BACKGROUND: Histone deacetylase 4 (HDAC4) regulates chondrocyte hypertrophy and bone formation. The aim of the present study was to explore the effects of HDAC4 on Interleukin 1 beta (IL-1β)-induced chondrocyte extracellular matrix degradation and whether it is regulated through the WNT family member 3A (WNT3A)/β-catenin signaling pathway. METHODS: Primary chondrocytes (CC) and human chondrosarcoma cells (SW1353 cells) were treated with IL-1β and the level of HDAC4 was assayed using Western blotting. Then, HDAC4 expression in the SW1353 cells was silenced using small interfering RNA to detect the effect of HDAC4 knockdown on the levels of matrix metalloproteinase 3 (MMP3) and MMP13 induced by IL-1β. After transfection with HDAC4 plasmids, the overexpression efficiency was examined using Real-time quantitative polymerase chain reaction (qRT-PCR) and the levels of MMP3 and MMP13 were assayed using Western blotting. After incubation with IL-1β, the translocation of β-catenin into the nucleus was observed using immunofluorescence staining in SW1353 cells to investigate the activation of the WNT3A/β-catenin signaling pathway. Finally, treatment with WNT3A and transfection with glycogen synthase kinase 3 beta (GSK3β) plasmids were assessed for their effects on HDAC4 levels using Western blotting. RESULTS: IL-1β downregulated HDAC4 levels in chondrocytes and SW1353 cells. Furthermore, HDAC4 knockdown increased the levels of MMP3 and MMP13, which contributed to the degradation of the extracellular matrix. Overexpression of HDAC4 inhibited IL-1β-induced increases in MMP3 and MMP13. IL-1β upregulated the levels of WNT3A, and WNT3A reduced HDAC4 levels in SW1353 cells. GSK-3β rescued IL-1β-induced downregulation of HDAC4 in SW1353 cells. CONCLUSION HDAC4 exerted an inhibitory effect on IL-1β-induced extracellular matrix degradation and was regulated partially by the WNT3A/β-catenin signaling pathway.
Cell Line, Tumor ; Cells, Cultured ; Chondrocytes/metabolism* ; Glycogen Synthase Kinase 3 beta/genetics* ; Histone Deacetylases/genetics* ; Humans ; Interleukin-1beta/pharmacology* ; Matrix Metalloproteinase 13/metabolism* ; Matrix Metalloproteinase 3 ; Repressor Proteins ; Wnt Signaling Pathway ; Wnt3A Protein/genetics* ; beta Catenin/metabolism*

BACKGROUNDBisdemethoxycurcumin (BDMC) is an active component of curcumin and a chemotherapeutic agent, which has been suggested to inhibit tumor growth, invasion and metastasis in multiple cancers. But its contribution and mechanism of action in invasion and metastasis of non-small cell lung cancer (NSCLC) are not very clear. Therefore, we tried to study the effects of BDMC on regulation of epithelial-to-mesenchymal transition (EMT), which is closely linked to tumor cell invasion and metastasis.

METHODSIn this study, we first induced transforming growth factor-β1 (TGF-β1) mediated EMT in highly metastatic lung cancer 95D cells. Thereafter, we studied the effects of BDMC on invasion and migration of 95D cells. In addition, EMT markers expressions were also analyzed by western blot and immunofluorescence assays. The contribution of Wnt inhibitory factor-1 (WIF-1) in regulating BDMC effects on TGF-β1 induced EMT were further analyzed by its overexpression and small interfering RNA knockdown studies.

RESULTSIt was observed that BDMC inhibited the TGF-β1 induced EMT in 95D cells. Furthermore, it also inhibited the Wnt signaling pathway by upregulating WIF-1 protein expression. In addition, WIF-1 manipulation studies further revealed that WIF-1 is a central molecule mediating BDMC response towards TGF-β1 induced EMT by regulating cell invasion and migration.

CONCLUSIONSOur study concluded that BDMC effects on TGF-β1 induced EMT in NSCLC are mediated through WIF-1 and elucidated a novel mechanism of EMT regulation by BDMC.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Blotting, Western ; Cell Line, Tumor ; Cell Movement ; drug effects ; genetics ; Curcumin ; analogs & derivatives ; pharmacology ; Epithelial-Mesenchymal Transition ; drug effects ; genetics ; Humans ; Lung Neoplasms ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

The Escherichia coli fadR protein product, a paradigm/prototypical FadR regulator, positively regulates fabA and fabB, the two critical genes for unsaturated fatty acid (UFA) biosynthesis. However the scenario in the other Ɣ-proteobacteria, such as Shewanella with the marine origin, is unusual in that Rodionov and coworkers predicted that only fabA (not fabB) has a binding site for FadR protein. It raised the possibility of fad regulon contraction. Here we report that this is the case. Sequence alignment of the FadR homologs revealed that the N-terminal DNA-binding domain exhibited remarkable similarity, whereas the ligand-accepting motif at C-terminus is relatively-less conserved. The FadR homologue of S. oneidensis (referred to FadR_she) was over-expressed and purified to homogeneity. Integrative evidence obtained by FPLC (fast protein liquid chromatography) and chemical cross-linking analyses elucidated that FadR_she protein can dimerize in solution, whose identity was determined by MALDI-TOF-MS. In vitro data from electrophoretic mobility shift assays suggested that FadR_she is almost functionally-exchangeable/equivalent to E. coli FadR (FadR_ec) in the ability of binding the E. coli fabA (and fabB) promoters. In an agreement with that of E. coli fabA, S. oneidensis fabA promoter bound both FadR_she and FadR_ec, and was disassociated specifically with the FadR regulatory protein upon the addition of long-chain acyl-CoA thioesters. To monitor in vivo effect exerted by FadR on Shewanella fabA expression, the native promoter of S. oneidensis fabA was fused to a LacZ reporter gene to engineer a chromosome fabA-lacZ transcriptional fusion in E. coli. As anticipated, the removal of fadR gene gave about 2-fold decrement of Shewanella fabA expression by β-gal activity, which is almost identical to the inhibitory level by the addition of oleate. Therefore, we concluded that fabA is contracted to be the only one member of fad regulon in the context of fatty acid synthesis in the marine bacteria Shewanella genus.
Amino Acid Sequence ; Bacterial Proteins ; chemistry ; metabolism ; Base Sequence ; Binding Sites ; DNA, Bacterial ; metabolism ; Escherichia coli ; genetics ; metabolism ; Fatty Acid Synthase, Type II ; genetics ; metabolism ; Fatty Acids ; biosynthesis ; Gene Expression Regulation, Bacterial ; drug effects ; Molecular Sequence Data ; Oleic Acid ; pharmacology ; Protein Binding ; drug effects ; Regulon ; genetics ; Repressor Proteins ; chemistry ; metabolism ; Shewanella ; genetics ; metabolism

OBJECTIVETo study the effects of E2F-1-silencing lentivirus vector on the growth and chemoresistance of subcutaneous human gastric cancer in nude mice.

METHODSThirty-six nude mice were inoculated subcutaneously with chemoresistant SGC-7901/DDP cells to establish subcutaneous tumor models of gastric carcinoma. The mice were randomly divided into E2F-1/RNAi-LV group, LV-scrRNAi group and PBS group (n = 12). E2F-1/RNAi-LV, LV-scrRNAi or PBS (0.1 ml per time) was injected into the mice, respectively, every two days. The nude mice received an intraperitoneal injection of cisplatin (25 mg/kg) every two days. The tumor volume was measured and histopathological changes of the tumors were observed by HE staining. The expressions of E2F-1, c-Myc, survivin, MDR1 and MRP were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Apoptosis in tumor xenografts was determined by in situ TUNEL labeling technique.

RESULTSThe mean tumor growth rate of the E2F-1/RNAi-LV group was significantly slower than that of the LV-scrRNAi and control groups (P < 0.05). The tumor volume of the E2F-1/RNAi-LV group was (745.13 ± 154.42)mm(3), significantly lower than that of the LV-scrRNAi and PBS groups (P < 0.05). Compared with that in the LV-scrRNAi and PBS groups, the expressions of mRNA and protein of E2F-1, c-Myc, survivin, MDR1 and MRP were significantly decreased in the E2F-1/RNAi-LV group (P < 0.05). The apoptotic rate in the E2F-1/RNAi-LV treatment group was (27.5 ± 9.7)%, significantly higher than (7.0 ± 1.1)% in the LV-scrRNAi group and (7.3 ± 1.2)% in the PBS group (P < 0.05).

CONCLUSIONIntra-tumoral injection of E2F-1/RNAi-LV shows significantly inhibitory effect on the tumor growth and chemoresistance of subcutaneous human gastric cancer in nude mice.

ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Cell Line, Tumor ; Cisplatin ; pharmacology ; Drug Resistance, Neoplasm ; E2F1 Transcription Factor ; genetics ; metabolism ; Female ; Gene Silencing ; Genetic Vectors ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; metabolism ; Lentivirus ; genetics ; Mice ; Mice, Nude ; Multidrug Resistance-Associated Proteins ; genetics ; metabolism ; Neoplasm Transplantation ; Proto-Oncogene Proteins c-myc ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Random Allocation ; Repressor Proteins ; genetics ; metabolism ; Stomach Neoplasms ; genetics ; metabolism ; pathology ; Transfection ; Tumor Burden

The differentiation of periodontal ligament (PDL) progenitor cells is important for maintaining the homeostasis of PDL tissue and alveolar bone. Vitamin C (VC), a water-soluble nutrient that cannot be biosynthesized by humans, is vital for mesenchymal stem cells differentiation and plays an important role in bone remodeling. Therefore, the objective of this study was to determine the function and mechanism of VC in PDL progenitor cells osteogenic differentiation at the molecular level. We demonstrated that VC could induce the osteogenic differentiation and maturation of PDL progenitor cell without other osteogenic agents. During the process, VC preferentially activated ERK1/2 but did not affect JNK or p38. Co-treatment with ERK inhibitor effectively decreased the Vitamin C-induced expression of Runx2. ERK inhibitor also abrogated Vitamin C-induced the minimized nodules formation. PELP1, a nuclear receptor co-regulator, was up-regulated under VC treatment. PELP1 knockdown inhibited ERK phosphorylation. The overexpression of PELP1 had a positive relationship with Runx2 expression. Taken together, we could make a conclude that VC induces the osteogenic differentiation of PDL progenitor cells via PELP1-ERK axis. Our finding implies that VC may have a potential in the regeneration medicine and application to periodontitis treatment.
Ascorbic Acid ; pharmacology ; Butadienes ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Co-Repressor Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Core Binding Factor Alpha 1 Subunit ; genetics ; metabolism ; Humans ; MAP Kinase Signaling System ; drug effects ; Mitogen-Activated Protein Kinase 1 ; antagonists & inhibitors ; metabolism ; Mitogen-Activated Protein Kinase 3 ; antagonists & inhibitors ; metabolism ; Nitriles ; pharmacology ; Periodontal Ligament ; cytology ; Phosphorylation ; drug effects ; RNA Interference ; RNA, Small Interfering ; metabolism ; Stem Cells ; cytology ; Transcription Factors ; antagonists & inhibitors ; genetics ; metabolism ; Up-Regulation ; drug effects

OBJECTIVETo investigate the high expression of HPV16L2N120E7E6 fusion protein by prokaryotic expression system, and evaluate its immunogenicity and antitumor efficacy in vaccinated mice.

METHODSThe HPV16L2N120E7E6 fusion gene, its codons were optimized to increase the expression of the protein, was constructed by overlap extension PCR and inserted into prokaryotic expression vector pET9a. Then the fusion protein was expressed by inducing with IPTG in E. coli strain BL21 (DE3) harboring with plasmid pETL2N120E7E6, and further detected by SDS-PAGE and Western-blot. Finally, the humoral and cellular immune responses were measured by ELISA and ELISPOT, respectively, in vaccinated mice with the purified HPV16L2N120E7E6 fusion protein, and the antitumor efficacy was assessed in mice using the TC-1 tumor challenge model.

RESULTSThe codon-optimized HPV16L2N120E7E6 fusion gene was highly expressed in E. coli strain BL21 (DE3) harboring with plasmid pETL2N120E7E6, and the amount of fusion protein was nearly 48.6% of the total bacterial protein. The purified fusion protein could induce high titer of specific antibody against L2, E7 and E6 in vaccinated mice. When accompanied with the adjuvant CpG, the fusion protein was able to elicit strong and moderate cellular immune responses in vaccinated mice against peptide HPV16E7(49-57) and peptide pools of HPV16E6, respectively. Furthermore, the tumor therapeutic experiment showed that HPV16L2N120E7E6 + CpG could prevent the tumor formation in 80.0% (8/10) vaccinated mice.

CONCLUSIONSThe data of this study suggest that HPV16L2N120E7E6 fusion protein could be a promising candidate vaccine for treatment of chronic HPV16 infection and post-operative adjuvant therapy for cervical cancer.

Adjuvants, Immunologic ; pharmacology ; Animals ; Cancer Vaccines ; immunology ; therapeutic use ; Capsid Proteins ; genetics ; immunology ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Codon ; Escherichia coli ; immunology ; metabolism ; Female ; Humans ; Immunization ; methods ; Immunotherapy ; methods ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Oligodeoxyribonucleotides ; immunology ; Oncogene Proteins, Viral ; genetics ; immunology ; metabolism ; Papillomavirus E7 Proteins ; genetics ; immunology ; metabolism ; Papillomavirus Vaccines ; immunology ; therapeutic use ; Plasmids ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Repressor Proteins ; genetics ; immunology ; metabolism

OBJECTIVE: To determine the effect of RNA interference with transferred pshRNA/PHB on the biological characteristics of paclitaxel-resistant ovarian cancer cell lines. METHODS: Western blot and real time-PCR were used to assay the expression of PHB protein and mRNA in SKOV3/Taxol-25 and SKOV3 cell lines. The SKOV3/Taxol-25 cell lines were transiently transfected by 3 target-specific small hairpin RNA (shRNA) interference fragments with fluorescent protein named the pshRNA427/PHB1, pshRNA248/PHB2, and pshRNA136/PHB3. The empty plasmid transfection via vehicle Lipofectamine2000 served as a negative control. The expression levels of PHB protein and mRNA were detected by Western blot and real time-PCR after the transfection for 48 h. The silence effect of PHB1 and PHB3 groups was obvious. PHB1, PHB3, and the negative control groups were used for the following experiments. MTT and flow cytometry assay were used to test the cell proliferation, IC50 of paclitaxel, and cell apoptosis in the 3 groups. RESULTS: The expression levels of PHB protein and mRNA (2(-ΔΔCt)) were significantly higher in SKOV3/Taxol-25 cell line than those in SKOV3 cell line (P<0.05). The expression levels of PHB protein and mRNA were significantly lower in the PHB1 and PHB3 groups than those in the negative control group (P<0.05). The cell proliferations in the PHB1 and PHB3 groups were obviously slower than those in the negative control group after transfection for 48 h and 72 h (P<0.05). The IC50 of paclitaxel in the PHB1 and PHB3 groups significantly decreased after transfection for 72 h compared with the negative control group(P<0.05). The cell apoptotic rate in the PHB1 and PHB3 groups significantly increased after transfection for 48 h compared with the negative control group (P<0.05). CONCLUSION The shRNA/PHB can effectively suppress the expression of PHB gene in paclitaxel-resistant ovarian cancer cell lines. The cell proliferation in paclitaxel-resistant cell lines with removed PHB gene is significantly reduced. The apoptotic rate and the paclitaxel sensitivity of resistant cell lines with removed PHB gene are significantly increased. PHB gene is related to paclitaxel-resistance and interfering PHB gene expression may reduce paclitaxel resistance in ovarian cancer.
Antineoplastic Agents, Phytogenic ; pharmacology ; Drug Resistance, Neoplasm ; genetics ; Female ; Humans ; Ovarian Neoplasms ; genetics ; metabolism ; pathology ; Paclitaxel ; pharmacology ; RNA Interference ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Repressor Proteins ; genetics ; metabolism ; Transfection ; Tumor Cells, Cultured

TGFβ/smad pathway is recognized as an important signal pathway to promote the pathogenesis of atherosclerosis (AS). Peroxisome proliferator-activated receptor γ (PPARγ) activation is considered to be important in modulating AS. Herein, we investigated the regulation of PPARγ on c-Ski, the repressor of TGFβ/smad pathway, in rat AS model and cultured vascular smooth muscle cells (VSMCs). c-Ski mRNA and protein expression were detected by real-time PCR and Western blot, respectively, in vivo and in vitro with treatment of PPARγ agonist rosiglitazone and antagonist GW9662. The proliferation and collagen secretion of VSMCs after c-Ski transfection were investigated. The underlying mechanism was further investigated by online program NUBIScan and luciferase reporter gene analysis. Results showed that both mRNA and protein expressions of c-Ski in the AS lesions was down-regulated in vivo, while in cultured VSMCs, c-Ski transfection significantly suppressed the proliferation and collagen secretion of rat VSMCs. Rosiglitazone significantly up-regulated mRNA and protein levels of c-Ski in VSMCs, which could be blocked by GW9662. Online NUBIScan analysis suggested possible PPARγ binding sites in the promoter region of c-Ski. In addition, luciferase activity of c-Ski reporter gene was also increased obviously in the presence of rosiglitazone. These results indicate that c-Ski is one of the newly found target genes of PPARγ and thus involved in the anti-AS effect of PPARγ.
Anilides ; pharmacology ; Animals ; Atherosclerosis ; physiopathology ; Cells, Cultured ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; metabolism ; PPAR gamma ; agonists ; antagonists & inhibitors ; physiology ; Proto-Oncogene Proteins ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Repressor Proteins ; genetics ; metabolism ; Signal Transduction ; Smad Proteins ; metabolism ; Thiazolidinediones ; pharmacology ; Transforming Growth Factor beta ; metabolism ; Up-Regulation

OBJECTIVETo investigate the inhibitory effect of survivin antisense oligodeoxynuleotides (ASODN) mediated by polyethylenimine (PEI) on hepatocelluar carcinoma SMMC-7721 cell proliferation and its effect on chemosensitivity to 5-FU in tumor-bearing mice.

METHODSThe inhibitory effect of PEI-ASODN on SMMC-7721 cell proliferation was assayed by WST-8 test, Trypan blue exclusion test, and cell clone formation assay. In mouse models of transplanted H22 cell hepatocarcinoma and ascites tumor, the effect of 5-FU combined with PEI-ASODN on the weight and volume of the subcutaneous tumors was examined. The tumor inhibition rate in the tumor-bearing mice was calculated and the average survival time recorded.

RESULTSSMMC-7721 cells incubated with different concentrations of PEI-ASODN for 48 h showed significantly reduced cell proliferation in comparison with the control cells, while PEI or ASODN alone produced no such inhibitory effect. Incubation of SMMC-7721 cells with 0.75 micromol/L PEI-ASODN for 24, 48, 72, and 96 h resulted in significantly suppressed cell proliferation, and a 7-day incubation of the cells with PEI-ASODN at different concentrations (0.25-0.75 micromol/L) significantly inhibited the cell clone formation. In the tumor-bearing mice, the tumor weight and volume were obviously reduced with a tumor inhibition rate of 56.91% and volume inhibition rate of 57.83%, significantly different from those in saline-treated mice (P<0.01). In the mice bearing ascites tumor, the average survival time was 22.0 days in saline group and 42.7 days in 5-FU+PEI-ASODN treatment group, showing a a life-prolonging rate of 94.09% in the latter group. A synergetic effect was noted between 5-FU and PEI-ASODN.

CONCLUSIONPEI-ASODN complex can significantly inhibit the proliferation of hepatocarcinoma SMMC-7721 cells and enhance 5-FU chemosensitivity of the tumor cells in vitro and transplanted H22 tumors in mice.

Animals ; Antimetabolites, Antineoplastic ; pharmacology ; therapeutic use ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drug Synergism ; Female ; Fluorouracil ; pharmacology ; therapeutic use ; Inhibitor of Apoptosis Proteins ; genetics ; pharmacology ; therapeutic use ; Liver Neoplasms, Experimental ; drug therapy ; pathology ; Male ; Mice ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; therapeutic use ; Repressor Proteins ; genetics ; pharmacology ; therapeutic use

BACKGROUND AND OBJECTIVEiASPP, an inhibitory member of the apoptosis-stimulating proteins of p53 (ASPP) family, has been found to be up-regulated in various human tumor types. This study was to construct an efficient doxycycline-regulated, lentiviral vector-mediated knockdown system for iASPP that will allow for inducible down-regulation of iASPP gene expression and preliminary functional analysis.

METHODSA pair of complementary oligos with hairpin structures targeting the iASPP gene and a negative control were synthesized, then ligated with pLVTHM vector and sequenced. The fragment containing the shRNA cassette was cloned to pLVCT-tTR-KRAB plasmid. The recombinant vectors were co-transfected with viral packaging mix into 293T cells, and viral supernatant was harvested to determine the titer. After treatment with or without doxycycline, HepG2 cells infected with virus were harvested and the expression of iASPP was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Its effects on tumor growth were characterized using MTS assay, soft agar colony formation, and flow cytometry analysis.

RESULTSThe lentiviral vector expressing shRNA that targets to the oncogene iASPP was constructed successfully. HepG2 infected with the lentivirus expressing shRNA against iASPP inhibited the expression of iASPP in the presence of doxycycline, which resulted in the repression of tumor cell proliferation and anchorage-independent growth potential.

CONCLUSIONSThe lentiviral vector-mediated tet-on system demonstrates efficient and inducible knockdown of iASPP in hepatocellular carcinoma cells. iASPP gene may be involved in tumorigenesis and progression of human tumors.

Apoptosis ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Proliferation ; Down-Regulation ; Doxycycline ; pharmacology ; Genetic Vectors ; Hep G2 Cells ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Lentivirus ; genetics ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Plasmids ; RNA Interference ; RNA, Small Interfering ; genetics ; Recombinant Proteins ; genetics ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Transfection