Comparison of the protective effects of truncated bFGF and native bFGF against murine lung carcinomaLuo, Zichao ; Peng, Xinyun ; Shi, Huashan ; Gong, Changyang ; Qian, Zhiyong ; Yang, Li
doi: 10.3892/ijmm.2011.676pmid: 21503566
Basic fibroblast growth factor (bFGF), an angiogenic factor, exhibits pro-angiogenic abilities by interacting with tyrosine kinase receptors and heparin-sulfated proteoglycan receptors. Here, we designed an N-, C-terminally truncated basic fibroblast growth factor (tbFGF) for immuno-therapy of murine lung carcinoma with PCEC hydrogel as adjuvant, comparing it with the wild-type bFGF. In vitro, tbFGF did not stimulate NIH-3T3 fibroblast proliferation. In vivo, after immunization, both tbFGF and bFGF were able to induce a robust bFGF-specific immune response. The protective anti-tumor investigation showed a significant inhibition of tumor growth and reduction of tumor vascularization detected by immunohistochemical staining and the alginate-encapsulated tumor cell assay in the tbFGF or the bFGF group. These data suggested that tbFGF can be used in the immunotherapy of tumors, without the risks associated with bFGF, which induces neovascularization in normal tissues.
Involvement of a novel GATA4 mutation in atrial septal defectsLiu, Xing-Yuan ; Wang, Juan ; Zheng, Jing-Hao ; Bai, Kai ; Liu, Zhong-Min ; Wang, Xiao-Zhou ; Liu, Xu ; Fang, Wei-Yi ; Yang, Yi-Qing
doi: 10.3892/ijmm.2011.638pmid: 21373748
Atrial septal defect (ASD) is one of the most common types of congenital heart disease and is associated with a significant increase in the morbidity and mortality of affected individuals. Accumulating evidence indicates that genetic defects play important roles in the pathogenesis of congenital ASD. However, ASD is genetically heterogeneous and the genetic determinants for ASD in the majority of the patients remain to be identified. In this study, the entire coding region of GATA4, a gene encoding a zinc-finger transcription factor crucial to embryogenesis, was initially sequenced in 120 unrelated patients with ASD. The available relatives of patients carrying the identified mutation and 200 ethnicity-matched unrelated control individuals were genotyped. The functional characteristics of the GATA4 mutant were compared to its wild-type counterpart using a luciferase reporter assay system. A novel heterozygous missense GATA4 mutation, p.G21V, was identified in 2 unrelated families with ASD, which was not detected in the control population nor reported in the human gene mutation database. Alignment of multiple GATA4 proteins displayed that the affected amino acid residue was highly conserved across species. Functional analysis showed that the p.G21V GATA4 mutation was associated with a decreased transcriptional activity. The findings underscore the pathogenic link between compromised GATA4 function and congenital ASD, providing new insight into the molecular mechanism involved in this common form of congenital cardiovascular anomalies.
Anti-tumor effect of luteolin is accompanied by AMP-activated protein kinase and nuclear factor-κB modulation in HepG2 hepatocarcinoma cellsHwang, Jin-Taek ; Park, Ock Jin ; Lee, Yun Kyung ; Sung, Mi Jeong ; Hur, Haeng Jeon ; Kim, Myung Sunny ; Ha, Joo Hun ; Kwon, Dae Young
doi: 10.3892/ijmm.2011.667pmid: 21468539
Luteolin, a plant-derived flavonoid, is thought to inhibit tumor growth. However, the precise molecular mechanisms by which luteolin inhibits cancer cell growth remain unclear. In the present study, we evaluated the role of AMP-activated protein kinase (AMPK) in the inhibition of cancer cell growth by luteolin in HepG2 hepatocarcinoma cells. AMPK is a metabolic sensor and may prevent carcinogenesis via modulation of signaling networks. We found that luteolin strongly induced cell death in HepG2 cells and dramatically reduced the tumor volume in a tumor xenograft model; both effects were accompanied by AMPK activation by luteolin. Luteolin also had a strong inhibitory effect on nuclear factor (NF)-κB. To determine the relationship between AMPK and NF-κB signaling, we used Compound C, a pharmacological AMPK inhibitor, and a dominant-negative form of AMPK. Our results indicated that inhibition of AMPK activity restored luteolin-inhibited NF-κB DNA-binding activity. These results suggest that AMPK activity is critical for the inhibition of cancer cell growth, possibly via modulation of NF-κB activity. We also showed that luteolin treatment causes the release of reactive oxygen species (ROS) and that these intracellular ROS in turn mediate AMPK-NF-κB signaling in HepG2 hepatocarcinoma cells. In conclusion, we propose that AMPK is a novel regulator of NF-κB in luteolin-induced cancer cell death. Furthermore, our results suggest that AMPK is an attractive target for cancer prevention by flavonoids.
Novel compound heterozygous mutations T2C and 1149insT in the KCNQ1 gene cause Jervell and Lange-Nielsen syndromeWang, Rong-Rong ; Li, Ning ; Zhang, Yin-Hui ; Wang, Lin-Lin ; Teng, Si-Yong ; Pu, Jie-Lin
doi: 10.3892/ijmm.2011.642pmid: 21380488
Mutations in the KCNQ1 gene account for more than 90% of the individuals with Jervell and Lange-Nielsen syndrome (JLNS). In this study, we identified and characterized two novel KCNQ1 mutations that caused JLNS. A 6-year-old deaf girl suffering from recurrent syncope had a documented electrocardiogram with polymorphic ventricular fibrillation since the age of 4 years. The baseline electrocardiogram showed a significantly prolonged corrected QT interval (524 msec). Genetic analysis revealed that the proband carried two heterozygous mutations of T2C and 1149insT in the KCNQ1 gene on separate alleles. Patch-clamp analysis demonstrated that the T2C mutation resulted in significant reduction in the slowly activated delayed rectifier current (IKs). Furthermore, western blot analysis and confocal imaging revealed that the T2C mutation produced a truncated protein with trafficking defects. In contrast, the 1149insT mutation failed to generate any measurable current, consistent with no protein expression in both the cell membrane and cytoplasm. Moreover, co-expression of the T2C and 1149insT mutations significantly reduced the peak tail current density to 8.27% of the wild-type (WT) current value, while co-transfected WT channels with either T2C or 1149insT mutant channels produced comparable current and channel kinetics to that of WT channels. Our study demonstrates that the compound heterozygous mutations T2C and 1149insT cause the ‘loss-of-function’ of the IKs that may account for the clinical phenotype of the proband. Multiple mechanisms have been involved in the pathogenesis of ‘loss-of-function’ of IKs.
Analysis and optimization of interactions between peptides mimicking the GD2 ganglioside and the monoclonal antibody 14G2aHorwacik, Irena ; Kurciński, Mateusz ; Bzowska, Małgorzata ; Kowalczyk, Aleksandra K. ; Czaplicki, Dominik K. ; Koliński, Andrzej K. ; Rokita, Hanna K.
doi: 10.3892/ijmm.2011.655pmid: 21455557
Overexpression of the GD2 ganglioside (GD2) is a hallmark of neuroblastoma. The antigen is used in neuroblastoma diagnosis and to target newly developed therapies to cancer cells. Peptide mimetics are novel approaches in the design of antigens for vaccine development. We previously reported the isolation of five GD2-mimicking peptides from the LX-8 phage display library with the monoclonal antibody (mAb) 14G2a. The goal of our current study was to analyze and optimize the binding of the peptide mimetics to the mAb 14G2a. Therefore, we performed further experiments and supported them with molecular modeling to investigate structure-activity relationships that are the basis for the observed mimicry of GD2 by our peptides. Here, we show that the peptides have overlapping binding sites on the mAb, 14G2a and restricted specificity, as they did not crossreact with other ganglioside-specific antibodies tested. In addition we demonstrate that the phage environment was involved in the process of selection of our peptides. The AAEGD sequence taken from the viral major coat protein, p8, and added to the C-termini of the peptides #65, #85 and #94 significantly improved their binding to the mAb, 14G2a. By application of analogs with amino acid substitutions and sequence truncations, we elucidated the structure-activity relationships necessary for the interactions between the 14G2a mAb and the peptide #94 (RCNPNMEPPRCF). We identified amino acids indispensable for the observed GD2-mimicry by #94 and confirmed a pivotal role of the disulphide bridge between the cysteine residues of #94 for binding to the mAb 14G2a. More importantly, we report five new peptides demonstrating a significant improvement of mAb 14G2a binding. The experimental data were supported and expanded with molecular modeling tools. Taken together, the experimental results and the in silico data allowed us to probe in detail the mechanism of the molecular mimicry of GD2 by the peptides. Additionally, we significantly optimized binding of the leading peptide sequence #94 to the mAb 14G2a. We can conclude that our findings add to the knowledge on factors governing selections of peptide mimetics from phage-display libraries.
Differential expression of microRNAs in cardiac myocytes compared to undifferentiated P19 cellsHu, De-Liang ; Liu, Yao-Qiu ; Chen, Fu-Kun ; Sheng, Yan-Hui ; Yang, Rong ; Kong, Xiang-Qing ; Cao, Ke-Jiang ; Zhang, Jin-Song ; Qian, Ling-Mei
doi: 10.3892/ijmm.2011.664pmid: 21455566
microRNA (miRNA) expression is tightly controlled in a tissue-specific and developmental stage-specific manner; some are highly and specifically expressed in cardiovascular tissues. miRNA expression profiling, using miRNA microarrays facilitates studying the biological function of miRNAs. We investigated changes in miRNA expression profiles during differentiation of P19 cells into cardiac myocytes in order to elucidate the mechanisms of heart development. The morphology of P19 cells during differentiation was observed using an inverted microscope. Western blot analysis was performed to detect cardiac troponin I (cTnI) expression. Total RNA was extracted from P19 cells for microarray and real-time quantitative reverse transcription-polymerase chain reaction (real-time qRT-PCR) analyses to determine the miRNA expression profile. The miRNA microarray revealed differential expression of 49 miRNAs, of which 26 were down-regulated and 23 were up-regulated in differentiated cardiac myocytes, compared to normal P19 cells. This was confirmed by real-time qRT-PCR. We also utilized target prediction analysis to identify gene targets. Some miRNAs may have important roles in cardiac development and congenital heart defects (CHDs). Further analysis of miRNA function to confirm their target genes during cardiac development will determine the potential for novel miRNA-based therapeutic strategies.
AICAR, an activator of AMPK, inhibits adipogenesis via the WNT/β-catenin pathway in 3T3-L1 adipocytesLee, Haeyong ; Kang, Ryunhwa ; Bae, Sungmin ; Yoon, Yoosik
doi: 10.3892/ijmm.2011.674pmid: 21491080
AMP-activated protein kinase (AMPK) is known to sense the cellular energy state and regulates various cellular energy metabolism pathways through its activation by AMP, an indicator of a low-energy state. 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), an activator of AMPK, efficiently inhibited the adipogenesis of 3T3-L1 cells. To elucidate its possible mechanism of action, the expression levels of β-catenin and other members of the WNT/β-catenin pathway were analyzed during the adipogenesis of 3T3-L1 cells in the presence or absence of AICAR. It was found that AICAR significantly enhanced β-catenin expression and its nuclear accumulation. Transfection of β-catenin small interfering RNA (siRNA) significantly prevented the effects of AICAR on the expression of various genes. The expression of the major genes of adipogenesis including the peroxisome proliferator-activated receptor (PPAR)γ, the CCAAT/enhancer binding protein (C/EPB)α, the fatty acid binding protein (FABP)4 and lipoprotein lipase (LPL), which were all reduced by AICAR treatment, were significantly recovered in β-catenin siRNA-transfected cells. Among the members of the WNT/β-catenin pathway, the expression of low density lipoprotein receptor-related protein (LRP)6, dishevelled (DVL)2 and DVL3 were significantly up-regulated by AICAR treatment, whereas the expression of AXIN was down-regulated. The present study provides compelling evidence that AICAR inhibits adipogenesis through the modulation of the WNT/β‑catenin pathway.
Effect of the release from mechanical stress on osteoclastogenesis in RAW264.7 cellsShibata, Kenjiro ; Yoshimura, Yoshitaka ; Kikuiri, Takashi ; Hasegawa, Tomokazu ; Taniguchi, Yumi ; Deyama, Yoshiaki ; Suzuki, Kuniaki ; Iida, Junichiro
doi: 10.3892/ijmm.2011.675pmid: 21491081
The effects of mechanical stress release on osteoclastogenesis may be as important as those of mechanical stress application. However, the direct effects of mechanical stress on the behavior of osteoclasts has not been thoroughly investigated and there is limited information on the results of the release from mechanical stress. In this study, the effects of mechanical stress application and its release on osteoclast differentiation were examined. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts derived from RAW264.7 cells were measured and the expression of osteoclast differentiation genes, which was altered in response to the release from mechanical stress according to the Flexercell tension system was evaluated by real-time PCR. Osteoclast differentiation and fusion were suppressed by mechanical stress application and were rapidly induced after mechanical stress release. The mRNA expression of the osteoclast specific genes, TRAP, matrix metalloproteinase-9 (MMP-9), cathepsin-K (cath-k), calcitonin receptor (CTR), ATPase H+ transporting vacuolar proton pump member I (ATP6i), chloride channel-7 (ClC7) and dendritic cell-specific transmembrane protein (DC-STAMP) was decreased with mechanical stress application, and increased up to 48 h after the release from it. These alterations in gene mRNA expression were associated with the number of osteoclasts and large osteoclasts. Inducible nitric oxide synthetase (iNOS) mRNA was increased with mechanical stress and decreased after its release. Nitric oxide (NO) production was increased with mechanical stress. Nuclear factor of activated T cells cytoplasmic (NFATc) family mRNAs were not altered with mechanical stress, but were up-regulated up to 48 h after the release from it. These findings indicate that the suppression of osteoclast differentiation and fusion induced by mechanical stress is the result of NO increase via iNOS, and that the promotion of osteoclast differentiation and fusion after the release from mechanical stress is related to the NFATc family genes, whose expression remained constant during mechanical stress but was up-regulated after the release from mechanical stress.