Hot water extract of Agaricus blazei Murrill specifically inhibits growth and induces apoptosis in human pancreatic cancer cells.

BACKGROUND: Pancreatic cancer is one of the most aggressive human malignancies. The development of a novel drug to treat pancreatic cancer is imperative, and it is thought that complementary and alternative medicine (CAM) could yield such a candidate. Agaricus blazei Murrill is a CAM that has been tested as an anticancer drug, but its efficacy against pancreatic cancer is poorly understood. To study the potential of A. blazei in the treatment of pancreatic cancer, we examined the effects of its hot water extract on the proliferation and global gene expression profile of human pancreatic cancer cells. METHODS: Three distinct human pancreatic cancer cell lines, MIAPaCa-2, PCI-35, and PK-8, and the immortalized human pancreatic duct-epithelial cell line, HPDE, were employed. The cells were incubated with the appropriate growth medium supplemented with the hot water extract of A. blazei at final concentrations of 0.005, 0.015%, or 0.045%, and cellular proliferation was assessed for five consecutive days using an MTT assay. Apoptosis was examined by using flow cytometry and the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Caspase-dependent apoptosis was assayed using immunoblotting. Global gene expression profiles were examined using a whole human genome 44 K microarray, and the microarray results were validated by using real-time reverse transcription PCR. RESULTS: The hot water extract of A. blazei significantly inhibited the proliferation of cultured pancreatic cancer cells through the induction of G0/G1 cell cycle arrest and caspase-dependent apoptosis; the effect was the smallest in HPDE cells. Furthermore, significant alterations in the global gene expression profiles of pancreatic cancer cells occurred following treatment with the hot water extract of A. blazei. Genes associated with kinetochore function, spindle formation, and centromere maintenance were particularly affected, as well as cyclins and cyclin-dependent kinases that are essential for cell cycle progression. In addition, proapoptotic genes were upregulated. CONCLUSIONS: The hot water extract of A. blazei may be useful for the treatment of pancreatic cancer and is a potential candidate for the isolation of novel, active compounds specific for mitotic spindle dysfunction.

Fabrication and characterization of microfluidic devices based on boron-modified epoxy resin using CO2 laser ablation for bio-analytical applications.

CO2 laser ablation is a rapid and precise technique for machining microfluidic devices. And also, low-cost epoxy resin (ER) proved the great feasibility of fabricating these devices using the CO2 laser ablation technique in our previous studies. However, such a technique has shown negative impacts on such ER-based microfluidics as rough surface microchannels, and thermal defects. Therefore, incorporating different proportions of boric acid (BA) into epoxy resin formulation was proposed to obviate the genesis of these drawbacks in ER-based microfluidics. The structural and optical properties of plain ER- and B-doped ER-based chips were characterized by Fourier transform infrared (FT-IR) and UV/Vis spectral analyses. Furthermore, their thermal properties were studied by thermo-gravimetric (TGA) and differential scanning calorimetric (DSC) analysis. A CO2 laser ablation machine was used in vector mode to draw the designed micro-channel pattern onto plain ER- and B-doped ER-based chips. The quality of microchannels engraved onto these chips was assessed using 3D laser microscopy. This microscopic examination showed a noticeable reduction in the surface roughness and negligible bulge heights in the laser-ablated micro-channels. On the other hand, overall and specific migration using gravimetric methods and gas chromatography-mass spectrometry (GC-MS), respectively, and PCR compatibility test were performed to explore the convenience of these micro-plates for the biological reactions. These findings validated the applicability of B-doped ER-based microfluidics in bio-analytical applications as a result of the effective role of boric acid in enhancing the thermal properties of these chips leading to get micro-channels with higher quality with no effect on the biological reactions.

Fe Nanoparticle Size Control of the Fe-MOF-Derived Catalyst Using a Solvothermal Method: Effect on FTS Activity and Olefin Production.

The design of a highly active Fe-supported catalyst with the optimum particle and pore size, dispersion, loading, and stability is essential for obtaining the desired product selectivity. This study employed a solvothermal method to prepare two Fe-MIL-88B metal-organic framework (MOF)-derived catalysts using triethylamine (TEA) or NaOH as deprotonation catalysts. The catalysts were analyzed using X-ray diffraction, N2-physisorption, Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, H2 temperature-programed reduction, and thermogravimetric analysis and were evaluated for the Fischer-Tropsch synthesis performance. It was evident that the catalyst preparation in the presence of TEA produces a higher MOF yield and smaller crystal size than those produced using NaOH. The pyrolysis of MOFs yielded catalysts with different Fe particle sizes of 6 and 35 nm for the preparation in the presence of TEA and NaOH, respectively. Also, both types of catalysts exhibited a high Fe loading (50%) and good stability after 100 h reaction time. The smaller particle size TEA catalyst showed higher activity and higher olefin yield, with 94% CO conversion and a higher olefin yield of 24% at a lower reaction temperature of 280 °C and 20 bar at H2/CO = 1. Moreover, the smaller particle size TEA catalyst exhibited higher Fe time yield and CH4 selectivity but with lower chain growth probability (α) and C5+ selectivity.

Successful gene therapy requires targeting the vast majority of cancer cells.

Suicide gene therapy using gene-directed enzyme prodrug therapy (GDEPT) is based on delivering a gene-encoded enzyme to cells that converts a nontoxic prodrug into its toxic metabolite. The bystander effect is thought to compensate for inefficiencies in delivery and expression because the produced toxic metabolite can spread to adjacent non-expressing cells.The purpose of this study was to assess the significance of bystander effect in GDEPT over the long term in vivo.We performed experiments using mixtures of yeast cytosine deaminase (yCD) expressing and empty vector (EV) containing cells. First, the bystander effect was assessed in various ratios of colon cancer cell lines RKO with yCD/EV in 2D and 3D culture. Next, tumors raised from RKO with yCD/EV in mice were treated with the prodrug 5-fluorocytosine (5-FC) for 42 days to assess bystander effect in vivo. Cell types constituting relapsed tumors were determined by 5-FC treatment and PCR.We were able to demonstrate bystander effect in both 2D and 3D. In mice, tumors initially regressed, but they all eventually recurred including those produced from 80% yCD expressing cells. Cells explanted from the recurrent tumors demonstrated that suicide gene expressing cells had been selected against during in vivo treatment with 5-FC.We conclude that gene therapy of malignant tumors in patients using the yCD/5-FC system will require targeting well over 80% of the malignant cells, and therefore will likely require improved bystander effect or repeated treatment.

Biphenotypic Differentiation of Pancreatic Cancer in 3-Dimensional Culture.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is the third most common cause of cancer death in the United States. Improved characterized models of PDAC are needed for drug screening. METHODS: We grew 4 established pancreatic cancer cell lines in hanging drop cultures to produce spheroids. We also grew organoids from explanted xenografted PDAC and surgically resected primary PDAC. We performed transmission and scanning electron microscopy and compared findings with those of the normal pancreatic duct. We also performed single-cell cloning to determine the potential options for differentiation. RESULTS: Spheroids contained tight junctions and desmosomes but lacked zymogen granules, as expected. The former features were present in normal pancreatic duct but absent from PDAC cell lines grown in standard 2-dimensional culture. Spheroids functionally excluded macromolecules in whole mounts. Cells on the surface of PDAC spheroids were carpeted by microvilli except for rare cells with prominent stereocilia. Carpets of microvilli were also seen in low passage organoids produced from xenografts and surgically resected human PDAC, in addition to normal human pancreatic duct. We performed single-cell cloning and resulting spheroids produced both cell phenotypes at the same approximate ratios as those from bulk cultures. CONCLUSIONS: Pancreatic cancer spheroids/organoids are capable of biphenotypic differentiation.

Down-regulation of osteopontin mediates a novel mechanism underlying the cytostatic activity of TGF-ß.

PURPOSE: Loss of a cytostatic response to TGF-ß has been implicated in multiple hyper-proliferative disorders, including cancer. Although several key genes involved in the cytostatic activity of TGF-ß have in the past been identified, its exact mode of action is yet to be elucidated. A comprehensive understanding of the mechanisms underlying the cytostatic activity of TGF-ß may open up new avenues for the development of therapeutic strategies. METHODS: Quantitative real-time RT-PCR was used to assess osteopontin (OPN) gene expression in human hepatoma-derived Huh-7 and lung adenocarcinoma-derived A549 cells. Reporter assays using an OPN promoter-luciferase construct and its mutated counterparts were performed to assess its transcriptional activity. Binding of Smad4 to the OPN gene promoter was investigated using chromatin immunoprecipitation (CHIP). The putative role of Smad4 in OPN gene expression down-regulation was also assessed using a shRNA-mediated knockdown strategy. The anti-proliferative effect of TGF-ß on different cancer-derived cell lines was determined using the cell proliferation reagent WST-1. RESULTS: We found that the OPN expression levels dose-dependently decreased in TGF-ß-treated Huh-7 and A549 cells. Our reporter assays indicated that this TGF-ß-induced repression occurred at the transcriptional level, and could largely be abrogated by disruption of an element (TIE2) similar to the TGF-ß inhibitory element found in other TGF-ß-repressed genes. Our CHIP assay revealed that the Smad protein complex specifically binds to the OPN gene promoter, and that the TGF-ß-mediated inhibition of OPN was lost upon shRNA-mediated knockdown of Smad4. Moreover, we found that the deregulation of OPN gene expression by TGF-ß occurred concomitantly with loss of the TGF-ß anti-proliferative response, whereas a neutralizing anti-OPN antibody partially restored this response. CONCLUSIONS: Our results indicate that the OPN gene is a direct target of Smad-mediated TGF-ß signaling, implying that OPN expression inhibition serves as a novel mechanism underlying the cytostatic activity of TGF-ß.

Identification of brefelamide as a novel inhibitor of osteopontin that suppresses invasion of A549 lung cancer cells.

The contribution of aberrant osteopontin (OPN) expression to tumor progression and metastasis has been documented in a wide spectrum of malignancies, and targeted inhibition of OPN has therefore emerged as an attractive strategy for cancer therapy. Transcription of OPN is regulated by various transcription factors, and our recently published study demonstrated that downregulation of OPN is an important event in the TGF­ß cytostatic program. We report here that brefelamide exerts an inhibitory effect on OPN expression and function in A549 human lung carcinoma cells. The promoter, RNA, and protein levels of OPN were decreased in brefelamide­treated A549 cells, which was accompanied by reduced invasive ability in vitro. OPN inhibition by brefelamide was largely abrogated by disruption of a putative TGF­ß inhibitory element in the OPN promoter. Treatment with brefelamide induced Smad4 expression, and knockdown of Smad4 by RNA interference partially diminished the inhibitory effect of brefelamide on OPN. These results indicate that brefelamide inhibited OPN­mediated cell invasion through restoration of the OPN repression by TGF­ß/Smad signaling. Together with the reported antiproliferative property, our findings suggest that brefelamide might serve as a potential candidate for the development of a new antitumor and antimetastatic agent.

Mutations of NOTCH3 in childhood pulmonary arterial hypertension.

Mutations of BMPR2 and other TGF-ß superfamily genes have been reported in pulmonary arterial hypertension (PAH). However, 60-90% of idiopathic PAH cases have no mutations in these genes. Recently, the expression of NOTCH3 was shown to be increased in the pulmonary artery smooth muscle cells of PAH patients. We sought to investigate NOTCH3 and its target genes in PAH patients and clarify the role of NOTCH3 signaling. We screened for mutations in NOTCH3, HES1, and HES5 in 41 PAH patients who had no mutations in BMPR2, ALK1, endoglin, SMAD1/4/8, BMPR1B, or Caveolin-1. Two novel missense mutations (c.2519 G>A p.G840E, c.2698 A>C p.T900P) in NOTCH3 were identified in two PAH patients. We performed functional analysis using stable cell lines expressing either wild-type or mutant NOTCH3. The protein-folding chaperone GRP78/BiP was colocalized with wild-type NOTCH3 in the endoplasmic reticulum, whereas the majority of GRP78/BiP was translocated into the nuclei of cells expressing mutant NOTCH3. Cell proliferation and viability were higher for cells expressing mutant NOTCH3 than for those expressing wild-type NOTCH3. We identified novel NOTCH3 mutations in PAH patients and revealed that these mutations were involved in cell proliferation and viability. NOTCH3 mutants induced an impairment in NOTCH3-HES5 signaling. The results may contribute to the elucidation of PAH pathogenesis.

Identification of CD44 as a downstream target of noncanonical NF-κB pathway activated by human T-cell leukemia virus type 1-encoded Tax protein.

Our previous study showed Human T-cell leukemia virus type 1 Tax induces osteopontin (OPN) expression by transactivating its promoter. As an extension, we investigated here the possible influence of Tax on CD44, an important receptor for OPN. Co-expression of Tax, but not its NF-κB-defective mutant, significantly increased the reporter gene expression directed by CD44 promoter. Tax-mediated CD44 activation was largely diminished by disrupting an element similar to the noncanonical κß site found in other IKKα target genes, and further, co-transfection of RelB siRNA abolished CD44 induction by Tax, suggesting an involvement of noncanonical NF-κB pathway in Tax-mediated transactivation. Consistently, chromatin immunoprecipitation revealed a specific interaction of CD44 promoter with RelB-containing complex. Together, these results indicate that D44 gene is one of the downstream target genes of aberrantly activated noncanonical NF-κB signaling by Tax, providing an additional line of evidence explaining how Tax-induced NF-κB signaling is integrated into a fate-determining cellular program.

Transactivation of human osteopontin promoter by human T-cell leukemia virus type 1-encoded Tax protein.

Osteopontin (OPN) is a cytokine that contributes substantially to the growth and metastasis in a wide spectrum of malignancies. We report here that OPN gene is transactivated by Tax protein of human T-cell leukemia virus type 1 (HTLV-1). Northern blot showed enhanced OPN gene expression in cells stably expressing Tax. Co-expression of Tax increased the reporter gene expression directed by OPN promoter. Tax-induced OPN activation was abrogated by treatment with LY294002 (PI3K inhibitor) or co-transfection with AKT siRNA, suggesting PI3K/AKT pathway is involved in Tax-mediated transactivation. Reporter assay with deletion mutants showed that the 5'-partial sequence between -765 and -660 of the OPN promoter is the region responsive to Tax, and further, disrupting the AP-1 site within this region abolished the OPN induction by Tax, indicating that Tax activation of OPN promoter is likely mediated by AP-1 site. This study suggests that OPN is one of the downstream mediators of aberrantly activated PI3K/AKT signaling by Tax, which may partially contribute to HTLV-1-associated leukemogenesis.

Mutation of junctophilin type 2 associated with hypertrophic cardiomyopathy.

Junctophilin subtypes, designated as JPH1 approximately 4, are protein components of junctional complexes and play essential roles in cellular Ca2+ signaling in excitable cells. Knockout mice lacking the cardiac-type Jph2 die of embryonic cardiac arrest, and the mutant cardiac myocytes exhibit impaired formation of peripheral couplings and arrhythmic Ca2+ signaling caused by functional uncoupling between dihydropyridine and ryanodine receptor channels. Based on these observations, we hypothesized that mutations of JPH2 could cause human genetic cardiac diseases. Among 195 Japanese patients (148 index cases and 47 affected family members) with hypertrophic cardiomyopathy (HCM), two heterozygous nonsynonymous nucleotide transitions, G505S and R436C, were newly found in JPH2. When Fisher's exact test was used to compare index cases with HCM to unrelated Japanese healthy controls in the frequencies of mutant alleles, only the G505S mutation showed statistical significance (4/296 HCM patients and 0/472 control individuals, P=0.022). This result was still significant after Bonferroni's correction for multiple comparisons (P=0.044). To the best of our knowledge, this is the first report on JPH2 mutation associated with HCM.

Down-regulation of viral replication by adenoviral-mediated expression of siRNA against cellular cofactors for hepatitis C virus.

Small interfering RNA (siRNA) is currently being evaluated not only as a powerful tool for functional genomics, but also as a potentially promising therapeutic agent for cancer and infectious diseases. Inhibitory effect of siRNA on viral replication has been demonstrated in multiple pathogenic viruses. However, because of the high sequence specificity of siRNA-mediated RNA degradation, antiviral efficacy of siRNA directed to viral genome will be largely limited by emergence of escape variants resistant to siRNA due to high mutation rates of virus, especially RNA viruses such as poliovirus and hepatitis C virus (HCV). To investigate the therapeutic feasibility of siRNAs specific for the putative cellular cofactors for HCV, we constructed adenovirus vectors expressing siRNAs against La, polypyrimidine tract-binding protein (PTB), subunit gamma of human eukaryotic initiation factors 2B (eIF2Bgamma), and human VAMP-associated protein of 33 kDa (hVAP-33). Adenoviral-mediated expression of siRNAs markedly diminished expression of the endogenous genes, and silencing of La, PTB, and hVAP-33 by siRNAs substantially blocked HCV replication in Huh-7 cells. Thus, our studies demonstrate the feasibility and potential of adenoviral-delivered siRNAs specific for cellular cofactors in combating HCV infection, which can be used either alone or in combination with siRNA against viral genome to prevent the escape of mutant variants and provide additive or synergistic anti-HCV effects.