Search

The present study was undertaken to investigate the mechanisms of peroxisome proliferator activated receptor-gamma (PPAR-gamma) ligand-induced apoptosis on human myeloid leukemia K562 and HL-60 cell lines. The results revealed that both 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) and troglitazone (TGZ) have significant anti-proliferation- and apoptosis-inducing effects on these two kinds of leukemia cells. Marked morphological changes of cell apoptosis including condensation of chromatin and nuclear fragmentation were observed clearly using Wright's and Hoechst 33258 staining. Reverse transcription-PCR and western blot analyses demonstrated that both survivin and bcl-2 expression were downregulated markedly, while bax expression was upregulated concurrently when apoptosis occurred. We therefore conclude that 15d-PGJ2 and TGZ have significant apoptosis effects on K562 and HL-60 cells in vitro, and that upregulation of bax as well as downregulation of survivin and bcl-2 expression may be the important apoptosis-inducing mechanisms. The results suggest that PPAR-gamma ligands may serve as potential therapeutic agents for both acute and chronic myeloid leukemia.

Severe aplastic anemia (SAA) is a disease with an autoimmune component. The susceptibility to the development of SAA is strongly associated with genes in the major histocompatibility complex (MHC). The gene for tumor necrosis factor-alpha (TNF-alpha) is encoded in the MHC locus and TNF-alpha is involved in the pathogenesis of SAA. A TNF-alpha variant with a polymorphism at position -308 in its promoter region (-308A), which is designated TNF2, has been demonstrated to be linked to a number of autoimmune diseases. In this study, the TNF-alpha -308 promoter polymorphism and HLA-DRB1 alleles were analyzed in 75 SAA patients, 55 mild aplastic anemia patients (MAA), and 128 controls. In SAA the phenotype frequencies of TNF2, HLA-DR3, and -DR2 were significantly higher in comparison to controls. Stratification analysis confirmed that the TNF2 allele contributes to the susceptibility to SAA independently of HLA-DR3 or -DR2. The results indicated that TNF2 might act as an independent risk factor for SAA.

Chronic autoimmune thrombocytopenic purpura (AITP) is characterized by platelet-specific autoantibody production that is influenced by enhanced antiplatelet T-helper cell reactivity. Costimulatory signals are absolutely required for T-cell activation and play key roles in the decision between tolerance and immunity. In this study we cultured T cells isolated from patients with chronic AITP to investigate the effects of the B7-blocking agent cytologic T-lymphocyte-associated antigen 4-immunoglobulin (CTLA4-Ig), and cyclosporin A (CsA), alone or in combination, on induction of platelet-specific T-cell anergy. The data showed that in most cases CTLA4-Ig and/or CsA could induce tolerance toward platelet antigens based on anergy. It could be overcome by stimulation with unrelated antigens, demonstrating its platelet specificity. The anergy is associated with lack of interleukin 2 (IL-2) and withheld by exogenous IL-2, emphasizing the pivotal role of IL-2 suppression in the induction of platelet-specific anergy. We also prospectively evaluated the efficacy of CsA therapy in patients with refractory AITP and observed that the response to CsA treatment in vivo was associated with the inhibiting sensitivity of platelet-reactive T cells to CsA in vitro. This suggests that the sensitivity of T cells to CsA in vitro could serve as a reliable parameter in predicting the efficacy of CsA for patients with refractory AITP. CTLA4-Ig may become a promising new therapeutic agent for the treatment of chronic AITP, and the combination of CTLA4-Ig and CsA would be a more powerful strategy for the management of refractory AITP.

Philadelphia-chromosome positive thrombocythemia without features of chronic myeloid leukemia in peripheral blood has been described before. However, there are few reports on Philadelphia-chromosome positive chronic idiopathic myelofibrosis. We present a case of Philadelphia positive chronic idiopathic myelofibrosis that had no obvious response to imatinib.

Ponicidin, an ent-kaurane diterpenoid derived from a constituent of the herbal supplement PC-SPES, Rabdosia rubescens, is recently reported to have anti-tumor effects on a large variety of cancers. In this study, we demonstrate that ponicidin exhibits cytotoxicity, induces apoptosis, disrupts the mitochondrial membrane potential, and triggers the activation of caspase-3, -8 and -9 in lung cancer A549 and GLC-82 cells. Ponicidin treatment of lung cancer cells caused downregulation of anti-apoptotic protein Bcl-2 and survivin as well as upregulaton of pro-apoptotic protein Bax in a time dependent manner when apoptosis ocurred. Ponicidin induced activation of caspase-3 can be blocked by a caspase-3-specific inhibitor z-DEVD-FMK Furthermore, the caspase-8-specific inhibitor z-IETD-FMK could block the ponicidin-induced activation of caspase-3, PARP cleavage, and prevented the release of cytochrome c from mitochondria into the cytoplasm. This indicate that activated caspase-8 initiates the release of cytochrome c during ponicidin-induced apoptosis. We therefore conclude that ponicidin has significant apoptosis-inducing effects by activation of caspase-3 -8, and -9 as well as downregulation of anti-apoptotic protein Bcl-2, survivin and upregulation of pro-apoptotic protein Bax, with caspase-8 acting as an upstream activator. The data offer a potential mechanism for ponicidin-induced apoptosis in lung cancer cells, suggesting that ponicidin may severve as an effective reagent for the treatment of lung cancer, and that in vivo anti-cancer effects as well as its potential clinical effectiveness need further investigation.

Oridonin, an ent-kaurane diterpenoid derived from the herbal Rabdosia rubescens, has been recently reported to have antitumor effects on a large variety of cancer cells. The present study was undertaken to investigate the in vitro antiproliferation and apoptosis inducing effects of oridonin on HPB-ALL cell lines and its mechanisms of action. HPB-ALL cells in culture medium in vitro were treated with different concentrations of oridonin (16-56 micromol/L). MTT assay was used to detect the cell growth inhibitory rate, and the cell viability was assessed by the trypan blue dye-exclusion method. Cell apoptosis and the mitochondrial membrane potential (delta psi m) were investigated by flow cytometry (FCM), Hoechst 33258 staining, and DNA fragmentation analysis. The expression of caspase-3 and different apoptosis modulators, including Fas and Bcl-2 family members, was analyzed by Western blotting. The results revealed that oridonin could significantly inhibit the growth of HPB-ALL cells and cause apoptosis, and the suppression was both time- and dose-dependent. After treatment with oridonin for 48 hr, the percentage of disruption of delta psi m gradually increased in a dose-dependent manner along with marked changes of cell apoptosis, and necrotic cells increased remarkably after the cells were treated with oridonin for 72 hr; Western blotting showed cleavage of the caspase-3 zymogen protein (32 kDa) with the appearance of its 20-kDa subunit when apoptosis occurred; expression of Bcl-2 and Bcl-XL was downregulated remarkably while expression of Bax and Bid was upregulated concurrently after the cells were treated with oridonin for 24 hr. Of note, the expressions of Fas and other Bcl-2 family members including Bak and Bad remained constant before and after apoptosis occurred. We therefore conclude that oridonin has significant antiproliferation effects on HPB-ALL cells by induction of apoptosis as well as directly causing cell necrosis and that oridonin-induced apoptosis on HPB-ALL cells is mainly related to the disruption of delta psi m and activation of caspase-3 as well as downregulation of anti-apoptotic protein Bcl-2, Bcl-XL, and upregulation of pro-apoptotic proteins Bax and Bid. The results indicate that oridonin may serve as a potential antileukemia reagent.

The anti-proliferation effects of oridonin on acute promyelocytic leukemia (APL) cells and its mechanisms were studied in vitro. NB4 cells as well as fresh leukemia cells obtained from APL patients in culture medium were treated with different concentrations of oridonin. Cell growth inhibition, apoptosis and related pathways were assessed by MTT assay as well as flow cytometry (FCM) and western blot analysis. The data revealed that oridonin (over 16 micromol/L) could inhibit the growth of NB4 cells by induction of apoptosis. Marked changes of cell apoptosis were observed very clearly by using electron microscopy and DNA fragmentation analysis after the cells exposed to oridonin for 48 h; Western blotting showed cleavage of the caspase-3 zymogen protein (32-kDa) with the appearance of its 20-kDa subunit as well as a cleaved 89-kDa fragment of 116-kDa PARP when apoptosis occurred. The expression of Bcl-2 was down-regulated remarkably accompanied by the disruption of the mitochondrial membrane potential (delta(psi)m). The anti-proliferative and apoptosis-inducing effects by oridonin in fresh APL cells were also found remarkably using Trypan Blue dye exclusion method and Wright's staining. We concluded that oridoning has significant anti-proliferative and apoptosis-inducing effects on NB4 cells by activation of caspase-3 and cleavage of PARP as well as by down regulation of Bcl-2 and disruption of the delta(psi)m. Furthermore, oridonin demonstrated apparent cell growth inhibition effects on fresh APL cells in vitro. The results indicated that oridonin may serve as a potential anti-leukemia reagent.

Ponicidin, an extract from the Chinese herb Rabdosia rubescens, is currently one of the most important traditional Chinese herbal medicines. Ponicidin has been reported to have anti-tumor effects on a large variety of malignant diseases. In this study, we investigated the anti-proliferation effects of ponicidin on human myeloid K562 and HL-60 cells. Cell viability was measured by MTT assay; cell apoptosis was assessed by flow cytometry, DNA fragmentation analysis and Hoechst 33258 staining. Caspase-3 and poly(ADP-ribose) polymerase (PARP) activation and Bax and Bcl-2 expression were detected by Western blot analysis. The results revealed that ponicidin could significantly inhibit the growth of K562 and HL-60 cells by induction of apoptosis. The suppression was both time- and dose-dependent. Cell apoptosis was observed clearly after the cells were treated with ponicidin for 48-72 h. Western blotting showed cleavage of the caspase-3 zymogen protein (32 kDa) with the appearance of its 17 kDa subunit, together with a cleaved 89-kDa fragment of 116 kDa PARP when apoptosis occurred. Bcl-2 expression was down-regulated while Bax expression up-regulated concurrently when the cells were treated with ponicidin for 24-48 h. Therefore, we conclude that ponicidin has significant anti-proliferation effects by induction of apoptosis on myeloid leukemia cells in vitro, down-regulation of Bcl-2, and up-regulation of Bax, and that activation of caspase-3 and PARP may be an important apoptosis-inducing mechanism. The results suggest that ponicidin may serve as a potential therapeutic agent for leukemia.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is one of the best characterized nuclear hormone receptors (NHRs) in the superfamily of ligand-activated transcriptional factors. PPAR-gamma ligands have recently been demonstrated to affect proliferation, differentiation and apoptosis of different cell types. The present study was undertaken to investigate PPAR-gamma ligands induced cell growth inhibition and its influence on matrix metalloproteinase MMP-9 and MMP-2 activities on leukemia K562 and HL-60 cells in vitro. The results revealed that PPAR-gamma expression was detectable in the two kinds of leukemia cells; Both 15-deoxy-delta(12,14)-prostaglandin J2(15d-PGJ2) and troglitazone (TGZ) have significant growth inhibition effects on these two kinds of leukemia cells. These two PPAR-gamma ligands could inhibit the leukemic cell adhesion to the extracellular matrix (ECM) proteins and the invasion through matrigel matrix. The expressions of MMP-9 and MMP-2 as well as their gelatinolytic activities in both HL-60 and K562 cells were inhibited by 15d-PGJ2 and TGZ significantly. We therefore conclude that PPAR-gamma ligands 15d-PGJ2 and TGZ have significant growth inhibition effects on myeloid leukemia cells in vitro, and that PPAR-gamma ligands can inhibit K562 and HL-60 cell adhesion to and invasion through ECM as well as downregulate MMP-9 and MMP-2 expressions. The data suggest that PPAR-gamma ligands may serve as potential anti-leukemia reagents.

The treatment of chronic idiopathic thrombocytopenic purpura (ITP) is difficult in those unresponsive to corticosteroids and/or splenectomy. We attempted to induce durable response in 21 patients with refractory ITP by applying mycophenolate mofetil (MMF) (1.5-2.0 g/d), a novel immunosuppressive agent. Overall response rate was 62% (13 of 21), including 24% (five of 21) in complete response (CR), 29% (six of 21) in partial response (PR), and 10% (two of 21) in minor response (MR). The response rates for non-splenectomized and splenectomized ITP patients were 64% (nine of 14) and 57% (four of seven), respectively (P0.05). 39% (five of 13) responders relapsed as a result of dose reduction or withdraw of MMF, and 61% (eight of 13) responders maintained their effectiveness for a median of 24 wk. Sustained response was observed in three patients in whom MMF was withdrawn. MMF was well tolerated with only slight nausea and diarrhea recorded in 3 of 21 cases. No premature withdrawal was found in this study. CD3+ peripheral blood mononuclear cells (PBMC) and CD19+ PBMC were significantly reduced 12 wk after MMF administration in the responders. Platelet-associated antibodies against glycoproteins GPIIb/IIIa were detected in 13 of 21 (62%) patients before MMF treatment, and antibody levels were significantly decreased in responders 12 wk after MMF administration. This suggested that MMF might correct the immunologic abnormalities underlying the destruction of circulating platelets in ITP. We conclude that MMF could be used as a second-line agent for the treatment of steroid-resistant ITP before or after splenectomy and thereby is worth of further evaluation in randomized studies.

To investigate the percentage of splenic CD(5)(+) B lymphocytes in chronic idiopathic thrombocytopenic purpura (IT) and the impact of splenic CD(5)(+) and CD(5)(-) B lymphocytes on the production of platelet glycoprotein (GP)-specific autoantibodies.Splenic CD(5)(+) B lymphocytes were identified by two-color flow cytometric analysis in eight patients. Four of the eight patients displayed plasma autoantibodies against both GPIIb/IIIa and GPIb/IX, and their splenic B lymphocytes were separated by Ficoll-Hypaque density gradient and sheep erythrocyte, and further purified by magnetic activate cell separation (MACS). Purified CD(5)(+) and CD(5)(-) B lymphocytes were cultured separately with or without staphylococcus aureus cowan I (SAC). GP specific autoantibodies in culture supernatants were measured by modified monoclonal antibody immobilization of platelet antigen assay (MAIPA).The percentage of splenic CD(5)(+) B lymphocytes in ITP patients was slightly higher than that in control with no statistical significance. MACS purified splenic CD(5)(+) and CD(5)(-) B lymphocytes from three out of four ITP patients produced high levels of anti-GPIIb/IIIa and anti-GPIb/IX antibodies. Culture supernatants of CD(5)(+) B lymphocytes from the other patient showed positive reaction only in GPIb/IX MAIPA. Culture supernatant of CD(5)(-)B lymphocytes from the same patient were double positive in both GPIIb/IIIa and GPIb/IX MAIPA.Both splenic CD(5)(+) and CD(5)(-) B lymphocytes produce platelet GP-specific autoantibodies in chronic ITP with similar antibody spectrum and titer, and may all play a role in the autoimmune pathogenesis of ITP.