GADD45A plays a protective role against temozolomide treatment in glioblastoma cells.

Glioblastoma multiforme (GBM) is one of the most aggressive cancers. Despite recent advances in multimodal therapies, high-grade glioma remains fatal. Temozolomide (TMZ) is an alkylating agent used worldwide for the clinical treatment of GBM; however, the innate and acquired resistance of GBM limits its application. Here, we found that TMZ inhibited the proliferation and induced the G2/M arrest of GBM cells. Therefore, we performed microarrays to identify the cell cycle- and apoptosis-related genes affected by TMZ. Notably, GADD45A was found to be up-regulated by TMZ in both cell cycle and apoptosis arrays. Furthermore, GADD45A knockdown (GADD45Akd) enhanced the cell growth arrest and cell death induced by TMZ, even in natural (T98) and adapted (TR-U373) TMZ-resistant cells. Interestingly, GADD45Akd decreased the expression of O6-methylguanine-DNA methyltransferase (MGMT) in TMZ-resistant cells (T98 and TR-U373). In MGMT-deficient/TMZ-sensitive cells (U87 and U373), GADD45Akd decreased TMZ-induced TP53 expression. Thus, in this study, we investigated the genes influenced by TMZ that were important in GBM therapy, and revealed that GADD45A plays a protective role against TMZ treatment which may through TP53-dependent and MGMT-dependent pathway in TMZ-sensitive and TMZ-resistant GBM, respectively. This protective role of GADD45A against TMZ treatment may provide a new therapeutic strategy for GBM treatment.

Evaluation of cellular retinoic acid binding protein 2 gene expression through the retinoic acid pathway by co-incubation of Blastocystis ST-1 with HT29 cells in vitro.

Blastocystis is a parasitic protist with a worldwide distribution that is commonly found in patients with colon and gastrointestinal pathological symptoms. Blastocystis infection has also commonly been reported in colorectal cancer and HIV/AIDS patients with gastrointestinal symptoms. To understand the pathway networks of gene regulation and the probable mechanisms influencing functions of HT-29 host cells in response to parasite infection, we examined the expression of 163 human oncogenes and kinases in human colon adenocarcinoma HT-29 cells co-incubated with Blastocystis by in-house cDNA microarray and PCR analysis. At least 10 genes were shown to be modified following Blastocystis co-incubation, including those with immunological, tumorigenesis, and antitumorigenesis functions. The expression of genes encoding cellular retinoic acid binding protein 2 (CRABP2) and proliferating cell nuclear antigen (PCNA) was markedly upregulated and downregulated, respectively. Reverse transcriptase-PCR validated the modified transcript expression of CRABP2 and other associated genes such as retinoic acid (RA)-related nuclear-receptor (RARα). Together, our data indicate that CRABP2, RARα, and PCNA expressions are involved in RA signaling regulatory networks that affect the growth, proliferation, and inflammation of HT-29 cells.

The Pathogenesis of Human Cervical Epithelium Cells Induced by Interacting with Trichomonas vaginalis.

BACKGROUND: Trichomonas vaginalis is a protozoan parasite that occurs in the urogenital-vaginal tract and is the primary causative agent of trichomoniasis, a common sexually transmitted disease in humans. The aggregation of this protozoan tends to destroy epithelial cells and induce pathogenesis. PRINCIPAL FINDINGS: This study cultured T. vaginalis and human cervical epithelial cells (Z172) under the same conditions in the experiments. Following co-culturing for ten hours, the protozoans became attached to Z172, such that the cells presented a round shape and underwent shrinkage. Time-lapse recording and flow cytometry on interacted Z172 revealed that 70% had been disrupted, 18% presented a necrosis-like morphology and 8% showed signs of apoptosis. Gene expression profiling revealed in the seven inflammatory Z172 genes as well as in T. vaginalis genes that code for adhesion proteins 65 and 65-1. SIGNIFICANCE: These results suggest that cytopathogenic effects progress while Z172 is in contact with T. vaginalis, and the resulting morphological changes can be categorized as disruption.

Ras induces experimental lung metastasis through up-regulation of RbAp46 to suppress RECK promoter activity.

BACKGROUND: Mutant Ras plays multiple functions in tumorigenesis including tumor formation and metastasis. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a metastasis inhibitor gene, suppresses matrix metalloproteinase (MMP) activity in the metastatic cascade. Clarifying the relationship between Ras and RECK and understanding the underlying molecular mechanism may lead to the development of better treatment for Ras-related tumors. METHODS: Suppression subtractive hybridization PCR (SSH PCR) was conducted to identify Ha-ras (val12) up-regulated genes in bladder cancer cells. Stable cell lines of human breast cancer (MCF-7-ras) and mouse NIH3T3 fibroblasts (7-4) harboring the inducible Ha-ras (val12) oncogene, which could be induced by isopropylthio-ß-D-galactoside (IPTG), were used to clarify the relationship between Ras and the up-regulated genes. Chromatin immunoprecipitation (ChIP) assay, DNA affinity precipitation assay (DAPA) and RECK reporter gene assay were utilized to confirm the complex formation and binding with promoters. RESULTS: Retinoblastoma binding protein-7 (RbAp46) was identified and confirmed as a Ha-ras (val12) up-regulated gene. RbAp46 could bind with histone deacetylase (HDAC1) and Sp1, followed by binding to RECK promoter at the Sp1 site resulting in repression of RECK expression. High expression of Ras protein accompanied with high RbAp46 and low RECK expression were detected in 75% (3/4) of the clinical bladder cancer tumor tissues compared to the adjacent normal parts. Ras induced RbAp46 expression increases invasion of the bladder cancer T24 cells and MMP-9 activity was increased, which was confirmed by specific lentiviral shRNAs inhibitors against Ras and RbAp46. Similarly, knockdown of RbAp46 expression in the stable NIH3T3 cells "7-4" by shRNA decreased Ras-related lung metastasis using a xenograft nude mice model. CONCLUSIONS: We confirmed that RbAp46 is a Ha-ras (val12) up-regulated gene and binds with HDAC1 and Sp1. Furthermore, RbAp46 binds to the RECK promoter at the Sp1 site via recruitment by Sp1. RECK is subsequently activated, leading to increased MMP9 activity, which may lead to increased metastasis in vivo. Our findings of Ras upregulation of RbAp46 may lead to revealing a novel mechanism of Ras-related tumor cell metastasis.

Antiangiogenesis as the novel mechanism for justicidin A in the anticancer effect on human bladder cancer.

Justicidin A (JA) is one of the methanol extracts of Justicia procumbens and was reported to induce apoptosis and inhibit the proliferation of human colon cancer cells. Using bladder cancer as a paradigm, this study was designed to identify the novel molecular basis underlying the antiangiogenic activities of JA and its potential in cancer therapy. Human bladder cancer cell lines (TSGH8301 and RT4) and immortalized uroepithelial cell lines (E6 and E7) were chosen to investigate the efficacy of JA in cell proliferation, apoptosis, and angiogenesis in vitro. The biological effects of JA treatment in vivo were examined using a xenograft tumor model in SCID mice. JA showed a dose-dependent and time-dependent inhibition of cell proliferation on TSGH8301 cancer cells, with IC50 values determined to be 0.44 µmol/l. Of interest, TSGH8301 cancer cells were more sensitive to JA than E7 immortalized uroepithelial cells, especially at lower concentrations. We further showed that JA inhibited the autocrine production of angiogenic factors and matrix-degrading enzymes in vitro and microvessel density in SCID mice in vivo (P< 0.01). Both differential cytotoxicity and angiogenesis inhibition of JA were confirmed by SCID mice experiments. Together, JA showed antiangiogenesis in vitro and in vivo through pleiotropic positive and negative regulators of angiogenesis molecules. The current investigation supports the potential of JA as an alternative chemoprevention agent for human bladder cancer.

Epithelial membrane protein 2 is a prognostic indictor for patients with urothelial carcinoma of the upper urinary tract.

Upper urinary tract urothelial carcinoma is a relatively uncommon disease and is diagnosed more frequently at advanced stages. The prognosis of these patients mainly has been related to tumor stage and grade. As a result, the definition of prognostic indicators enabling precise patient selection is mandatory for neoadjuvant or adjuvant therapies. The epithelial membrane protein (EMP2) was identified as one of the up-regulated genes by isoflavones. EMP2 overexpression suppressed foci formation, anchorage-independent growth in vitro, and tumorigenicity in severe combined immunodeficiency mice (all P < 0.05). In addition, a cross-talk between EMP2 and integrins αV and ß3 was shown in the regulation of cell adhesion and migration. Higher EMP2 expression was associated with a better progression-free survival (P = 0.008) and cancer-related death (P < 0.001). EMP2 was identified as a tumor-suppressor gene in urinary tract urothelial carcinoma and may be an innovative co-targeting candidate for designing integrin-based cancer therapy.

Transcriptional activation of the Axl and PDGFR-α by c-Met through a ras- and Src-independent mechanism in human bladder cancer.

BACKGROUND: A cross-talk between different receptor tyrosine kinases (RTKs) plays an important role in the pathogenesis of human cancers. METHODS: Both NIH-Met5 and T24-Met3 cell lines harboring an inducible human c-Met gene were established. C-Met-related RTKs were screened by RTK microarray analysis. The cross-talk of RTKs was demonstrated by Western blotting and confirmed by small interfering RNA (siRNA) silencing, followed by elucidation of the underlying mechanism. The impact of this cross-talk on biological function was demonstrated by Trans-well migration assay. Finally, the potential clinical importance was examined in a cohort of 65 cases of locally advanced and metastatic bladder cancer patients. RESULTS: A positive association of Axl or platelet-derived growth factor receptor-alpha (PDGFR-α) with c-Met expression was demonstrated at translational level, and confirmed by specific siRNA knock-down. The transactivation of c-Met on Axl or PDGFR-α in vitro was through a ras- and Src-independent activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathway. In human bladder cancer, co-expression of these RTKs was associated with poor patient survival (p < 0.05), and overexpression of c-Met/Axl/PDGFR-α or c-Met alone showed the most significant correlation with poor survival (p < 0.01). CONCLUSIONS: In addition to c-Met, the cross-talk with Axl and/or PDGFR-α also contributes to the progression of human bladder cancer. Evaluation of Axl and PDGFR-α expression status may identify a subset of c-Met-positive bladder cancer patients who may require co-targeting therapy.

Decreased proinflammatory cytokines production in children with complicated parapneumonic pleural effusion after intrapleural fibrinolytic treatment.

Intrapleural fibrinolytic therapy (IFT) provides clinical benefit in the treatment of complicated pleural parapneumonic effusion (CPE). Whether IFT influences the proinflammatory cytokines production and fibrinlytic activity is currently unclear. Therefore, we collected pleural effusion samples from CPE patients with IFT (study group) and patients without IFT (control group). A membrane human inflammatory cytokines array kit was used to compare the difference of targeted cytokine production between these two groups. Enzyme-linked immunosorbent assay (ELISA) methods were used for quantitative analysis of targeted cytokines and fibrinolytic enzymes. The results showed there were no significant differences between the study (n = 16) and control (n = 14) groups in patients' demographic data. After fibrinolytic therapy, the patients in the study group had significant lower plasminogen activator inhibitor (PAI) level (732.36+/-254.09 ng/mL vs 1,509.36+/-1,340.11 ng/mL, p<0.05) and higher urokinase plasminogen activator (u-PA) level (75.56+/-41.70 ng/mL vs 6.87+/-5.07 ng/mL, p<0.05) than they did before treatment. Moreover, the tissue inhibitors of metalloproteinase-2 (TIMP-2) (1,560.03+/-403.49 pg/mL vs 3,686.45+/-1,263.83 pg/mL, p<0.05) and inflammatory chemokine, regulated on activation normal T-cell expressed and secreted/chemokine (C-C motif) ligand 5 (RANTES), (293.58+/-212.93 pg/mL vs 749.27+/-53.79 pg/mL, p<0.05), were also significantly lower in the study group after fibrinolytic therapy, but not in the control group. In conclusion, intrapleural fibrinolytic treatment with urokinase could enhance fibrinolytic activity and decrease TIMP-2 and RANTES production.

Ha-ras oncogene-induced Stat3 phosphorylation enhances oncogenicity of the cell.

The ras oncogene needs a second factor to induce transformation and tumorigenicity of the cell. In this study, we show that mouse fibroblast 7-4-Stat3C cells overexpressing both Ha-ras(val12) oncogene and active-form Stat3 (Stat3C) showed higher colony formation in soft agar and xenograft tumor growth in BALB/c mice. Further studies show that both serine-727 and tyrosine-705 of Stat3 were phosphorylated while Ha-ras was overexpressed. Interleukin-6 (IL-6)-induced phosphorylation of tyrosine-705 and serine-727, as well as DNA-binding and transcriptional activity of Stat3 were further enhanced by Ha-ras overexpression. In addition, overexpression of Stat3C in 7-4-Stat3C cells prevented the cells from morphological change and apoptosis triggered by the Ha-ras oncogene under serum-depleted conditions. We demonstrate that Ha-ras and Stat3 acting together synergistically induce Stat3 phosphorylation at serine-727 phosphorylation and cyclin D1 expression and further enhance transformation and tumorigenicity of the cell. Ha-ras-induced Stat3 phosphorylation at serine-727 plays a pivotal role in transcriptional activation of cyclin D1 and suppression of cell apoptosis. The effect of Ha-ras on Stat3 phosphorylation at serine-727 was also detected in human bladder (T24) and lung (H460) cancer cells. Stat3 phosphorylation at serine-727 is important in Ras-related tumorigenesis.

Magnetic-bead-based microfluidic system for ribonucleic acid extraction and reverse transcription processes.

This paper presents a new integrated microfluidic chip that automatically performs ribonucleic acid (RNA) extraction and reverse transcription (RT) processes. The microfluidic system consists of a microfluidic control module and a magnetic bio-separator. The microfluidic control module can perform pumping and mixing of small amount of fluids and subsequent purification and concentration of RNA samples by incorporating with the magnetic bio-separator consisting of 2-dimension twisted microcoils. Notably, the magnetic bio-separators are developed either to generate the required magnetic field to perform the separation of magnetic beads or to work as a micro-heater to control the temperature field for the following RT process. Experimental results show that the total RNA can be successfully purified and extracted by using magnetic beads and the subsequent RT processing of the RNA can be performed automatically. Total RNA is successfully extracted and purified from T98 cells utilizing the microfluidic system, which is comparable with the conventional methods. The whole automatic procedure of RNA sample extraction only takes 35 min, which is much faster than the conventional method (more than 2 h). As a whole, the developed microfluidic system may provide a powerful platform for rapid RNA extraction and RT processes for further biomedical applications.

Prohibitin in the pathogenesis of transitional cell bladder cancer.

Our profiling experiment demonstrated that prohibitin 1 (PHB) was ubiquitously expressed in uroepithelial and urothelial carcinoma cell lines and exhibited a trend toward a positive relationship with tumor progression. The aim of this study was, therefore, to examine the potential role of PHB in multistage bladder carcinogenesis and predicting patient outcome. Immunohistochemical staining showed that PHB was overexpressed in 141 out of 167 cases (84.4%) of bladder cancer. This expression was positively related to met receptor overexpression (p = 0.04) and to multiple tumors (p = 0.05). Independent factors in predicting patient survival were multiple tumors (p = 0.002), muscle invasion (p = 0.003), and met overexpression (p = 0.05) in a multivariate analysis. Interestingly, patients with superficial bladder cancer overexpressing both PHB and met had a significantly lower recurrence-free survival rate than those not expressing PHB (p = 0.04). Taken together, our findings showed that PHB was activated at an early stage of carcinogenesis and that it may play a synergistic role with met in the progression of human bladder cancer. In addition, we demonstrated that genistein and justicidin A, natural chemoprevention agents, could suppress the expression of PHB in vitro. Thus, targeting PHB would be a useful approach for treating and preventing human bladder cancer.

Dengue viruses can infect human primary lung epithelia as well as lung carcinoma cells, and can also induce the secretion of IL-6 and RANTES.

Dengue viruses (DENV) are herein demonstrated for the first time as being able to infect and replicate in human primary lung epithelium and various lung cancer cell lines. The detection of dengue virus particles and viral negative strand RNA synthesis in the cell, in conjunction with the release of viral progenies in culture supernatants, support the notion that lung cells are susceptible to dengue virus infection. The replication efficiency of DENV in lung cancer cells from high to low is: DEN-2 (dengue virus type-2), DEN-3, DEN-4 and DEN-1. Moreover, the susceptibility of the six lung cancer cell lines to DEN-2 infection is: SW1573>A549>H1435; H23; H520; Bes2B. DEN-2 infection significantly increased the expression levels of IL-6 and RANTES in four of the six lung cancer cell lines, which is consistent with the high expression levels of these molecules in DHF/DSS patients. IL-6 expression induced by DEN-2 infection was NF-kappaB dependent. In summary, our results indicate that lung epithelial cell is a possible target of dengue viruses and IL-6 and RANTES may play pivotal roles in lung related immuno-pathogenesis.

Dominant-negative Rac1 suppresses Ras-induced apoptosis possibly through activation of NFkappaB in Ha-ras oncogene-transformed NIH/3T3 cells.

We investigated the involvement of Rac1 in Ha-ras-overexpression-induced apoptosis using a murine NIH/3T3-derived cell line (designated 7-4), which contains an inducible Ha-ras oncogene under the regulation of Escherichia coli lac operator-repressor system. Ha-ras overexpression was induced by isopropyl beta-D-thiogalactoside (IPTG). To reveal the role of endogenous Rac1, the dominant negative Rac1(Asn17) gene was transfected into the 7-4 cells. Using two cell lines 7-4 Racd2 and 7-4 Racd3 (7-4 derivates) stably expressing Rac1(Asn17), we demonstrate that suppression of Rac1 activity blocked Ha-ras-overexpression-induced apoptosis under a serum-depleted condition, indicating that Rac1 activity is required for a Ras-mediated apoptosis pathway. Cell-cycle analysis revealed that dominant-negative Rac1 partially shifted cell population from S-phase to G0/G1 phase in the cells overexpressing Ha-ras. In contrast to other reports, we showed activation of the transcription factor NFkappaB in the two cell lines expressing dominant-negative Rac1. All together, our results demonstrate that Ha-ras-overexpression- induced apoptosis can be blocked by dominant-negative Rac1, possibly through decreased S-phase accumulation and increased NFkappaB activity.

Identifying the factors and signal pathways necessary for anchorage-independent growth of Ha-ras oncogene-transformed NIH/3T3 cells.

Ha-ras(Val 12) overexpression was positively correlated with colony formation by NIH/3T3 derivative "2-12" cells harboring an inducible Ha-ras(Val 12) transgene. The ras-farnesylation inhibitor, Lovastatin, completely suppressed colony formation at higher dosages. However, Ha-ras oncogene overexpression alone could not stimulate colony formation under serum-deprived conditions, suggesting that ras is required but not sufficient for supporting colony formation. Substituting cow colostrum (AC-2) for serum did not result in colony formation from 2-12 cells in soft agar, suggesting the colostrum lacked or contained insufficient amounts of factors that stimulate colony formation. Supplementation of AC-2-containing medium with growth factors, such as insulin-like growth factor-1 (IGF-1), partially restored the capability of anchorage-independent cell growth induced by Ha-ras overexpression. Consistently, antibodies specific for IGF-1 receptors only partially blocked colony formation from 2-12 cells. The data indicate that multiple factors, including IGF-1, are required for Ha-ras-dependent colony formation. Signal transduction studies revealed that, under Ha-ras overexpression conditions, IGF-1 utilizes phosphatidyl inositol 3-kinase and NF-kappaB to transduce colony formation-related signaling.

Comparative study of enterovirus 71 infection of human cell lines.

The cell tropism of enterovirus 71 (Enteroviridae) in neuronal, glial and laryngeal cells. The 4643 strain, an enterovirus 71 isolate from a patient in Taiwan, was used to infect three human cell lines representing neuronal cells (SK-N-SH, neuroblastoma), glial cells (U373MG, glioblastoma), and laryngeal cells (HEp-2, larynx epidermoid carcinoma). Immunofluorescent staining and transmission electron microscopy (TEM) were used to detect mature enterovirus 71 4643 virions in these cell lines. The three cell lines were also compared for presence of virus-mediated cytopathic effect (CPE), synthesis of infected cell-specific proteins, viral (-) RNA, and virus replication rate. Virus particles were detected by TEM, and viral replication increased over time, indicating the existence and release of mature viruses from all three infected cell lines. The most severe CPE and the highest viral replication rate were observed in the SK-N-SH cells. Further screening of the infected cell lines by microarray analysis revealed that the neuron growth factor receptor (NGFR) gene was uniquely upregulated in infected SK-N-SH cells, implying that the receptor encoded by this gene may be involved in cell tropism. The data show that neurons are vulnerable to enterovirus 71 4643 infection and are consistent with the clinical observation that enterovirus 71 4643 targets mainly neuronal cells but is also found in many organs in conjunction with an inflammatory reaction.

In vitro action of carboxyfullerene.

Fullerene compounds have avid reactivity with free radicals and are regarded as 'radical sponges'. The trimalonic acid derivative of fullerene is one of the water-soluble compounds that has been synthesized and found to be an effective antioxidant both in vivo and in vitro. Carboxyfullerene has been shown to be effective in the treatment of both Gram-positive and -negative infections, although its mode of action is poorly understood. We determined the MIC and minimal bactericidal concentration of carboxyfullerene for 20 isolates, including Staphylococcus spp., Streptococcus spp., Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi and Klebsiella pneumoniae. We further investigated the action of carboxyfullerene using transmission electron microscopy (TEM), anticarboxyfullerene antibody binding assay and a membrane perturbation assay. All Gram-positive species were inhibited by < or = 50 mg/L of carboxyfullerene, whereas Gram-negative species were not inhibited, even at 500 mg/L carboxyfullerene. Bactericidal activity was demonstrated only for Gram-positive species, particularly for Streptococcus pyogenes A-20, which was killed rapidly. Intercalation of carboxyfullerene into the cell wall of staphylococci and streptococci was demonstrated by TEM and anti-carboxyfullerene binding assay. Damage to the cell membrane in Gram-positive, but not Gram-negative, bacteria was confirmed by the membrane perturbation assay. These findings indicate that the action of carboxyfullerene on Gram-positive bacteria is achieved by insertion into the cell wall and destruction of membrane integrity.