OBJECTIVETo explore the effects of resveratrol-induced apoptosis and autophagy in T-cell acute lymphoblastic leukemia (T-ALL) cells and potential molecular mechanisms.

METHODSThe anti-proliferation effect of resveratrol-induced, apoptosis and autophagy on T-ALL cells were detected by using MTT test, immunofluorescence, electronic microscope, and flow cytometry, respectively. Western blotting was performed for detecting changes of apoptosis-associated proteins, cell cycle regulatory proteins and state of activation of Akt, mTOR, p70S6K, 4E-BP1, and p38-MAPK.

RESULTSResveratrol inhibited the proliferation and induced apoptosis and autophagy in T-ALL cells in a dose and time-dependent manner. It also induced cell cycle arrest at G0/G1 phase via up regulating cyclin-dependent kinase (CDK) inhibitors p21 and p27 and down regulating cyclin A and cyclin D1. Western blotting revealed that resveratrol significantly decreased the expression of antiapoptotic proteins (Mcl-1 and Bcl-2) and increased the expression of proapoptotic proteins (Bax, Bim, and Bad), and induced cleaved-caspase-3 in a time-dependent manner. Significant increase in ratio of LC3-II/LC3-I and Beclin 1 was also detected. Furthermore, resveratrol induced significant dephosphorylation of Akt, mTOR, p70S6K, and 4E-BP1, but enhanced specific phosphorylation of p38-MAPK which could be blocked by SB203580. When autophagy was suppressed by 3-MA, apoptosis in T-ALL cells induced by resveratrol was enhanced.

CONCLUSIONOur findings have suggested that resveratrol induces cell cycle arrest, apoptosis, and autophagy in T-ALL cells through inhibiting Akt/mTOR/p70S6K/4E-BP1 and activating p38-MAPK signaling pathways. Autophagy might play a role as a self-defense mechanism in T-ALL cells treated by resveratrol. Therefore, the reasonable inhibition of autophagy in T-ALL cells may serve as a promising strategy for resveratrol induced apoptosis and can be used as adjuvant chemotherapy for T-ALL.

Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Autophagy ; drug effects ; Cell Culture Techniques ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Flow Cytometry ; Humans ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; enzymology ; pathology ; Proto-Oncogene Proteins c-akt ; antagonists & inhibitors ; Stilbenes ; pharmacology ; T-Lymphocytes ; drug effects ; enzymology ; ultrastructure ; TOR Serine-Threonine Kinases ; antagonists & inhibitors ; p38 Mitogen-Activated Protein Kinases ; metabolism

Potential toxicological interactions of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and/or dibuthyl phthalate (DBP) on ozone were investigated after 32- and 52-wk exposures using hprt mutation assay. Male and female B6C3F1 mice exposed to ozone (0.5 ppm), NNK (1.0 mg/kg), DBP (5,000 ppm), and two or three combinations of these toxicants 6 h per day for 32- and 52-wk showed increases in the frequencies of TG rlymphocytes compared to the control groups. Additive interactions were noted from two combination groups compared to the ozone alone in both sexes of 32- and 52-wk studies. The most common specific mutation type in the hprt genes of test materials-treated male and female mice was transversion with very few transition. The results indicate that such dominant transversion may be responsible for toxicity and combined exposure to ozone, NNK, and DBP induces additive genotoxicities compared to ozone alone.
Animals ; Carcinogens/*toxicity ; DNA Mutational Analysis ; Dibutyl Phthalate/*toxicity ; Drug Combinations ; Female ; Hypoxanthine Phosphoribosyltransferase/*genetics ; Male ; Mice ; Mutagenicity Tests ; *Mutation/drug effects ; Nitrosamines/*toxicity ; Ozone/*toxicity ; Reverse Transcriptase Polymerase Chain Reaction ; T-Lymphocytes/drug effects/enzymology

OBJECTIVEEscape from the body's immune response is a basic characteristic of lung cancer, and indoleamine-2,3-dioxygenase (IDO) plays a key role in mediating immune escape of non-small-cell lung cancer, which leads to recurrence and metastasis. Feiji Recipe, a compound Chinese herbal medicine, has the effect of stabilizing lesions and prolonging survival in patients with lung cancer. The purpose of this study was to investigate the mechanisms underlying the anticancer properties of Feiji Recipe.

METHODSAn orthotopic transplant model of mouse Lewis lung cancer, with stable expression of IDO gene, was established in C57BL/6 mice. Optical imaging was used to observe the effects of Feiji Recipe in the treatment of lung cancer in vivo. The effects of Feiji Recipe on the proliferation of mouse Lewis lung cancer cell line 2LL, 2LL-enhanced green fluorescent protein (2LL-EGFP) and 2LL-EGFP-IDO were investigated, and the apoptosis of T-cells was examined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide using flow cytometry. Chemical composition of Feiji Recipe was validated by high-performance liquid chromatography.

RESULTSCompared to the control group, the survival of animals treated with Feiji Recipe was significantly prolonged (P = 0.0074), and the IDO protein level decreased (P = 0.0072); moreover, the percentages of CD4CD25 T-cells and Foxp3 T-cells were significantly decreased (P < 0.05). The molecular mechanism of Feiji Recipe against lung cancer may relate to the regulation of immune cells, such as T-cells and regulatory T-cells.

CONCLUSIONThe molecular mechanism of Feiji Recipe in treatment of lung cancer is to restore the function of T-cells in the cancer microenvironment through interfering with the IDO pathway.

Animals ; Apoptosis ; drug effects ; Carcinoma, Lewis Lung ; drug therapy ; enzymology ; immunology ; physiopathology ; Cell Proliferation ; drug effects ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Growth Inhibitors ; administration & dosage ; Humans ; Indoleamine-Pyrrole 2,3,-Dioxygenase ; genetics ; immunology ; Lung Neoplasms ; drug therapy ; enzymology ; immunology ; physiopathology ; Male ; Mice ; Mice, Inbred C57BL ; T-Lymphocytes, Regulatory ; drug effects ; immunology

OBJECTIVETo investigate the influence of endothelial dysfunction induced by inoculated dendritic cells (DCs) loaded heat shock protein 60 (HSP60) in apolipoprotein (Apo) E-null mice, and the effect of Puerarin on it.

METHODSHSP60 DC (DChsp) acquired after prepared bone marrow-derived DCs of ApoE-null mice and treated with HSP60. In vitro, the function of DCs and the effect of Puerarin were detected. While in vivo, ApoE-null mice fed with high-cholesterol forage were divided into two groups and intravenous inoculated with DCh-sp or normal saline via vein twice respectively. The mice in the two groups were subdivided into the Puerarin group and non-treated group, and they were injected intraperitoneally with Puerarin and normal saline at the beginning of inoculation and the following 3 weeks, respectively. In addition, C57BL/6 mice without inoculation were taken as the normal control group. Two weeks after the last time inoculated, the response of T lymphocytes to HSP60 and endothelial-dependent diastolic function of aortic ring were detected.

RESULTSHSP60 could promote DCs expressed CD86 and stimulate T lymphocytes proliferation in vitro, while Puerarin had significantly inhibitory effect. After inoculated, DChsp activated inflammatory response in vivo and aggravated endothelium-dependent dilation in mice. Puerarin could significantly inhibit inflammatory reaction caused by DChsp and improve endothelium dilation.

CONCLUSIONHsp60 could activate DCs in vitro and in vivo, Puerarin could significantly inhibit specific immunity induced by HSP60 and improve vascular endothelium-dependent dilation.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Apolipoproteins E ; genetics ; B7-2 Antigen ; immunology ; Cell Proliferation ; drug effects ; Chaperonin 60 ; metabolism ; Dendritic Cells ; drug effects ; enzymology ; immunology ; Endothelium, Vascular ; drug effects ; physiology ; Immunity ; drug effects ; Inflammation ; chemically induced ; Isoflavones ; pharmacology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Protective Agents ; pharmacology ; T-Lymphocytes ; drug effects ; Vasodilator Agents ; pharmacology

We evaluated the effectiveness of rhamnogalacturonan II (RG-II)-stimulated bone marrow-derived dendritic cells (BMDCs) vaccination on the induction of antitumor immunity in a mouse lymphoma model using EG7-lymphoma cells expressing ovalbumin (OVA). BMDCs treated with RG-II had an activated phenotype. RG-II induced interleukin (IL)-12, IL-1beta, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) production during dendritic cell (DC) maturation. BMDCs stimulated with RG-II facilitate the proliferation of CD8+ T cells. Using BMDCs from the mice deficient in Toll-like receptors (TLRs), we revealed that RG-II activity is dependent on TLR4. RG-II showed a preventive effect of immunization with OVA-pulsed BMDCs against EG7 lymphoma. These results suggested that RG-II expedites the DC-based immune response through the TLR4 signaling pathway.
Acute-Phase Proteins/metabolism ; Adaptor Proteins, Vesicular Transport/metabolism ; Animals ; Antigens, CD14/metabolism ; Bone Marrow Cells/cytology/drug effects ; CD8-Positive T-Lymphocytes/*immunology ; Carrier Proteins/metabolism ; Cell Differentiation/drug effects ; Cell Nucleus/drug effects/metabolism ; Cell Proliferation/drug effects ; Cytokines/biosynthesis ; Dendritic Cells/cytology/drug effects/enzymology/*immunology ; Enzyme Activation/drug effects ; Lymphocyte Activation/*drug effects ; Membrane Glycoproteins/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mitogen-Activated Protein Kinases/metabolism ; Myeloid Differentiation Factor 88/metabolism ; NF-kappa B/metabolism ; Neoplasms/immunology/*pathology ; Pectins/*pharmacology ; Phenotype ; Protein Transport/drug effects ; Receptors, Chemokine/metabolism ; Signal Transduction/drug effects ; T-Lymphocytes, Cytotoxic/cytology/drug effects ; Toll-Like Receptor 4/*agonists/metabolism

OBJECTIVETo study the anti-tumor immunotherapeutic effect induced by the suicidalcancer vaccine FC/TK, and to evaluate the safety of this vaccine.

METHODSThe suicidal cancer vaccine, named FC/TK, was prepared by fusion of suicide gene (HSVI,-TK gene) -modified ovarian carcinoma NuTu-19 cells with rat bone marrow-derived dendritic cells (DCs). The morphology of FC/TK was evaluated by scanning electron microscopy. The stimulatory effect of FC/TK on T cells was determined by T cell proliferation assay. In immunotherapeutic studies in vivo, Fischer344 rats were injected subcutaneously with NuTu-19 cells, followed by treatment of FC/TK on days 7 and 14, compared to controls treated with irradiated FC/TK, FC or PBS, respectively. Tumor incidence and volume were measured in 90 days after challenge. To determine the killing effect of FC/TK in vivo, TUNEL assays were applied to detect apoptotic cell death in spleen of vaccinated rats with prodrug ganciclovir administration.

RESULTSFC/TK cells were of irregular shape with surface membrane processes. Compared to the control groups, FC/TK significantly promoted T cell proliferation (P <0.01). The rats vaccinated with FC/TK and FC significantly inhibited the tumor growth compared to rats vaccinated with irradiated FC/TK (P <0.05) or with PBS ( P <0.01). The immunotherapeutic effect induced by FC/TK was similar to that using FC. Fluorescence microscopy showed that fluorescein-stained FC/TK cells migrated into spleen also showed to be TUNEL-positive, suggesting that the FC/TK cells were killed by ganciclovir in vivo.

CONCLUSIONOur data indicate that suicidal cancer vaccine is an effective and safe therapy for ovarian carcinoma and may serve as a broadly applicable approach for other cancer vaccines in the future.

Animals ; Apoptosis ; drug effects ; Cancer Vaccines ; immunology ; Cell Fusion ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dendritic Cells ; cytology ; immunology ; Female ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; Herpesvirus 1, Human ; enzymology ; genetics ; Immunotherapy ; methods ; Microscopy, Electron, Scanning ; Microscopy, Fluorescence ; Neoplasms, Experimental ; enzymology ; pathology ; therapy ; Ovarian Neoplasms ; enzymology ; pathology ; therapy ; Rats ; Rats, Inbred F344 ; Survival Analysis ; T-Lymphocytes ; drug effects ; metabolism ; pathology ; Thymidine Kinase ; genetics ; metabolism ; Transfection

OBJECTIVETo explore relationship between effect of Lamivudine in the treatment of cirrhotic patients with uncompensated hepatitis B with hepatitis B virus (HBV)genotypes and HBV specific cytotoxic T lymphocytes (CTL).

METHODS80 cases of uncompensated cirrhotic hepatitis B (40 cases with genotype B and 40 with genotype C), HBV DNA positive, HBeAg positive and human leukocyte antigen (HLA)-A2 positive,were treated with Lamivudine 100 mg/d, one year later, its effect and relationship with HBV genotypes and HBV specific CTL were observed.

RESULTSHBV DNA turned negative:40 cases with genotype B turned negative (100%). In the 9th and 10th month of treatment, there was one case with genotype C had YMDD variation respectively and Adefovir dipivoxil was used for treatment, of the rest 38 cases, HBV DNA of 26 cases (68.42%) turned negative,HBV DNA negative rate of patients with genotype is lower than that of patients with genotype B, chi2 = 14.91, P < 0.01. HBeAg turned negative: 18 cases with genotype B (45%) turned negative, more than that of patients with genotype C (7 cases, 18.42%), chi2 = 6.32, P < 0.05. Peripheral blood HBV specific CTL level: before treatment, it was (0.33 +/- 0.03)% of patients with genotype B,higher than that of patients with genotype C [(0.11 +/- 0.02)%], t = 8.12, P < 0.001. 1 year after treatment: it was (0.44 +/- 0.04)% of patients with genotype B, higher than that before treatment, t = 4.01, P < 0.001, it was also higher than that of patients with genotype C 1 year after treatment [(0.23 +/- 0.03)%], t = 5.63, P < 0.01, alanine amino-transferase (ALT) returned to normal: 38 cases with genotype B (95%) returned to normal, more than that of patients with genotype C (28 cases, 73.68%), X2 = 6.79, P < 0.01.

CONCLUSIONEffect of Lamivudinein the treatment of cirrhotic patients with uncompensated hepatitis B is better in patients with genotype B than patients with genotype C, its mechanism may be related to lower level of HBV specific CTL in patients with genotype C than patients with genotype B.

Adult ; Aged ; Alanine Transaminase ; metabolism ; Antiviral Agents ; therapeutic use ; Female ; Genotype ; Hepatitis B ; drug therapy ; enzymology ; immunology ; virology ; Hepatitis B virus ; drug effects ; genetics ; isolation & purification ; Humans ; Lamivudine ; therapeutic use ; Liver Cirrhosis ; drug therapy ; enzymology ; immunology ; virology ; Male ; Middle Aged ; T-Lymphocytes, Cytotoxic ; immunology

OBJECTIVETo explore and evaluate the activities of composite extract from Salvia Yunnanensis and in cell cultures (DS-MEF) for inhibition of human immuno-deficiency virus type 1 (HIV-1) in vitro and in cell cultures.

METHODSThe inhibitory activity of DS-MEF on HIV-1 reverse transcriptase (RT), protease (PR) and integrase (IN) were detected in vitro with radionuclide 3H incorporation, fluorescence assay and enzyme-linked immunosorbent assay respectively. The human T-lymphocyte MT-4 cell line, human T-lymphocyte H 9 cell line chronically infected with HIV-1 IIIB, and the fresh peripheral blood mononuclear cell (PBMC) of healthy persons as well as the laboratory passed HIV-1 IIIB and the clinically isolated HIV-1 AZT sensitive 018a or resistant 018c infected cell cultures were used for evaluating the cytotoxicities and inhibitory activities of DS-MEF on HIV-1 P 24 antigen. The acute toxicities of DS-MEF on KM mice were determined by gastric gavages and intraperitoneal injections with various dosages.

RESULTSThe IC50 of DS-MEF for inhibiting HIV-1 IN, RT and PR were 2.59 +/- 0.50 mg/L, 27.39 +/- 11.18 mg/L and 9.38 +/- 2.45 mg/L respectively. In MT-4 cell cultures infected with HIV-1 III, TC50 were 13.19 +/- 6.07 mg/L, IC50 and SI of anti-HIV-1 activity were 0.224 +/- 0.163 mg/L and 58.7; in chronically infected H 9 cell cultures, TC50 were 18.11 +/- 9.84 mg/L, IC50 on HIV-1 P 24 antigen and SI were l7.230 +/- 21.114 mg/L and 1.1 respectively; TC50 in HIV-1 infected PBMC cultures were 288.70 +/- 0.08 mg/L; IC50 on AZT sensitive HIV-1 018a: 26.42 +/- 11.16 mg/L, and SI: 10.9; On AZT resistant HIV-1 018c, IC50: 27.87 +/-5.35 mg/L, and SI: 10.4. Moreover, DS-MEF showed synergistic effect with AZT or nevirapine (NVP) on HIV-1 IIIB in MT-4 cell cultures, the respective combination index was 0.78 or 0.67. DS-MEF showed no acute toxicity in KM mice with the dosage up to 20 g/kg via gastrogavage, and the 50% lethal dose (LD50) via intraperitoneal injection was 1.18 g/kg.

CONCLUSIONDS-MEF is a promising anti- HIV-1 agent with low toxicity in mice and possesses multi-targets and effective activities.

Animals ; Anti-HIV Agents ; pharmacology ; therapeutic use ; Cell Line ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; HIV Infections ; drug therapy ; virology ; HIV-1 ; drug effects ; enzymology ; physiology ; Humans ; Leukocytes, Mononuclear ; virology ; Male ; Mice ; Mice, Inbred BALB C ; Salvia ; chemistry ; T-Lymphocytes ; virology ; Viral Proteins ; antagonists & inhibitors ; Virus Replication ; drug effects

OBJECTIVETo study the effect of dendritic cells (DC) stimulated with K562 cell lysate in inducing specific cytotoxic T lymphocytes (CTL) against K562 cells in vitro.

METHODSThe DCs were derived from healthy human peripheral blood monocytes in the presence of granulocyte-macrophage colony-stimulating factor, interleukin (IL)-4 and tumor necrosis factor (TNF) alpha. The T cells were stimulated by DCs loaded with freeze-thawed K562 cells and T-cell cytotoxicities were measured by lactate dehydrogenase (LDH) assay.

RESULTSThe DCs could be successfully obtained from peripheral blood monocyte after the culture. Mixed lymphocyte reactions induced by the antigen-loaded DC were much stronger than those induced by human peripheral blood monocytes (P<0.05). At the effector to target ratio of 10:1 and 20:1, cytotoxicities against K562 cells by CTL derived from culture with the antigen-loaded DCs were the strongest (P<0.05).

CONCLUSIONCTL derived from DCs pulsed with K562 cell lysate show effective and specific cytotoxicity against K562 cells.

Antigens ; immunology ; Cytotoxicity, Immunologic ; immunology ; Dendritic Cells ; drug effects ; immunology ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Interleukin-4 ; pharmacology ; K562 Cells ; L-Lactate Dehydrogenase ; metabolism ; Lymphocyte Activation ; immunology ; Lymphocyte Culture Test, Mixed ; T-Lymphocytes, Cytotoxic ; enzymology ; immunology ; Tumor Necrosis Factor-alpha ; pharmacology

BACKGROUNDOverwhelming evidences on chronic myeloid leukemia (CML) indicate that patients harbor quiescent CML stem cells that are responsible for blast crisis. While the hematopoietic stem cell (HSC) origin of CML was first suggested over 30 years ago, recently CML-initiating cells beyond HSCs are also being investigated.

METHODSWe have previously isolated fetal liver kinase-1-positive (Flk1(+)) cells carrying the BCR/ABL fusion gene from the bone marrow of Ph(+) patients with hemangioblast property. In this study, we isolated CML patient-derived Flk1(+)CD31(-)CD34(-) mesenchymal stem cells (MSCs) and detected their biological characteristics and immunological regulation using fluorescence in situ hybridization (FISH) analysis, fluorescence activated cell sorting (FACS), enzyme-linked immunoadsorbent assay, mixed lymphocyte reaction assays; then we compared these characters with those of the healthy donors.

RESULTSCML patient-derived Flk1(+)CD31(-)CD34(-) MSCs had normal morphology, phenotype and karyotype while appeared impaired in immuno-modulatory function. The capacity of patient Flk1(+)CD31(-)CD34(-) MSCs to inhibit T lymphocyte activation and proliferation was impaired in vitro.

CONCLUSIONSCML patient-derived MSCs have impaired immuno-modulatory functions, suggesting that the dysregulation of hematopoiesis and immune response may originate from MSCs rather than hematopoietic stem cells (HSCs). MSCs might be a potential target for developing efficacious treatment for CML.

Adolescent ; Adult ; Antigens, CD34 ; genetics ; metabolism ; Apoptosis ; drug effects ; Blotting, Western ; Cell Cycle ; drug effects ; Cells, Cultured ; Enzyme-Linked Immunosorbent Assay ; Female ; Flow Cytometry ; Fusion Proteins, bcr-abl ; genetics ; metabolism ; Humans ; Immunomodulation ; In Situ Hybridization, Fluorescence ; Karyotype ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; enzymology ; immunology ; metabolism ; Male ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Mesenchymal Stromal Cells ; cytology ; immunology ; Middle Aged ; Platelet Endothelial Cell Adhesion Molecule-1 ; genetics ; metabolism ; T-Lymphocytes ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; metabolism ; Young Adult