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1.
J Zhejiang Univ Sci B ; 20(4): 310-321, 2019.
Article in English | MEDLINE | ID: mdl-30932376

ABSTRACT

OBJECTIVE: Reactive oxygen species (ROS) are involved in a variety of biological phenomena and serve both deleterious and beneficial roles. ROS quantification and assessment of reaction networks are desirable but difficult because of their short half-life and high reactivity. Here, we describe a pro-oxidative model in a single human lung carcinoma SPC-A-1 cell that was created by application of extracellular H2O2 stimuli. METHODS: Modified microfluidics and imaging techniques were used to determine O2 •- levels and construct an O2 •- reaction network. To elucidate the consequences of increased O2 •- input, the mitochondria were given a central role in the oxidative stress mode, by manipulating mitochondria-interrelated cytosolic Ca2+ levels, mitochondrial Ca2+ uptake, auto-amplification of intracellular ROS and the intrinsic apoptotic pathway. RESULTS AND CONCLUSIONS: Results from a modified microchip demonstrated that 1 mmol/L H2O2 induced a rapid increase in cellular O2 •- levels (>27 vs. >406 amol in 20 min), leading to increased cellular oxidizing power (evaluated by ROS levels) and decreased reducing power (evaluated by glutathione (GSH) levels). In addition, we examined the dynamics of cytosolic Ca2+ and mitochondrial Ca2+ by confocal laser scanning microscopy and confirmed that Ca2+ stores in the endoplasmic reticulum were the primary source of H2O2-induced cytosolic Ca2+ bursts. It is clear that mitochondria have pivotal roles in determining how exogenous oxidative stress affects cell fate. The stress response involves the transfer of Ca2+ signals between organelles, ROS auto-amplification, mitochondrial dysfunction, and a caspase-dependent apoptotic pathway.


Subject(s)
Hydrogen Peroxide/chemistry , Mitochondria/metabolism , Oxidative Stress , Reactive Oxygen Species/metabolism , Superoxides/chemistry , Apoptosis , Calcium/metabolism , Calcium Signaling , Caspases/metabolism , Cell Line, Tumor , Cell Lineage , Cytosol/metabolism , Glutathione/metabolism , Humans , Oxidation-Reduction , Signal Transduction
2.
Genes Dev ; 31(3): 247-259, 2017 02 01.
Article in English | MEDLINE | ID: mdl-28223311

ABSTRACT

Tumor infiltrated type II (M2) macrophages promote tumorigenesis by suppressing immune clearance, promoting proliferation, and stimulating angiogenesis. Interestingly, macrophages were also found to enrich in small foci of altered hepatocytes containing liver tumor-initiating cells (TICs). However, whether and how TICs specifically recruit macrophages and the function of these macrophages in tumor initiation remain unknown due to technical difficulties. In this study, by generating genetically defined liver TICs, we demonstrate that TICs actively recruit M2 macrophages from as early as the single-cell stage. Elimination of TIC-associated macrophages (TICAMs) abolishes tumorigenesis in a manner dependent on the immune system. Mechanistically, activation of the Hippo pathway effector Yes-associated protein (YAP) underlies macrophage recruitment by TICs. These results demonstrate for the first time that macrophages play a decisive role in the survival of single TICs in vivo and provide a proof of principle for TIC elimination by targeting YAP or M2 macrophages.


Subject(s)
Carcinoma, Hepatocellular/immunology , Cell Transformation, Neoplastic/immunology , Hepatocytes/immunology , Liver Neoplasms/immunology , Macrophages/immunology , Neoplastic Stem Cells/immunology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Communication/immunology , Cell Cycle Proteins , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Cells, Cultured , Hepatocyte Growth Factor/physiology , Hepatocytes/metabolism , Hepatocytes/pathology , Homeodomain Proteins/physiology , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Macrophages/cytology , Male , Mice , Mice, Inbred ICR , Mice, Knockout , Neoplastic Stem Cells/cytology , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/physiology , Proto-Oncogene Proteins/physiology , Serine-Threonine Kinase 3 , Tumor Suppressor Protein p53/physiology , YAP-Signaling Proteins
3.
Cell Biol Int ; 40(7): 770-8, 2016 Jul.
Article in English | MEDLINE | ID: mdl-27079177

ABSTRACT

Oleanolic acid (OA), a plant-derived pentacyclic terpenoid, is known to have hepatoprotective effects. In this study, we found that OA induced autophagic cell death in multiple human gastric cancer cell lines. Moreover, OA-induced autophagy was shown for the first time in human gastric cancer cells, evidenced by the formation of GFP-RFP-LC3 puncta and autophagosomes. OA suppressed phospho-mTOR through inhibition of the PI3 K/AKT and ERK/p38 MAPK signalling pathways and through activation of the AMPK signalling pathway. Furthermore, we found that OA-induced cytotoxicity and autophagy could be blocked by the autophagy inhibitor 3-methyladenine or via siRNA targeting Beclin-1. Our in vivo research showed that OA delayed the formation of MGC-803 tumours in an autophagy-dependent manner. These results reveal a novel mechanism for OA in gastric cancer cells and suggest that OA could be a novel agent in the treatment of gastric cancer.


Subject(s)
Oleanolic Acid/pharmacology , Stomach Neoplasms/drug therapy , Animals , Apoptosis/drug effects , Apoptosis Regulatory Proteins/metabolism , Autophagy/drug effects , Beclin-1/genetics , Cell Line, Tumor , Female , Humans , MAP Kinase Signaling System/drug effects , Mice , Mice, Inbred BALB C , Mice, Nude , Proto-Oncogene Proteins c-akt/metabolism , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , Random Allocation , Signal Transduction/drug effects , Stomach Neoplasms/metabolism , Stomach Neoplasms/pathology , TOR Serine-Threonine Kinases/metabolism , Transfection , Xenograft Model Antitumor Assays
4.
Cell Res ; 25(9): 997-1012, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26272168

ABSTRACT

Organ size determination is one of the most intriguing unsolved mysteries in biology. Aberrant activation of the major effector and transcription co-activator YAP in the Hippo pathway causes drastic organ enlargement in development and underlies tumorigenesis in many human cancers. However, how robust YAP activation is achieved during organ size control remains elusive. Here we report that the YAP signaling is sustained through a novel microRNA-dependent positive feedback loop. miR-130a, which is directly induced by YAP, could effectively repress VGLL4, an inhibitor of YAP activity, thereby amplifying the YAP signals. Inhibition of miR-130a reversed liver size enlargement induced by Hippo pathway inactivation and blocked YAP-induced tumorigenesis. Furthermore, the Drosophila Hippo pathway target bantam functionally mimics miR-130a by repressing the VGLL4 homolog SdBP/Tgi. These findings reveal an evolutionarily conserved positive feedback mechanism underlying robustness of the Hippo pathway in size control and tumorigenesis.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Carcinogenesis , MicroRNAs/metabolism , Phosphoproteins/metabolism , Acyltransferases , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/genetics , Animals , Base Sequence , Cell Line, Tumor , Cell Proliferation , Cysteine-Rich Protein 61/antagonists & inhibitors , Cysteine-Rich Protein 61/genetics , Cysteine-Rich Protein 61/metabolism , Drosophila/metabolism , Drosophila Proteins/metabolism , Hep G2 Cells , Humans , Male , Mice , Mice, Inbred ICR , Mice, Nude , MicroRNAs/antagonists & inhibitors , Organ Size/genetics , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/genetics , Sequence Alignment , Signal Transduction , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Transcription Factors/metabolism , Transplantation, Heterologous , YAP-Signaling Proteins
5.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 42(5): 504-10, 2013 Sep.
Article in Chinese | MEDLINE | ID: mdl-24167130

ABSTRACT

OBJECTIVE: To investigate the effect of luteolin on cell growth and apoptosis of HepG2 cells in vitro. METHODS: Cultured HepG2,HL60,A549 and LO2 cells were treated with luteolin for different doses (0 µg/ml,2.5 µg/ml,10 µg/ml and 20 µg/ml) and varied times (0 h,24 h,48 h and 72 h). Cell viability was measured with MTT assay and IC50 was calculated. The reactive oxygen species (ROS) levels in HepG2 cells treated with luteolin for 6 h and 12 h were measured with flow cytometry. Cell apoptosis of HepG2 cells treated with luteolin for 24h was examined with flow cytometry and Annexin V-FITC/PI. Expression levels of apoptosis pathway proteins (p53,ASPP2 and iASPP) in HepG2 cells were detected with western blot and the dose and time-effect was analyzed. RESULTS: Luteolin effectively inhibited tumor cell proliferation in a dose-and time-dependent manner,and the inhibition rates of 20 µg/ml Luteolin for 72 h were 39.34%,62.90%,57.57% and 62.90% to LO2,HepG2, HL60 and A549 cells,respectively. The intracellular ROS level was decreased in HepG2 cells by 13.88% and 41.11% after being treated with luteolin for 6 h and 12 h,respectively. The apoptosis rate of HepG2 cells treated with luteolin for 24 h was 14.43%,and western blot showed that luteolin reduced the expression level of iASPP by 77.07% and up-regulated the expression of p53 by 179.37% and ASPP2 by 725.02% in HepG2 cells treated with luteolin for 12 h. CONCLUSION: Luteolin has ant-proliferative and pro-apoptotic activity on hepatoma HepG2 cells, which is associated with the altered expression of pro-apoptotic factors and decreased ROS level in HepG2 cells.


Subject(s)
Apoptosis/drug effects , Luteolin/pharmacology , Hep G2 Cells , Humans , Luteolin/administration & dosage , Reactive Oxygen Species/metabolism
6.
Biochem Biophys Res Commun ; 431(2): 169-75, 2013 Feb 08.
Article in English | MEDLINE | ID: mdl-23313479

ABSTRACT

Nutlin3, a non-genotoxic agonist of p53, is currently in phase II clinical trials for cancer treatment. However, its effects on normal tissues and cell types remain largely to be determined. Drugs that can selectively target cancer cells as well as cooperate with the p53 pathway are thus greatly needed. Iron-superoxide dismutase (Fe-SOD) is a potential candidate as it selectively targets cancer cells by eliminating the abnormally high levels of reactive oxygen species (ROS) in cancer cells; it also inhibits cancer cell growth by induction of p27. Here, we show evidence that modulating redox and ROS homeostasis cooperates with Nutlin3 to selectively inhibit cancer cells in vitro and in vivo. Co-treatment of Fe-SOD and Nutlin3 showed synergistic inhibition on cancer cells in vitro, and the induction of p27 appeared to be involved. No effects were observed on normal cells. In addition, such co-treatment further exhibited synergistic inhibition on tumor growth in vivo in a murine B16 xenograft model, while the individual treatments only achieved very limited inhibition. Thus, Fe-SOD cooperated with Nutlin3 to selectively inhibit cancer cells in vitro and in vivo.


Subject(s)
Imidazoles/administration & dosage , Neoplasms/drug therapy , Piperazines/administration & dosage , Superoxide Dismutase/administration & dosage , Animals , Cell Proliferation/drug effects , Cyclin-Dependent Kinase Inhibitor p27/biosynthesis , Drug Synergism , Female , Hep G2 Cells , Humans , Liposomes , MCF-7 Cells , Melanoma, Experimental/drug therapy , Mice , Reactive Oxygen Species/metabolism , Skin Neoplasms/drug therapy , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Xenograft Model Antitumor Assays
8.
Chem Biol Interact ; 193(3): 246-53, 2011 Sep 30.
Article in English | MEDLINE | ID: mdl-21835169

ABSTRACT

FLZ is a synthetic novel squamosamide derivative and has previously been proved to be a potential drug for Parkinson's disease and Alzheimer's disease. FLZ has strong antioxidant activity, which implies that FLZ could eliminate excessive intracellular reactive oxygen species (ROS) in tumor cells and induce a pathway related to low cellular ROS levels, thereby inhibiting tumor cells proliferation. However, few reports have focused on the antitumor effects of FLZ. In this study, we investigated the antitumor efficacy of FLZ in HepG2 cells and the mechanism of cell growth inhibition. FLZ effectively inhibited HepG2 cell proliferation in a dose- and time-dependent manner; meanwhile, it was minimally toxic to normal cells. FLZ induced a significant decrease in oxidative stress through elimination of excessive intracellular ROS and strengthening of the glutathione antioxidant system. In addition, FLZ can effectively attenuate redundant [Ca(2+)](i), thereby avoiding uncontrolled amplification by Ca(2+)/ROS positive feedback. Furthermore, Western blot showed that FLZ inhibited phosphorylation of Akt and retinoblastoma protein (Rb), down-regulated the expressions of cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2), and enhanced the expression of CDK inhibitor p27(kip1), while did not affect CDK4 expression. These results suggest that FLZ has potent anti-proliferative activity against malignant human hepatoma cells via modulation of the expression or activation of cell-cycle regulatory proteins, which are associated with decreased Ca(2+)/ROS levels, and indicate that FLZ is a potential liver cancer drug worthy of further research and development.


Subject(s)
Acrylamides/pharmacology , Antineoplastic Agents/pharmacology , Caffeic Acids/pharmacology , Calcium/metabolism , Carcinoma, Hepatocellular/drug therapy , Cell Cycle Proteins/metabolism , Liver Neoplasms/drug therapy , Reactive Oxygen Species/metabolism , Acrylamides/chemistry , Acrylamides/therapeutic use , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Caffeic Acids/chemistry , Caffeic Acids/therapeutic use , Carcinoma, Hepatocellular/metabolism , Cell Proliferation/drug effects , Cyclin D1/metabolism , Cyclin E/metabolism , Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase Inhibitor p27/metabolism , G1 Phase , Glutathione/metabolism , Hep G2 Cells , Humans , Liver Neoplasms/metabolism , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Retinoblastoma Protein/metabolism
9.
Chem Biol Interact ; 183(3): 341-8, 2010 Feb 12.
Article in English | MEDLINE | ID: mdl-20018182

ABSTRACT

3-(4-(Benzo[d]thiazol-2-yl)-1-phenyl-1H-pyrazol-3-yl) phenyl acetate (DPB-5) is a synthetic benzothiazole derivative. In the present study, we revealed that DPB-5 had strong cytotoxicity to induce cell apoptosis, which was mediated by ROS. And DPB-5 was more cytotoxic toward hepatoma cells than toward normal hepatic cells, which was resulted from the greater susceptibility of the malignant cells to ROS. DBP-5 caused massive ROS accumulation and GSH decrease, which lead to MMP disruption, caspase activation and finally induced cell apoptosis. Additionally, rotenone, an inhibitor of mitochondria electron transport system, effectively blocked the ROS elevated effect of DPB-5, which suggested that DPB-5-induced ROS generated from the mitochondria. Further studies showed that DPB-5-induced cell apoptosis through caspases-cascade, but failed to activate caspase-9. Hence, we concluded that DPB-5-induced Hep G2 cells apoptosis via a ROS-mediated pathway which was caspase-dependent but did not rely on caspase-9.


Subject(s)
Apoptosis , Benzothiazoles/toxicity , Pyrazoles/toxicity , Reactive Oxygen Species/metabolism , Benzothiazoles/chemistry , Caspase 9/metabolism , Drug Screening Assays, Antitumor , Glutathione/metabolism , Hep G2 Cells , Humans , Membrane Potential, Mitochondrial/drug effects , Pyrazoles/chemistry
10.
Chem Biol Interact ; 180(3): 383-8, 2009 Aug 14.
Article in English | MEDLINE | ID: mdl-19442654

ABSTRACT

Reactive oxygen species (ROS) are constantly generated and eliminated in the biological system and play important roles in a variety of physiological and pathological processes. Previous studies indicate that modulation of cellular ROS affects cell proliferation. Thymosin alpha 1 (Talpha1) is a naturally occurring thymic peptide and has previously been shown to be a potential therapy for some immunodeficiencies, malignancies, and infections. However, few reports have focused on manipulation of cellular ROS level effects of Talpha1. In this study, the Talpha1-treated leukomonocytes, which were isolated from mice spleens, exhibited a higher ROS level and a lower reduced glutathione (GSH) level; however, HepG2 cells treated with Talpha1 exhibited lower ROS level and higher GSH level. In addition, after treatment with Talpha1, the population of leukomonocytes in the G(2) phase increased, resulting in a slight increase in viability. However, in Talpha1-treated HepG2 cells, the cell cycle was delayed in the G(1) phase, thereby inhibiting tumor cell proliferation; in addition, dephosphorylation of the serine/threonine kinase Akt was detected. In conclusion, we show that Talpha1 has potent anti-proliferative activity against malignant human hepatoma cells and proliferative activity against leukomonocytes associated with manipulation of oxidative stress levels which indicates the potential of Talpha1 as an antitumor drug.


Subject(s)
Antineoplastic Agents/pharmacology , Reactive Oxygen Species/metabolism , Thymosin/analogs & derivatives , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , G1 Phase , G2 Phase , Glutathione/metabolism , Humans , Mice , Monocytes/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Thymalfasin , Thymosin/pharmacology
11.
J Biomed Biotechnol ; 2008: 736060, 2008.
Article in English | MEDLINE | ID: mdl-18645619

ABSTRACT

Human thymosin alpha 1 (Talpha1) is an important peptide in the development and senescence of immunological competence in human, and many studies have reported the expression of this peptide. In this study, we designed and synthesized the Talpha1 gene according to the E. coli codon usage preference and constructed a 6xTalpha1 concatemer. The latter was inserted into an E. coli expression vector pET-22b (+), and transformed into E. coli BL21 (DE3). After induction with IPTG, the concatemer protein was successfully expressed in E. coli then cleaved by hydroxylamine to release the Talpha1 monomer. Gly-SDS-PAGE and mass spectrometry confirmed that the recombinant protein was cleaved as intended. The bioactivity of the Talpha1 monomer was analyzed by lymphocyte proliferation and by mitochondrial activity in two different tumor cell lines. This study provides a description of the preparation of a bioactive Talpha1, which may prove useful in future biomedical research.


Subject(s)
Hydroxylamine/pharmacology , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Thymosin/analogs & derivatives , Amino Acid Sequence , Animals , Cell Line, Tumor , Cell Proliferation , Cells, Cultured , Escherichia coli/genetics , Formazans/metabolism , Gene Expression/drug effects , Humans , Inclusion Bodies/chemistry , Isopropyl Thiogalactoside/pharmacology , Lymphocytes/cytology , Lymphocytes/drug effects , Lymphocytes/physiology , Mice , Mice, Inbred BALB C , Mitochondria/drug effects , Mitochondria/physiology , Molecular Sequence Data , Molecular Weight , Peptide Fragments/chemistry , Peptide Fragments/genetics , Protein Structure, Secondary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Tetrazolium Salts/metabolism , Thymalfasin , Thymosin/chemical synthesis , Thymosin/chemistry , Thymosin/genetics , Thymosin/metabolism
12.
Biol Pharm Bull ; 31(6): 1075-8, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18520033

ABSTRACT

Two anthraquinones which inhibit activity of the Src tyrosine kinase were isolated from a water extract of Hedyotis diffusa WILLD. and identified as 2-hydroxy-3-methylanthraquinone (compound 1) and 1-methoxy-2-hydroxyanthraquinone (compound 2). Both compounds showed inhibitory activity against protein tyrosine kinases v-src and pp60src and arrested the growth of SPC-1-A, Bcap37 and HepG2 cancer cells. Observation of mitochondrial membrane potential collapse and caspase-3 activation following treatment with the compounds indicates that their apoptotic induction activity may act via the mitochondrial apoptotic pathway. Compared with compound 2, compound 1 is more active as an antagonist of Src kinase, which might account for its higher potency to induce growth arrest and apoptosis. These results provide a deeper insight into the functions of these two simple anthraquinones and the anti-tumour pathway of Hedyotis diffusa WILLD.


Subject(s)
Anthraquinones/pharmacology , Antineoplastic Agents, Phytogenic , Apoptosis/drug effects , Hedyotis/chemistry , Annexin A5 , Bisbenzimidazole , Caspase 3/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Coloring Agents , Fluorescein-5-isothiocyanate/analogs & derivatives , Fluorescent Dyes , Humans , Magnetic Resonance Spectroscopy , Mass Spectrometry , Membrane Potentials/drug effects , Mitochondrial Membranes/drug effects , Prohibitins , Rhodamine 123 , Spectrophotometry, Infrared , Spectrophotometry, Ultraviolet , Tetrazolium Salts , Thiazoles , src-Family Kinases/antagonists & inhibitors
13.
Cell Biol Int ; 32(3): 337-43, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18262806

ABSTRACT

Sarsasapogenin is a steroidal sapogenin with antitumor properties. To explain the mechanism of its apoptotic effect, mitochondrial activity was assessed via a 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Flow cytometry (FCM) was used to estimate the changes in mitochondrial membrane potential (MMP), reactive oxygen species (ROS) generation, and cellular-reduced glutathione (GSH) level. Laser scanning confocal microscope (LSCM) recorded instantaneous ROS burst after application of sarsasapogenin. Western blotting was used to determine the expression level and intracellular distribution of cytochrome c (cyt c). It is demonstrated that during apoptosis, ROS burst acted as an early event followed by depolarization of MMP, prolonged ROS generation, and significantly declined GSH level. Cyt c was upregulated and released from mitochondria to cytosol during the process. These findings show that a mitochondrial ROS burst is an early upstream apoptotic signal which may trigger the mitochondrial apoptotic pathway and play a vital role in sarsasapogenin-induced HepG2 cell apoptosis.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Carcinoma, Hepatocellular/drug therapy , Liver Neoplasms/drug therapy , Mitochondria/drug effects , Reactive Oxygen Species/metabolism , Spirostans/pharmacology , Blotting, Western , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cytochromes c/metabolism , Flow Cytometry , Glutathione/metabolism , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Membrane Potential, Mitochondrial/drug effects , Microscopy, Confocal , Mitochondria/enzymology , Mitochondria/metabolism , Mitochondria/pathology , Oxidative Stress/drug effects , Time Factors
14.
Appl Microbiol Biotechnol ; 76(1): 193-201, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17387468

ABSTRACT

The coding sequence for an iron superoxide dismutase (fe-sod) was amplified from the Nostoc commune genome. Recombinant Fe-SOD was overexpressed in Escherichia coli, accounting for approximately 76% of total bacterial protein. Fe-SOD was purified from bacterial lysate by Ni-NTA column chromatography and used to generate an anti-SOD antibody. The purified Fe-SOD was encapsulated in liposomes and delivered to HepG2 liver tumor cells to eliminate cellular superoxide anions. The SOD-loaded cells exhibited lower reactive oxygen species (ROS) levels and higher reduced glutathione (GSH) levels. In Fe-SOD-treated cells, the cell cycle was delayed in the G(1) phase, and HepG2 cell growth slowed in association with dephosphorylation of the serine-threonine kinase Akt. Low-dose H(2)O(2) stimulated Akt phosphorylation, implying that Akt activation in HepG2 cells is redox-sensitive. Akt phosphorylation was abrogated by phosphatidylinositol 3-kinase (PI3K) inhibitors, suggesting that PI3K is an upstream mediator of Akt activation in HepG2 cells. This study provides insight into recombinant Fe-SOD-induced signaling mechanisms in liver tumor cells and suggests the feasibility of using Fe-SOD as an antitumor agent.


Subject(s)
Cell Proliferation/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Recombinant Proteins/pharmacology , Superoxide Dismutase/pharmacology , Cell Line, Tumor/cytology , Humans , Nostoc commune/metabolism , Oxidation-Reduction , Oxidative Stress/physiology , Protein Serine-Threonine Kinases/metabolism , Reactive Oxygen Species/metabolism , Recombinant Proteins/biosynthesis , Signal Transduction , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism
15.
J Chromatogr A ; 1135(1): 109-14, 2006 Nov 24.
Article in English | MEDLINE | ID: mdl-17005186

ABSTRACT

An intracellular derivatization method mediated by liposome was developed for single cell analysis with chip-based capillary electrophoresis (CE) and laser-induced fluorescence (LIF) detection. Liposomes with an average diameter of 100 nm were produced from phosphatidylcholine to encapsulate fluorescent dyes by an ultrasonic method. The encapsulation yield and the vesicle density were determined to be 46+/-5% and 8.8 x 10(14)/mL, respectively. The amount of fluorescent dye that entered the cells was dependent on the duration of incubating cells with liposomes, liposome density, and concentration of the dye solution encapsulated in liposomes. The described method introduced cell membrane nonpermeable fluorescent dyes into living cells without reducing cell viability. Single cell analysis using microfluidic chip-based CE revealed that liposome-membrane fusion occurred after entrance of liposomes into the cells, with release of encapsulated fluorescence dyes and labeling of intracellular species.


Subject(s)
Cells , Electrophoresis, Capillary/methods , Fluorometry/methods , Liposomes/chemistry , Staining and Labeling , Cells/chemistry , Cells/metabolism , Cells/ultrastructure , Fluorescence , Fluorescent Dyes/chemistry , Lasers , Reproducibility of Results , Sensitivity and Specificity
16.
J Zhejiang Univ Sci B ; 7(1): 13-9, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16365920

ABSTRACT

v-Src is a non-receptor protein tyrosine kinase involved in many signal transduction pathways and closely related to the activation and development of cancers. We present here the expression, purification, and bioactivity of a GST (glutathione S-transferase)-fused v-Src from a bacterial expression system. Different culture conditions were examined in an isopropyl beta-D-thiogalactopyranoside (IPTG)-regulated expression, and the fused protein was purified using GSH (glutathione) affinity chromatography. ELISA (enzyme-linked immunosorbent assay) was employed to determine the phosphorylation kinase activity of the GST-fused v-Src. This strategy seems to be more promising than the insect cell system or other eukaryotic systems employed in earlier Src expression.


Subject(s)
Glutathione Transferase/biosynthesis , Oncogene Protein pp60(v-src)/biosynthesis , Oncogene Protein pp60(v-src)/chemistry , Protein Engineering/methods , Saccharomyces cerevisiae/metabolism , Bacterial Proteins/biosynthesis , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Glutathione Transferase/genetics , Glutathione Transferase/isolation & purification , Oncogene Protein pp60(v-src)/genetics , Oncogene Protein pp60(v-src)/isolation & purification , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Saccharomyces cerevisiae/genetics
17.
J Zhejiang Univ Sci B ; 6(8): 832-7, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16052719

ABSTRACT

Total RNA was isolated from the hybridoma cell line (LC-1), which secretes anti-lung adenocarcinoma monoclonal antibody, and was transferred into cDNA. Based on the FR1 (framework region 1) and FR4 conserved regions of LC-1 gene, the variable regions of heavy chain (Vh) and light chain (Vl) were amplified, and the Vh and modified Vl were connected to single chain Fv (ScFv) by SOE-PCR (splice overlap extension PCR). The modified ScFv was fused with green fluorescent protein (GFP) and introduced into E. coli JM109. The fusion protein induced by IPTG (Isopropylthiogalactoside) was about 57000 on a 10% SDS-PAGE gel (10% Sds Polyacrylamide Gel Electrophoresis), and primarily manifested as inclusion bodies. The renatured protein purified by Ni-NTA Superflow resins showed ability to bind to antigen on SPC-A-1 lung adenocarcinoma. In addition, the induced host cells fluoresced bright green under 395 nm wavelength, which indicated that the expected protein with dual activity was expressed in the prokaryotic system. The ScFv with GFP tag used in this research can be applied as a new reagent to detect immunological dye, and provide a feasible way to detect adenocarcinoma in a clinical setting.


Subject(s)
Adenocarcinoma/immunology , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/immunology , Cloning, Molecular/methods , Hybridomas/metabolism , Immunoglobulin Fragments/biosynthesis , Immunoglobulin Fragments/immunology , Lung Neoplasms/immunology , Protein Engineering/methods , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/isolation & purification , Cell Line , Cell Line, Tumor , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Humans , Immunoglobulin Fragments/genetics , Immunoglobulin Fragments/isolation & purification , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/isolation & purification
18.
Shi Yan Sheng Wu Xue Bao ; 37(3): 227-31, 2004 Jun.
Article in Chinese | MEDLINE | ID: mdl-15323425

ABSTRACT

Onconase, a ribonuclease purified from Rana pipiens oocytes, has cytotoxic activity against several tumor cell lines in vitro. With the characteristic of containing four pairs of disulfide bonds internal and N terminal sequence attributing mostly to its biological functions, it is difficult to obtain the active Onconase from Escherichia coli expression system with normal strategy. Here, we fused the cDNA coding for Onconase in frame with the pelB signal sequence in pET22b(+) expression vector. Onconase can be effectively expressed in inclusion body without additional residues at N terminal under the proper inducing condition. After refolding and purifying process, we can get the recombined Onconase which has a similar ribonuclease activity as the one isolated from oocytes. The recombined Onconase has a pronounced effect on proliferation of Hut-78 cells (IC50=0.5 micromol/L).


Subject(s)
Escherichia coli/metabolism , Ribonucleases/metabolism , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Humans , Liver/metabolism , Mice , Protein Synthesis Inhibitors/pharmacology , RNA/metabolism , Ribonucleases/genetics , Ribonucleases/pharmacology
19.
J Zhejiang Univ Sci ; 5(2): 164-72, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14674027

ABSTRACT

Beta-1,4-galactosyltransferase (beta4Gal-T) (EC 2.4.1.38) plays a multifunctional role in many aspects of normal cell physiology. By now, several dozens of beta4Gal-T genes have been cloned, separated from mouse, chick, bovine, human, etc. This paper presents the cloning and GST-fused expression of mouse beta4Gal-T gene in Escherichia coli (E. coli). The target gene was cloned by PCR, followed by identification by DNA sequencing and expression in E.coli with isopropyl-beta-D-thiogalactoside (IPTG) gradient concentrations, products of which were separated on SDS-PAGE showing that the target protein had the same molecular weight as that of mouse beta4Gal-T. The transcriptional product of beta4Gal-T gene was proved by Western hybridization analysis to be due to GST-fusion.


Subject(s)
Cloning, Molecular/methods , Escherichia coli/enzymology , Escherichia coli/genetics , Glutathione Transferase/biosynthesis , Glutathione Transferase/genetics , N-Acetyllactosamine Synthase/biosynthesis , N-Acetyllactosamine Synthase/genetics , Amino Acid Sequence , Animals , Gene Expression Regulation, Bacterial/physiology , Gene Expression Regulation, Enzymologic/physiology , Mice , Molecular Sequence Data , Molecular Weight , N-Acetyllactosamine Synthase/chemistry , Phylogeny , Recombinant Fusion Proteins/biosynthesis , Sequence Homology, Amino Acid , Transfection/methods
20.
Shi Yan Sheng Wu Xue Bao ; 37(5): 339-43, 2004 Oct.
Article in Chinese | MEDLINE | ID: mdl-15636360

ABSTRACT

We established a mouse melanoma model expressing beta-galactosidase for the study of tumor immunotherapy. The recombinant vector p3gal was constructed by inserting a beta-galactosidase gene into the MCS of plasmid pcDNA3. The vector then transfected the B16 cells. Through selection with 500 microg/ml G418 and in situ X-Gal staining, the melanoma cell line galB16, stably expressing beta-galactosidase was obtained. The melanoma model was successfully established after inoculation in mouse with galB16 cells. In situ X-Gal staining showed that the tumor cells expressed beta-galactosidase in vivo. With the model, we designed animal experiments for mouse tumor immunotherapy. Twenty mice were randomly assigned to four parallel groups. They received i.m. injection with saline, DNA vaccine p3gal (100 microg/mouse), adjuvant CpG 1826 (20 microg/mouse), or p3gal+CpG 1826 respectively. Our result suggested that the DNA vaccine containing beta-galactosidase gene could protect mice against the galB16 tumor challenge. In addition, when combining with the adjuvant CpG 1826, the effect was increased prominently.


Subject(s)
Cancer Vaccines/genetics , Cancer Vaccines/immunology , Melanoma, Experimental/immunology , Vaccines, DNA/genetics , Vaccines, DNA/immunology , beta-Galactosidase/metabolism , Adjuvants, Immunologic , Animals , Cell Line, Tumor , Genetic Vectors/genetics , Melanoma, Experimental/genetics , Melanoma, Experimental/metabolism , Mice , Plasmids/genetics , Random Allocation , beta-Galactosidase/genetics
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