Ren-Wei Huang

Peroxisome proliferator activated receptor-γ ligands induced cell growth inhibition and its influence on matrix metalloproteinase activity in human myeloid leukemia cells

Peroxisome proliferator-activated receptor-gamma (PPAR-γ) is one of the best characterized nuclear hormone receptors (NHRs) in the superfamily of ligand-activated transcriptional factors. PPAR-γ ligands have recently been demonstrated to affect proliferation, differentiation and apoptosis of different cell types. The present study was undertaken to investigate PPAR-γ 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-γ 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-γ 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-γ ligands 15d-PGJ2 and TGZ have significant growth inhibition effects on myeloid leukemia cells in vitro, and that PPAR-γ 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-γ ligands may serve as potential anti-leukemia reagents.

Down-Regulation of Telomerase Activity and Activation of Caspase-3 Are Responsible for Tanshinone I-Induced Apoptosis in Monocyte Leukemia Cells in Vitro

Tanshinone I (Tan-I) is a diterpene quinone extracted from the traditional herbal medicine Salvia miltiorrhiza Bunge. Recently, Tan-I has been reported to have anti-tumor effects. In this study, we investigated the growth inhibition and apoptosis inducing effects of Tan-I on three kinds of monocytic leukemia cells (U937, THP-1 and SHI 1). Cell viability was measured by MTT assay. Cell apoptosis was assessed by flow cytometry (FCM) and AnnexinV/PI staining. Reverse transcriptase polymerase chain reaction (RT-PCR) and PCR–enzyme-linked immunosorbent assay (ELISA) were used to detect human telomerase reverse transcriptase (hTERT) expression and telomerase activity before and after apoptosis. The activity of caspase-3 was determined by Caspase colorimetric assay kit and Western blot analysis. Expression of the anti-apoptotic gene Survivin was assayed by Western blot and Real-time RT-PCR using the ABI PRISM 7500 Sequence Detection System. The results revealed that Tan-I could inhibit the growth of these three kinds of leukemia cells and cause apoptosis in a time- and dose-dependent manner. After treatment by Tan-I for 48 h, Western blotting showed cleavage of the caspase-3 zymogen protein with the appearance of its 17-kD subunit, and a 89-kD cleavage product of poly (ADP-ribose) polymerase (PARP), a known substrate of caspase-3, was also found clearly. The expression of hTERT mRNA as well as activity of telomerase were decreased concurrently in a dose-dependent manner. Moreover, Real-time RT-PCR and Western blot revealed a significant down-regulation of Survivin. We therefore conclude that the induction of apoptosis by Tan-I in monocytic leukemia U937 THP-1 and SHI 1 cells is highly correlated with activation of caspase-3 and decreasing of hTERT mRNA expression and telomerase activity as well as down-regulation of Survivin expression. To our knowledge, this is the first report about the effects of Tan-I on monocytic leukemia cells.

Downregulation of cyclooxygenase-2 expression and activation of caspase-3 are involved in peroxisome proliferator-activated receptor-γ agonists induced apoptosis in human monocyte leukemia cells in vitro

Peroxisome proliferator-activated receptor-γ (PPAR-γ) is a transcription factor important in fat metabolism and PPAR-γ agonists were recently demonstrated to affect proliferation, differentiation, and apoptosis of different cell types. In the present study, two PPAR-γ agonists, 15-deoxy-delta (12,14)-prostaglandin J2 (15d-PGJ2) and a synthetic PPAR-γ agonist troglitazone (TGZ), were used to investigate activated PPAR-γ-induced apoptosis on human monocyte leukemia U937 and Mono Mac 6 cells in vitro. The results showed that both U937 and Mono Mac 6 cells demonstrated constitutive activation of COX-2 expression; treatment by 15d-PGJ2 and TGZ could induce apoptosis remarkably in human monocyte leukemia cells by disruption of mitochondrial membrane potential, activation of caspase-3, and causing cleavage of the caspase substrate poly (ADP-ribose) polymerase (PARP). Further studies revealed that treatment by both 15d-PGJ2 and TGZ remarkably downregulated COX-2 expression in these two kind of monocyte leukemia cells as measured by reverse transcriptase PCR (RT-PCR) and Western blot. Furthermore, the expression of Bcl-2 and Bcl-Xl and Mcl-1 was downregulated while Bax expression was upregulated concurrently after the cells were treated by these two agonists, and no variations were found in other Bcl-2 family members such as Bak, Bid, and Bad. Taken together, our results demonstrate for the first time that downregulation of cyclooxygenase-2 expression, disruption of mitochondrial membrane potential, activation of caspase-3, downregulation of Bcl-2, Bcl-Xl, and Mcl-1, and upregulation of Bax are involved in PPAR-γ agonists-induced apoptosis in these two human monocyte leukemia cells.

Anti-proliferative Effects of Oridonin on SPC-A-1 Cells and its Mechanism of Action

Oridonin, an extract from the Chinese herb Rabdosia rubescens, is currently one of the most important traditional Chinese herbal medicines. We investigated the anti-proliferative effect of oridonin on the lung cancer cell line SPC-A-1 and its mechanism of action. Growth inhibition was measured using a microculture tetrazolium assay and apoptosis was measured by several standard methods. Western blot analysis measured the expression of bcl-2 and bax proteins. Oridonin (> 28 μmol/l) inhibited the growth of SPC-A-1 cells and induced apoptosis. Marked morphological changes indicative of apoptosis were observed, especially in cells treated with oridonin for 48–60 h. Western blot analysis revealed downregulation of bcl-2 and upregulation of bax proteins following treatment with oridonin for 48 h. We conclude that oridonin demonstrated anti-proliferative and apoptosis-inducing effects on SPC-A-1 cells in vitro, and that changes in bcl-2 and bax protein levels may play an important role in its mechanism of action.

Inhibition of Lymphoma Cell Proliferation by Peroxisomal Proliferator-Activated Receptor-γ Ligands via Wnt Signaling Pathway

Peroxisome proliferator-activated receptor gamma (PPARγ) plays an important role in regulating energy balance, glucose and lipid metabolisms and inflammation. PPARγ also exerts multiple anti-cancer effects including tumor growth and angiogenesis inhibition, induction of cell differentiation, and apoptosis. Perturbed Wnt/β-catenin signaling likely plays a key role in tumorigenesis and the interaction between PPARγ and the transcriptional regulator β-catenin maybe important in this process. Phosphorylation of β-catenin by GSK-3β inactivates it and suppresses tumor cell proliferation and self-renewal of tumor stem cells. In combination with Frizzled, Wnt suppresses GSK-3β and causes degradation of β-catenin and activation of many tumor proliferation factors. In the present study, we investigated the effects of PPARγ agonist rosiglitazone (RGZ) and PPARγ antagonist GW9662 on the growth, mitotic cycle, and apoptosis of human lymphoma cell line, Raji cells. We also studied the influence of PPARγ ligands on the expression of β-catenin and GSK-3β in Raji cells to reveal whether Wnt/GSK-3β/β-catenin signaling pathways are involved in PPARγ ligands triggered Raji cell apoptosis. Results showed that both RGZ and GW9662 can inhibit the growth of Raji cells by inducing apoptosis and arresting cell cycle; however, there was no correlation between these effects and expression of PPARγ. Both the PPARγ ligands, RGZ and GW9662, appear to reciprocally regulate the mRNA and protein expressions of GSK-3β, which promotes apoptosis, and of β-catenin, which blocks apoptosis. These results suggest that PPARγ ligands mediate their effects via Wnt/GSK-3β/β-catenin signaling on Raji cell proliferation and survival.

PPARγ Agonist Suppresses TLR4 Expression and TNF-α Production in LPS Stimulated Monocyte Leukemia Cells

It has been well established that inflammation plays a critical role in cancer. Chronic inflammation promotes tumorgenesis and metastasis, which suggests that anti-inflammation drugs could act as a tumor suppressor. It is known that the peroxisome proliferator-activated receptor γ (PPARγ) has been implicated in anti-inflammatory responses; however, the anti-tumor effects of PPARγ have not been intensively investigated. In this study, we examined the effects of PPARγ in cancer. We show that the activation of PPARγ by its agonist rosiglitazone (RGZ) reduces cell proliferation rate in inflammatory and tumor-derived U937 cells. Treatment of RGZ suppresses the expression Toll-like receptor 4 (TLR4) and decreases the production of TNF-α in LPS treated U937 cells. This suggests that NF-κB signaling may be involved in anti-tumor effect of RGZ. Our results demonstrate a role of PPARγ in regulation of NF-κB signaling by modulating TLR4 expression and TNF-α production.

Antibody-directed Double Suicide Gene Therapy Targeting of MUC1- Positive Leukemia Cells In Vitro and In Vivo

Our aim was to specifically transfer the cytosine deaminase (CD) and thymidine kinase (TK) genes into mucin 1 (MUC1)-positive leukemia cells by anti-MUC1 antibody directed infection of replication-defective lentivirus and to evaluate the targeted cytotoxicity of double suicide genes to leukemia. The target gene vector (containing CD and TK) and envelope (containing GFP and anti-MUC1) and packaging plasmids were cotransfected into 293T cells to produce the recombinant lentivirus. Suicide genes in virus-infected leukemia cells (U937, Jurkat, and K562) were detected by western blot. The cytotoxicity and bystander effect in vitro and the therapeutic effect in vivo were detected after treatment with the prodrugs. The results revealed that combined treatment with prodrug 5-fluorocytosine (5-FC) and ganciclovir (GCV) inhibited leukemia cell growth and caused significant bystander effect than treatment with either prodrug alone. TK/GCV treatment alone induced degeneration and cell death while the effect of CD/5-FC alone mainly caused vacuolar degeneration and necrosis. The addictive effects of combinatorial use of GCV and 5-FC mainly induced swelling of the mitochondria followed by necrosis of the leukemia cells. In vivo experiments revealed that both single and combinatorial prodrug treatments could prolong the survival time of leukemic mice. In summary, anti-MUC1 antibody directed lentiviral vector successfully transduced dual suicide genes and exerted targeted cytotoxicity against MUC1 positive leukemia cells. This targeted lentiviral dual suicide gene delivering system provides a promising approach for clinical treatment of leukemia in future.

Acute promyelocytic leukaemia with a PML-RARA insertional translocation and a chromosome 21 abnormality in XYY syndrome: case report.

The concomitant presence of the XYY syndrome with haematological malignancies is rare. This report presents a case of acute promyelocytic leukaemia (APL) with the promyelocytic leukaemia-retinoic acid receptor alpha (PML-RARA) gene insertional translocation and a chromosome 21 abnormality in a 29-year-old XYY male patient. Karyotype analysis revealed an abnormal karyotype of 47,XYY [14]/46,XYY,–21[16]. Fluorescence in situ hybridization and reverse transcription–polymerase chain reaction analysis showed the existence of a PML-RARA fusion gene. The patient was treated by all-trans retinoic acid (ATRA) and chemotherapy. Laboratory results revealed that the coagulopathy improved and the patient achieved complete remission, based on bone-marrow morphology. The patient then received sequential monthly therapy using arsenic trioxide, followed by ATRA, followed by chemotherapy; he has survived disease-free for 36 months. Our findings suggest that the additional chromosomal abnormalities involving the sex chromosomes and chromosome 21 did not affect the prognosis of APL, and that the sequential treatment strategy had a good clinical effect without being associated with severe side-effects.

The expression and clinical significance of CDX2 and WT1 gene in children acute lymphoblastic leukemia

Aims: To investigate the distribution of food allergens in pediatric allergic rhinitis. Methods: 106 children diagnosed with allergic rhinitis were reviewed and divided into two age groups, including preschooler (2-5 years old) group, 48 cases; and school children group (6-13 years), 58 cases. Allergen-specific IgE for the food allergens and inhalant allergens in patient’s serum was determined using the AllergyScreen system (Mediwiss Analytic GmbH, Germany). Results: Out of 106 AR children, 88 (83.02%) were tested positive for food allergen-specific serum IgE (sIgG), 97 (91.51%) positive for inhalant allergen-specific sIgE. Among these children, 79 (74.53%) were positive for both food and inhalant allergens, 9 (8.49%) were positive only for food allergens, and 18 (16.98%) were negative for food allergens but positive for inhalant allergens. Out of 88 children positive for food allergens, 34 (32.08%) were positive for single food allergen, 54 (50.94%) were positive for multiple food allergens. The top 6 food allergens, in the order of their positivity rates, were milk (52.83%), eggs (30.19%), beef (26.42%), cashew (26.42%), lamb (12.26%) and crabs (10.38%). The food allergen-positive rate in the preschooler group was higher than that in the school children group (P<0.05). Conclusion: Majority of the children with allergic rhinitis are also positive for food allergens, suggesting that food allergens might be one of the main causes for pediatric allergic rhinitis.The present study was undertaken to ascertain the affinity of the amino acid D-Tryptophan (D-Trp) towards the particular receptors on the melanophores of a major carp Labeo rohita (Ham.). D-Trp, which is sweet in taste, in the dose range of 4.9 x 10 -16 M to 4.9 x 10 -4 M has induced a dose-dependent aggregation (MSI 3.72 ± 0.15 to 1.09 ± 0.02) in the isolated scale melanophores of the dorso-lateral region of this fish. The precise binding of the ligand D-Trp with its receptors is partially dependent, in eliciting its actions, on the release of neurotransmitters. The melanophores do not get desensitized under 90 min incubation within the agonist D-Trp. The general α adrenoceptors as well as the α 2 adrenoceptors are involved in the tastant (D-Trp) induced aggregation of the melanophores. D-Trp demonstrates that its aggregatory effects are mediated with the intervention of the β adrenoceptors. Inhibition of the D-Trp induced aggregatory effect has also been observed when the melanophores were pretreated with the antibiotic neomycin. The aggregating nature of D-Trp suggests that it could be binding with the receptors of aggregation causing ligands.

Cytogenetic Analysis of 55 Cases of Philadelphia Chromosome (Ph+) Leukemia

In this work, we isolated and characterized the first factor X activator from Bothropoides jararaca (= Bothrops jararaca ) venom, Bojaractivase X, which is probably involved in the genesis of the envenomation process. Bojaractivase X was purified by a combination of gel filtration and ion exchange chromatographies on Superdex HR 75 10/30 and HiTrap SP FF, respectively. Bojaractivase X consists of a single polypeptide chain with molecular mass of 28 kDa determined by SDS-PAGE. The enzyme shows maximum activity on factor X activation at pH 7.0 and 37°C. The activator converts factor X to its active form, factor X a , in the presence of Ca 2+ ions. Bojaractivase X also degrades the Aα, Bβ and γ -chain of fibrinogen molecule as well as casein. Inhibition of Bojaractivase X amidolytic activity by benzamidine suggests that it is a serine proteinase. Mass spectrometry analysis showed similarity between Bojaractivase X and a venom serine protease homolog from B. jararacussu .C. parapsilosis and C. orthopsilosis emerged as fungal pathogen with significant worldwide prevalence, particularly in causing nosocomial and skin infections. In this study, we aimed to develop molecular assay based on real-time PCR for sensitive and accurate detection of C. parapsilosis and C. orthopsilosis . A pair of primers that specifically target on both of these yeast species was designed and real-time PCR amplification assay was optimized using EvaGreen as the DNA binding dye. The optimized assay could detect and quantify up to 1 pg concentration of C. parapsilosis and C. orthopsilosis DNA with amplification efficiency of 104% and 103%, respectively. Both the designed primers and the quantitative assay will have a great potential as molecular diagnosis tool for early detection of fungal infection caused by either C. parapsilosis or C. orthopsilosis , which merits future clinical study prior to use in diagnosis.Sirenomelia or the “mermaid syndrome” is a rare entity. Malformations of almost every system have been reported in sirenomelia and it is invariably incompatible with survival; most babies are stillborn, or die shortly after birth. Isolated levocardia is an extremely rare condition in which the heart is located in the normal position while abdominal viscera are inverted .We report a case of symelia unipus having situs inversus of abdominal viscera with isolated levocardia, a ventricular septal defect, bilateral genitourinary agenesis and thoracic vertebral defects.[Abstract] Objective To explore the expression of IL-27, Th17 cells and their related cytokines IL- 17 in peripheral blood of patients with allergic rhinitis (allergic rhinitis, AR). Method 18 Cases of allergic rhinitis patients (10 males, 8 females) whose allergen was dust mite were collected from April to June 2012 as the AR group, and 10 cases of healthy volunteers (4 males, 6 females) without allergic diseases were put into the control group. IL-27 and IL-17 levels in serum of peripheral blood of the two groups were detected by ELISA, and the percentage of Th17 cell was detected by flow cytometry. Result IL-27 levels of AR group and control group were (21.69 ± 12.62) pg / ml and (53.10 ± 12.55) pg / ml respectively, and the difference was statistically significant (P <0.01); IL-17 levels of AR group and control group were (672.82±63.45) pg / ml and (576.62±22.81) pg / ml respectively, and the difference was statistically significant (P <0.01); Th17 cell percentage of AR group was 1.76 ± 0.60%, and in the control group it was 0.59 ± 0.17%. The difference between the two groups was statistically significant (P <0. 01). IL-27 was negatively correlated to Th17 cell and IL-17 (r was -0.361 and -0.435 respectively, P <0 05). Conclusion The reduction of IL-27 level in the peripheral blood of patients with allergic rhinitis, the increase of Th17 cells percentage and IL-17 level, as well as the negative correlation of IL-27 with Th17 cell and IL-17 suggest that decline of IL-27 suppression to Th17 cell may play an important role in the pathogenesis of allergic rhinitis.