ABSTRACT
Fucoidan is one of the main bioactive components of polysaccharides. The current study was focused on the anti-tumor effects of fucoidan on human heptoma cell line HepG2 and the possible mechanisms. Fucoidan treatment resulted in cell cycle arrest and apoptosis of HepG2 cells in a dose-dependent manner detected by MTT assay, flow cytometry and fluorescent microscopy. The results of flow cytometric analysis revealed that fucoidan induced G2/M arrest in the cell cycle progression. Hoechst 33258 and Annexin V/PI staining results showed that the apoptotic cell number was increased, which was associated with a dose-dependent up-regulation of Bax and down-regulation of Bcl-2 and p-Stat3. In parallel, the up-regulation of p53 and the increase in reactive oxygen species were also observed, which may play important roles in the inhibition of HepG2 growth by fucoidan. In the meantime, Cyclin B1 and CDK1 were down-regulated by fucoidan treatment. Down-regulation of p-Stat3 by fucoidan resulted in apoptosis and an increase in ROS in response to fucoidan exposure. We therefore concluded that fucoidan induces apoptosis through the down-regulation of p-Stat3. These results suggest that fucoidan may be used as a novel anti-cancer agent for hepatocarcinoma.
Subject(s)
Humans , Antineoplastic Agents , Pharmacology , Apoptosis , Blotting, Western , CDC2 Protein Kinase , Genetics , Metabolism , Cyclin B1 , Genetics , Metabolism , Dose-Response Relationship, Drug , Down-Regulation , Flow Cytometry , G2 Phase Cell Cycle Checkpoints , Genetics , Gene Expression Regulation, Neoplastic , Hep G2 Cells , Hepatoblastoma , Genetics , Metabolism , Pathology , Liver Neoplasms , Genetics , Metabolism , Pathology , Microscopy, Fluorescence , Polysaccharides , Pharmacology , Proto-Oncogene Proteins c-bcl-2 , Genetics , Metabolism , Reactive Oxygen Species , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , STAT3 Transcription Factor , Genetics , Metabolism , Tumor Suppressor Protein p53 , Genetics , Metabolism , bcl-2-Associated X Protein , Genetics , MetabolismABSTRACT
Icaritin, a prenylflavonoid derivative from Epimedium Genus, has been shown to exhibit many pharmacological and biological activities. However, the function and the underlying mechanisms of icaritin in human non-small cell lung cancer have not been fully elucidated. The purpose of this study was to investigate the anticancer effects of icaritin on A549 cells and explore the underlying molecular mechanism. The cell viability after icaritin treatment was tested by MTT assay. The cell cycle distribution, apoptosis and reactive oxygen species (ROS) levels were analyzed by flow cytometry. The mRNA and protein expression levels of the genes involved in proliferation and apoptosis were respectively detected by RT-PCR and Western blotting. The results demonstrated that icaritin induced cell cycle arrest at S phase, and down-regulated the expression levels of S regulatory proteins such as Cyclin A and CDK2. Icaritin also induced cell apoptosis characterized by positive Hoechst 33258 staining, accumulation of the Annexin V-positive cells, increased ROS level and alteration in Bcl-2 family proteins expression. Moreover, icaritin induced sustained phosphorylation of ERK and p38 MAPK. These findings suggested that icaritin might be a new potent inhibitor by inducing S phase arrest and apoptosis in human lung carcinoma A549 cells.
Subject(s)
Humans , Antineoplastic Agents, Phytogenic , Pharmacology , Apoptosis , Cell Line, Tumor , Flavonoids , Pharmacology , Lung Neoplasms , Drug Therapy , Metabolism , Pathology , MAP Kinase Signaling System , Neoplasm Proteins , Reactive Oxygen Species , Metabolism , S Phase Cell Cycle CheckpointsABSTRACT
Icaritin, a prenylflavonoid derivative from Epimedium Genus, has been shown to exhibit many pharmacological and biological activities. However, the function and the underlying mechanisms of icaritin in human non-small cell lung cancer have not been fully elucidated. The purpose of this study was to investigate the anticancer effects of icaritin on A549 cells and explore the underlying molecular mechanism. The cell viability after icaritin treatment was tested by MTT assay. The cell cycle distribution, apoptosis and reactive oxygen species (ROS) levels were analyzed by flow cytometry. The mRNA and protein expression levels of the genes involved in proliferation and apoptosis were respectively detected by RT-PCR and Western blotting. The results demonstrated that icaritin induced cell cycle arrest at S phase, and down-regulated the expression levels of S regulatory proteins such as Cyclin A and CDK2. Icaritin also induced cell apoptosis characterized by positive Hoechst 33258 staining, accumulation of the Annexin V-positive cells, increased ROS level and alteration in Bcl-2 family proteins expression. Moreover, icaritin induced sustained phosphorylation of ERK and p38 MAPK. These findings suggested that icaritin might be a new potent inhibitor by inducing S phase arrest and apoptosis in human lung carcinoma A549 cells.
ABSTRACT
Fucoidan is one of the main bioactive components of polysaccharides. The current study was focused on the anti-tumor effects of fucoidan on human heptoma cell line HepG2 and the possible mechanisms. Fucoidan treatment resulted in cell cycle arrest and apoptosis of HepG2 cells in a dose-dependent manner detected by MTT assay, flow cytometry and fluorescent microscopy. The results of flow cytometric analysis revealed that fucoidan induced G2/M arrest in the cell cycle progression. Hoechst 33258 and Annexin V/PI staining results showed that the apoptotic cell number was increased, which was associated with a dose-dependent up-regulation of Bax and down-regulation of Bcl-2 and p-Stat3. In parallel, the up-regulation of p53 and the increase in reactive oxygen species were also observed, which may play important roles in the inhibition of HepG2 growth by fucoidan. In the meantime, Cyclin B1 and CDK1 were down-regulated by fucoidan treatment. Down-regulation of p-Stat3 by fucoidan resulted in apoptosis and an increase in ROS in response to fucoidan exposure. We therefore concluded that fucoidan induces apoptosis through the down-regulation of p-Stat3. These results suggest that fucoidan may be used as a novel anti-cancer agent for hepatocarcinoma.
ABSTRACT
Previous studies have shown that STAT3 plays a vital role in the genesis and progression of cancer. In this study, we investigated the relationship between the JAK2/STAT3 signalling pathway and germacrone-induced apoptosis in HepG2 cells. HepG2 cells were incubated with germacrone for 24 h, the protein expression of p-STAT3, STAT3, p-JAK2 and JAK2 was detected by Western Blotting, and RT-PCR was used to determine the expression of STAT3, p53, Bcl-2 and Bax at transcriptional levels. Besides that, HepG2 cells were pre-treated with AG490 or IL-6 for 2 h, and then incubated with germacrone for 24 h. The expression of p-JAK2, JAK2, p-STAT3, STAT3, p53, Bax and Bcl-2 was detected by Western blotting. The activity of HepG2 cells was tested by MTT assay. The apoptosis of HepG2 cells and levels of reactive oxygen species (ROS) were flow cytometrically measured. The results showed that germacrone exposure decreased p-STAT3 and p-JAK2 and regulated expression of p53 and Bcl-2 family members at the same time. Moreover, IL-6 enhanced the activation of the JAK2/STAT3 signalling pathway and therefore attenuated the germacrone-induced apoptosis. Suppression of JAK2/STAT3 signalling pathway by AG490, an inhibitor of JAK2, resulted in apoptosis and an increase in ROS in response to germacrone exposure. We therefore conclude that germacrone induces apoptosis through the JAK2/STAT3 signalling pathway.
ABSTRACT
Fucoidan is an active component of seaweed, which inhibits proliferation and induces apoptosis of several tumor cells while the detailed mechanisms underlying this process are still not clear. In this study, the effect of Fucoidan on the proliferation and apoptosis of human breast cancer MCF-7 cells and the molecular mechanism of Fucoidan action were investigated. Viable cell number of MCF-7 cells was decreased by Fucoidan treatment in a dose-dependent manner as measured by MTT assay. Fucoidan treatment resulted in G1 phase arrest of MCF-7 cells as revealed by flow cytometry, which was associated with the decrease in the gene expression of cyclin D1 and CDK-4. Annexin V/PI staining results showed that the number of apoptotic cells was associated with regulation of cytochrome C, caspase-8, Bax and Bcl-2 at transcriptional and translational levels. Both morphologic observation and Hoechst 33258 assay results confirmed the pro-apoptotic effect of Fucoidan. Meanwhile, the ROS production was also increased by Fucoidan treatment, which suggested that Fucoidan induced oxidative damage in MCF-7 cells. The results of present study demonstrated that Fucoidan could induce G1 phase arrest and apoptosis in MCF-7 cells through regulating the cell cycle and apoptosis-related genes or proteins expression, and ROS generation is also involved in these processes.
ABSTRACT
Previous studies have shown that STAT3 plays a vital role in the genesis and progression of cancer. In this study, we investigated the relationship between the JAK2/STAT3 signalling pathway and germacrone-induced apoptosis in HepG2 cells. HepG2 cells were incubated with germacrone for 24 h, the protein expression of p-STAT3, STAT3, p-JAK2 and JAK2 was detected by Western Blotting, and RT-PCR was used to determine the expression of STAT3, p53, Bcl-2 and Bax at transcriptional levels. Besides that, HepG2 cells were pre-treated with AG490 or IL-6 for 2 h, and then incubated with germacrone for 24 h. The expression of p-JAK2, JAK2, p-STAT3, STAT3, p53, Bax and Bcl-2 was detected by Western blotting. The activity of HepG2 cells was tested by MTT assay. The apoptosis of HepG2 cells and levels of reactive oxygen species (ROS) were flow cytometrically measured. The results showed that germacrone exposure decreased p-STAT3 and p-JAK2 and regulated expression of p53 and Bcl-2 family members at the same time. Moreover, IL-6 enhanced the activation of the JAK2/STAT3 signalling pathway and therefore attenuated the germacrone-induced apoptosis. Suppression of JAK2/STAT3 signalling pathway by AG490, an inhibitor of JAK2, resulted in apoptosis and an increase in ROS in response to germacrone exposure. We therefore conclude that germacrone induces apoptosis through the JAK2/STAT3 signalling pathway.
Subject(s)
Humans , Apoptosis , Dose-Response Relationship, Drug , Drugs, Chinese Herbal , Hep G2 Cells , Janus Kinase 2 , Metabolism , STAT3 Transcription Factor , Metabolism , Sesquiterpenes, Germacrane , Signal TransductionABSTRACT
Fucoidan is an active component of seaweed, which inhibits proliferation and induces apoptosis of several tumor cells while the detailed mechanisms underlying this process are still not clear. In this study, the effect of Fucoidan on the proliferation and apoptosis of human breast cancer MCF-7 cells and the molecular mechanism of Fucoidan action were investigated. Viable cell number of MCF-7 cells was decreased by Fucoidan treatment in a dose-dependent manner as measured by MTT assay. Fucoidan treatment resulted in G1 phase arrest of MCF-7 cells as revealed by flow cytometry, which was associated with the decrease in the gene expression of cyclin D1 and CDK-4. Annexin V/PI staining results showed that the number of apoptotic cells was associated with regulation of cytochrome C, caspase-8, Bax and Bcl-2 at transcriptional and translational levels. Both morphologic observation and Hoechst 33258 assay results confirmed the pro-apoptotic effect of Fucoidan. Meanwhile, the ROS production was also increased by Fucoidan treatment, which suggested that Fucoidan induced oxidative damage in MCF-7 cells. The results of present study demonstrated that Fucoidan could induce G1 phase arrest and apoptosis in MCF-7 cells through regulating the cell cycle and apoptosis-related genes or proteins expression, and ROS generation is also involved in these processes.
Subject(s)
Humans , Antineoplastic Agents , Chemistry , Pharmacology , Apoptosis , Genetics , Blotting, Western , Breast Neoplasms , Genetics , Metabolism , Pathology , Caspase 8 , Genetics , Metabolism , Caspases , Genetics , Metabolism , Cell Proliferation , Cell Size , Cyclin D1 , Genetics , Metabolism , Cyclin-Dependent Kinase 4 , Genetics , Metabolism , Cytochromes c , Genetics , Metabolism , Dose-Response Relationship, Drug , Fucus , Chemistry , G1 Phase Cell Cycle Checkpoints , Genetics , Gene Expression Regulation, Neoplastic , MCF-7 Cells , Microscopy, Fluorescence , Molecular Structure , Polysaccharides , Chemistry , Pharmacology , Proto-Oncogene Proteins c-bcl-2 , Genetics , Metabolism , Reactive Oxygen Species , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , bcl-2-Associated X Protein , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To obtain specific anti-epidermal growth factor receptor variant III (EGFRvIII) single chain antibody (ScFv) by phage antibody library display system.</p><p><b>METHODS</b>The total RNA was extracted from the spleen B cells of BALB/c mice immunized with pep-3-OVA protein, and the first-strand cDNA was synthesized by reverse transcription. Antibody VH and VL gene fragments were amplified and joined to a ScFv gene with the linker. The ScFv gene was ligated into the phagemid vector pCANTAB5E, which was transformed into competent E. coli TG1. The transformed cells were then infected with M13KO7 helper phage to yield the recombinant phage to construct the phage ScFv library. Pep-3-BSA protein was used to screen the phage antibody library and ELISA carried out to characterize the activity of the antibody.</p><p><b>RESULTS</b>The VH and VL gene fragments of the antibody were about 350 bp and 320 bp in length as analyzed by agarose gel electrophoresis. The ScFv gene was 780 bp, consistent with the expected length. The recombinant phagemid with ScFv gene insert was rescued, and an immune phage ScFv library with the content of 5.0x10(6) was constructed. The recombinant ScFv phage had a titer of 3.0x10(4) cfu/ml, and the fourth phage harvest yielded 56 times as much as that of the first one. SDS-PAGE demonstrated a molecular mass of the soluble ScFv of about 28 kD. ELISA results indicated good specificity of the ScFv to bind EGFRvIII.</p><p><b>CONCLUSION</b>An immune phage ScFv library is successfully constructed, and the ScFv antibody fragment is capable of specific binding to EGFRvIII.</p>
Subject(s)
Animals , Mice , Amino Acid Sequence , Antibody Specificity , Base Sequence , Immunoglobulin Fragments , Genetics , Allergy and Immunology , Immunoglobulin Heavy Chains , Genetics , Immunoglobulin Light Chains , Genetics , Mice, Inbred BALB C , Molecular Sequence Data , Mutant Proteins , Genetics , Allergy and Immunology , Peptide Library , ErbB Receptors , Genetics , Allergy and Immunology , Single-Chain Antibodies , Genetics , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVES</b>To investigate the impact of AAV-encoding NT4-TAT-His-PR39 fusion gene expression on HIF-1alpha level in ECV304 cultured under hypoxic condition (1%O(2)) and on angiogenesis in hypoxic chick embryo.</p><p><b>METHODS</b>PR39 cDNA was connected with NT4, TAT, 6 x His cDNA by molecular biology methods. The recombinant AAV vector was obtained by three plasmid co-transfection in 293 cells. Then ECV304 were respectively infected with AAV-NT4-TAT-His-PR39, 6 x His expression and HIF-1alpha level in ECV304 were detected by immunocytochemistry. The chicken embryos were randomized into the AAV-PR39, EV and PBS groups (n = 10 each) subject to hypoxia (5%O(2), n = 15) or normoxia environments (n = 15), the vessel density of the chicken chorioallantoic membrane (CAM) were measured by Image Pro Plus (IPP) software.</p><p><b>RESULTS</b>The expression of 6 x His protein was detected in AAV-PR39 infected ECV304 cells. HIF-1alpha protein activity was significantly increased in AAV-PR39 infected ECV304 underwent hypoxia compared to PBS and non-infected ECV304 groups (P < 0.05). The vessel density of chicken CAM in hypoxia environment but not in normoxia environment was also significantly higher in AAV-PR39 group than in EV group and PBS group (all P < 0.05).</p><p><b>CONCLUSION</b>AAV-encoding NT4-TAT-His-PR39 fusion gene expression significantly increased HIF-1alpha level in ECV304 exposed to hypoxia and promoted angiogenesis in hypoxic chicken embryo.</p>
Subject(s)
Animals , Chick Embryo , Antimicrobial Cationic Peptides , Genetics , Cell Line , Dependovirus , Genetics , Gene Fusion , Gene Transfer Techniques , Genes, Viral , Hypoxia , Genetics , Neovascularization, Physiologic , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To construct a prokaryotic expression vector for apoptin and prepare polyclonal antibody of apoptin.</p><p><b>METHODS</b>Apoptin gene amplified from pGEM-T/Apoptin plasmid by PCR was cloned into pET-28a (+). E.coli BL21 (DE3) was transformed by the recombinant plasmid, and apoptin protein expression induced by IPTG was analyzed by SDS-PAGE. BALB/c mice were immunized with the protein and the titer of the antibody was determined using indirect enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>Apoptin gene was successfully cloned into pET-28a (+), and the expression of a protein with relative molecular mass of about 17 000 was identified by SDA-PAGE. After 5 immunizations of the mice with the protein, the blood antibody titer reached 1:5x10(5).</p><p><b>CONCLUSION</b>The prokaryotic expression vector for apoptin is successfully constructed and the polyclonal antibody of apoptin is obtained, which allows further functional study of apoptin.</p>
Subject(s)
Animals , Mice , Antibodies , Blood , Genetics , Allergy and Immunology , Capsid Proteins , Genetics , Allergy and Immunology , Cloning, Molecular , Escherichia coli , Metabolism , Gene Expression , Genetic Vectors , Genome , Mice, Inbred BALB C , PlasmidsABSTRACT
<p><b>UNLABELLED</b>OBJECTIVE To construct a recombinant adenovirus Ad.NT4p53(N15)Ant and explore its cytotoxic effect against hepatocellular carcinoma HepG2 cells in vitro.</p><p><b>METHODS</b>The recombinant adenovirus containing the fusion gene of neurotrophin 4 (NT4)signal peptide, N-terminal residues (12-26) of p53 and 17 amino acid Drosophila homeobox protein Antennapedia (Ant) was constructed by gene cloning protocol. The effect of this fusion gene on HepG2 cells was evaluated by MTT assay, PI staining and flow cytometry.</p><p><b>RESULTS</b>The fusion gene Ad.NT4p53(N15)Ant was successfully constructed, as verified by restriction endonuclease digestion and PCR. Ad.NT4p53(N15)Ant could strongly suppress the growth of HepG2 cells (with a growth inhibition rate of 63.3% 48 h after infection) without affecting NIH-3T3 cells. Flow cytometry showed that Ad.NT4p53(N15)Ant could induce obvious apoptosis of HepG2 cells.</p><p><b>CONCLUSION</b>The recombinant adenovirus containing NT4p53(N15)Ant fusion gene can inhibit the growth the of HepG2 cells in vitro partially by inducing cell apoptosis.</p>
Subject(s)
Animals , Humans , Mice , Adenoviridae , Genetics , Physiology , Apoptosis , Genetics , Carcinoma, Hepatocellular , Genetics , Pathology , DNA, Recombinant , Genetics , Genetic Engineering , Methods , Hep G2 Cells , Liver Neoplasms , Genetics , Pathology , NIH 3T3 Cells , Plasmids , Genetics , Recombinant Fusion Proteins , Genetics , Tumor Suppressor Protein p53 , Genetics , Viral LoadABSTRACT
<p><b>OBJECTIVE</b>To construct a recombinant retroviral vector for RNA interference targeting human telomerase reverse transcriptase (hTERT).</p><p><b>METHODS</b>The sequences coding for enhanced fluorescence protein (EGFP), U6 promoter and a small interfering RNA (siRNA) targeting hTERT were amplified by PCR, respectively, and sub-cloned sequentially into the retroviral shuttle plasmid pLXSN to construct the plasmid pLXSN-EGFP-U6-siTERT. The recombinant expression plasmid was identified by restriction enzyme digestion and sequencing. Fluorescence microscopy and flow cytometry were employed to analyze EGFP expression in NIH3T3 transfected with the recombinant plasmid, and MMT assay was performed to evaluate the growth inhibition of Hela cells resulting from RNA interference mediated by the plasmid.</p><p><b>RESULTS</b>Sequence analysis and restriction enzyme digestion showed that the recombinant expression plasmid pLXSN-EGFP-U6-siTERT was constructed successfully. Twenty-four hours after transfection of NIH3T3 cells with the recombinant plasmid, the expression rate of EGFP reached 24.1% as shown by flow cytometry. MTT assay demonstrated a cell death rate of 53.2% 72 h after transfection of Hela cells with the plasmid.</p><p><b>CONCLUSION</b>The successful construction of the recombinant retroviral plasmid mediating potent cell growth inhibition suggests the great potential of RNA interference technique in suppressing hTERT expression in mammalian tumor cells.</p>
Subject(s)
Animals , Humans , Mice , Cloning, Molecular , Flow Cytometry , Genetic Vectors , Genetics , Green Fluorescent Proteins , Genetics , HeLa Cells , Microscopy, Fluorescence , NIH 3T3 Cells , RNA Interference , RNA, Messenger , Genetics , RNA, Small Interfering , Genetics , Recombinant Fusion Proteins , Genetics , Retroviridae , Genetics , Telomerase , GeneticsABSTRACT
<p><b>OBJECTIVES</b>To examine the expression and purification of the TGFbeta1 vaccine from prokaryotic expression system and to determine the antigenicity of the fusion protein of recombinant vector pET28a/ HBcAg1-71-TGFbeta132-HBcAg89-144.</p><p><b>METHODS</b>The reconstructed vector pGEMEX-1/CTC was subcloned to pET28a and transformed into E. coli BL21 (DE3). The recombinant 6xHis- HBcAg1-71- TGFbeta132- HBcAg89-144 was to be expressed after induction by IPTG and purified with Ni-NTA-His affinity chromatography. The detection of the formation of core-like particles was done under an electron microscope and of their antigenity by using ELISA and Western blot.</p><p><b>RESULTS</b>A 2.46 x 10(4) protein was obtained by optimizing the conditions for both expression and purification. The protein had the TGFbeta1 antigenicity but not a HBc antigenity and the formed core-like particles were bigger than natural core particles.</p><p><b>CONCLUSION</b>The recombinant fusion protein in the prokaryotic expressed system can be used as an anti-TGFbeta1 vaccine to inhibit hepatic fibrosis.</p>