The expressions of carbonic anhydrase 9 and vascular endothelial growth factor in astrocytic tumors predict a poor prognosisYoo, Heon ; Sohn, Seil ; Nam, Byung Ho; Min, Hye Sook; Jung, Eugene Sook; Shin, Sang Hoon; Gwak, Ho-Shin Hoon; Lee, Seung Hoon
doi: 10.3892/ijmm_00000427pmid: 20514415
Hypoxia in the tumor microenvironment triggers a variety of genetic and adoptive responses that regulate tumor growth. Tumor hypoxia is often associated with a malignant phenotype, resistance to therapy, and poor survival. The objectives of this study were to evaluate the expressions of carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF) in astrocytic gliomas and to relate patterns of expression with prognosis, that is with histological grade and survival. We investigated 78 World Health Organization (WHO) grade II, III, and IV astrocytic gliomas. CA9 expression was examined in paraffin-embedded sections by immunohistochemistry. Fourteen tumors were grade II, 30 were grade III, and 34 were grade IV. It was found that CA9 expression was significantly associated with a higher-grade histology (p<0.001). There were 3 CA9 positive tumors in grade II (21.4%), 10 in grade III (33.3%), and 27 in grade IV (79.4%). For all tumors and WHO grade II, overall survival was found to be significantly dependent on CA9 expression (p=0.004, p=0.01). Furthermore, VEGF expression was found to be significantly related to tumor grade (p=0.02) and tended to be related to overall survival (p=0.1). However, no relation was found between the expression of CA9 and VEGF (p=0.17). Nevertheless, the expressions of CA9 and VEGF were found to be associated with tumor grade and possibly with survival. Further studies on a larger patient population are needed to determine the correlation between the expressions of CA9, and VEGF in astrocytic gliomas and clinical outcome.
Differential expression of genes in co-cultured versus separately cultured fibroblasts and epithelial cells from human benign hyperplastic prostate tissuesYang, Xiaoli ; Xuan, Qiang ; Mo, Linjian ; Huang, Fengyu ; Pang, Youhong ; He, Min ; Lin, Weixiong ; Li, Qingdi Quentin; Mo, Zengnan Quentin
doi: 10.3892/ijmm_00000429pmid: N/A
The prostate is composed mainly of epithelial and stromal cells, whose dynamic interaction is vital to a broad array of cellular processes, including proliferation, differentiation, growth, and apoptosis. To understand intercellular communication in the development and progression of prostatic diseases, we examined gene expression in tissues from five patients diagnosed with benign prostatic hyperplasia (BPH). Fibroblasts and epithelial cells derived from these tissues were grown in a primary co-culture system that retains many characteristics of the intact human prostate. The mRNA levels of expressed genes as assessed by differential-display reverse transcription-PCR revealed that 110 genes were differentially expressed in co-cultured fibroblasts and epithelial cells, compared with expression in separately cultured cells. Eighty-four of these were confirmed by reverse Northern blotting, and 68 were successfully sequenced. Of the sequenced genes, 43 were differentially expressed in epithelial cells (37 upregulated, 6 downregulated), and 25 were differentially expressed in fibroblasts (6 upregulated, 19 downregulated) in co-cultures versus separate cultures. Semi-quantitative RT-PCR analysis of 12 genes with known functions showed that five of these were differentially expressed in co-cultured cells. Human kallikrein gene 7 (KLK7) was markedly upregulated in co-cultured compared with separately cultured epithelial cells (P<0.001), whereas S100 calcium binding protein A11, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, cyclin I, and latexin were significantly downregulated in co-cultured fibroblasts (P<0.05). Quantitative real-time RT-PCR and Western blot analysis confirmed KLK7 up-regulation at both the mRNA and protein levels, respectively. Thus, epithelial-stromal cell interaction and communication are likely to be important in BPH. Epithelial cells and fibroblasts may interplay coordinately or collaboratively to influence cellular growth and death through dynamically differential gene expression in response to physiological and pathophysiological changes.
Knockdown of the Dickkopf 3 gene induces apoptosis in a lung adenocarcinomaJung, Il Lae; Kang, Hyo Jin; Kim, Kug Chan; Kim, In Gyu
doi: 10.3892/ijmm_00000431pmid: 20514419
The expression of the Wnt-antagonist Dickkopf gene (DKK) is downregulated in several types of tumors as a consequence of epigenetic DNA modification; four DKK members, DKK1, DKK2, DKK3, and DKK4, have been identified. In this study, we investigated another function of DKK3 in non-small cell lung cancer H460 cells, in which DKK3 was hypermethylated (44%) but still expressed, by interfering with DKK3 expression using DKK3-silencing RNA (SiRNA). We found that knockdown of DKK3 expression by DKK3 SiRNA transfection led to the detachment of H460 cells from the bottom of the culture plate and caused apoptosis. The expression of cyclin-dependent kinases D1 and E were increased by DKK3 knockdown, indicating that cells with blocked DKK3 expression entered the apoptotic pathway. We also found that the intracellular level of reactive oxygen species was higher in cells with blocked DKK3 expression than in normal H460 cells, and levels of p53, p21, and Bax were also increased by the gene knockdown. These results indicate that DKK3 acts as an antiapoptotic molecule by decreasing the intracellular level of reactive oxygen species.
Theaflavin digallate inactivates plasminogen activator inhibitor: Could tea help in Alzheimer's disease and obesity?Skrzypczak-Jankun, Ewa ; Jankun, Jerzy
doi: 10.3892/ijmm_00000433pmid: 20514421
Proteolysis in general and particularly the serine proteases are causally involved in many physiological processes and different diseases. Recently it was reported that plasminogen activator inhibitor type one (PAI-1) inactivation can alleviate the symptoms of Alzheimer's disease and reduce the body weight of obese individuals. In our broad search for natural compounds and their derivatives that can inhibit PAI-1, we include the polyphenols of teas since teas (green and black) or their components have been reported to alleviate the symptoms of both obesity and Alzheimer's disease. Inactivation of PAI-1 was measured in human plasma using thromboelastography. We used known PAI-1 inhibitor PAI039 ({1-benzyl-5-(4-(trifluoromethoxy) phenyl)-1H-indol-3-yl}(oxo)acetic acid) as a positive control and (-)-epigallo-catechin-3-gallate (EGCG), its prodrug octaacetate EGCG (OcAc EGCG) and theaflavin digallate (TH(2)) as potential PAI-1 inhibitors. We found that inactivation of PAI-1 in plasma by EGCG and OcAc EGCG was low or very low. However, TH(2) inactivated PAI-1 in a concentration-dependent manner with an IC50 of 18 µM which is equal to or better than the IC50 reported for known PAI-1 inhibitor PAI039. Clearly TH(2) inhibits PAI-1 and might play a role in slowing down the progression of Alzheimer's disease or obesity by a PAI-1-dependent pathway. While the clinical value of TH(2) has not been proven, long-term prospective studies assessing its efficacy are warranted due to the benign nature of the substance.
A promising alternative anti-HBV agent: The targeted ribonucleaseLi, Yinghui ; Zhao, Ya ; Liu, Jun ; Huang, Yuxiao ; Liu, Zhongxiang ; Xue, Caifang
doi: 10.3892/ijmm_00000434pmid: 20514422
HBV-targeted ribonuclease (TR) is a fusion of HBV core protein (HBVc) and human eosinophil-derived neurotoxin (hEDN). Introduction of TR by transfection or transduction into HepG2.2.15 cells (a cell model of HBV infection) revealed that it significantly reduces serological markers of HBV replication (including HBsAg, HBeAg and HBV DNA) in cell supernatants, suggesting that the targeted ribonuclease inhibits HBV replication. To further our understanding of the molecular mechanism of the anti-HBV effect of TR, we expressed TR in E. coli and found that purified TR possesses RNase activity and targeting activity. Furthermore, the antiviral effect of TR depends both on an enzymatically active hEDN and on the core domain. In or out of HepG2.2.15 cells, TR coassembles with the wild-type capsid protein into particles with internal hEDN domains. Our data suggest an intracellular ribonuclease activation mechanism that, owing to the characteristics of HBV morphogenesis, is highly virus specific. HBV may therefore be particularly vulnerable to the capsid-targeted viral inactivation approach.
The role of VIT1/FBXO11 in the regulation of apoptosis and tyrosinase export from endoplasmic reticulum in cultured melanocytesGuan, Cuiping ; Lin, Fuquan ; Zhou, Miaoni ; Hong, Weisong ; Fu, Lifang ; Xu, Wen ; Liu, Dongyin ; Wan, Yinsheng ; Xu, Aie
doi: 10.3892/ijmm_00000435pmid: 20514423
Our previous study has shown that VIT1 gene in Chinese vitiligo patients is de facto the FBXO11 gene, and the silencing of that gene has an impact on the ultrastructure of melanocytes. In this study, we further identified the role of the FBXO11 gene in melanocytes and the relationship between dilated endoplasmic reticulum (ER) and tyrosinase by inhibition and overexpression of FBXO11 gene. Cell proliferation, apoptosis, cycle and migration of melanocytes were examined when the FBXO11 gene was silenced or overexpressed. The results showed that FBXO11 gene promoted cell proliferation and suppressed cell apoptosis, and yet had little effect on cell migration. Obvious swelling of ER was found in the cells transfected with siRNA of FBXO11 gene. Interestingly, protein level of tyrosinase was extraordinarily high following inhibition of FBXO11 gene. Further examination revealed that tyrosinase and calreticulin were co-localized in ER of transfected cells following siRNA of FBXO11 gene, suggesting that tyrosinase could not be exported from ER effectively. Collectively, our results support the notion that FBXO11 plays an important role in regulating proliferation and apoptosis of melanocytes, and functional export of tyrosinase from ER in vitiligo melanocytes.
Permanent up-regulation of regulatory T-lymphocytes in patients with head and neck cancerSchott, Anne K.; Pries, Ralph K.; Wollenberg, Barbara K.
doi: 10.3892/ijmm_00000436pmid: 20514424
Various immune functions of different types of immune cells are strongly impaired in patients with head and neck squamous cell carcinoma (HNSCC). Regulatory T-lymphocyte cells (Tregs) have been suggested to be involved in the immunomodulation of immune responses and contribute to HNSCC progression and immune escape. ‘Naturally’ occurring CD4+CD25+ Tregs represent a small fraction within the different subsets of regulatory T cells, which are known to inhibit numerous immune functions of different types of immune cells. In this study, the cellular ratio of CD4+CD25high Tregs to the entire population of CD4+ T-lymphocytes was analyzed with respect to different stages of tumor progression and disease. Our data indicate a significantly high increased abundance of CD4+CD25highCD127low Tregs in the peripheral blood of patients with HNSCC, which in addition show modulated expression levels of various functional proteins. Surprisingly, increased Treg levels were found even in patients with no active disease several years after tumor resection, with no significant correlation to the individual tumor stage. Additionally, increased levels of chemokine CCL22, which mediates migration of Tregs to the tumor, and upregulation of the corresponding receptor protein CCR4 were observed in HNSCC. Our data strongly suggest that HNSCC leads to a permanent shift of Treg levels with hardly recognizable recovery rates.