. Expression of PI3K/Akt signaling molecules and anti-apoptotic protein myeloid cell leukemia-1 (Mcl-1) was assessed by western blot analysis and/or reverse transcription real-time quantitative PCR (RT-qPCR). MM xenografts were used to evaluate antitumor effect of combined therapy with HHT and LY294002. Adhesion to BM stromal cells rendered MM cells resistant to HHT whereas silencing Mcl-1 partly reversed the resistance. LY294002 induced apoptosis in MM cells and potentiated the antimyeloma effects of HHT by inhibiting the PI3K/Akt signal pathway which was abnormally activated during adhesion. LY294002 also enhanced the antimyeloma effect of HHT in in vivo xenograft models. These findings suggest that activation of PI3K/Akt signal pathway was responsible for the resistance to HHT in MM cells adhered to stromal cells. LY294002 can potentiate the antimyeloma activity of HHT both in vitro and in vivo, which may represent a new clinical treatment in MM.