Todd Hsu

Anti-inflammatory properties of phenolic compounds and crude extract from Porphyra dentata.

ETHNOPHARMACOLOGICAL RELEVANCE Porphyra dentata, a red edible seaweed, has long been used worldwide in folk medicine for the treatment of inflammatory diseases such as hypersensitivity, lymphadenitis, bronchitis. AIMS OF STUDY To clarify the anti-inflammatory role of Porphyra dentata crude extract and its identified phenolic compounds by investigating their effect on the nitric oxide (NO)/inducible nitric oxide synthase (iNOS) transcription pathway in macrophage RAW 264.7 cells. MATERIALS AND METHODS Porphyra dentata crude extract was prepared with methanol. High performance liquid chromatography (HPLC) hyphenated to electrospray ionization mass spectrometry (ESI-MS) and UV detection were utilized to analyze the extract fingerprints. Nitrite measurement, iNOS promoter activity and nuclear factor-kappaB (NF-kappaB) enhancer activity were used to assess the anti-inflammatory effect in lipopolysaccharide (LPS) challenged mouse RAW 264.7 cell line. RESULTS Phenolic compounds (catechol, rutin and hesperidin) were identified in the crude extract of Porphyra dentata. The crude extract and the phenolic compounds inhibited the production of NO in LPS-stimulated RAW 264.7 cells. Catechol was a more potent suppressor of the up-regulation of iNOS promoter and NF-kappaB enhancer than rutin and yet, hesperidin alone failed to inhibit either activity. CONCLUSION Our results indicate that catechol and rutin, but not hesperidin, are primary bioactive phenolic compounds in the crude extract to suppress NO production in LPS-stimulated macrophages via NF-kappaB-dependent iNOS gene transcription. The data also explain the anti-inflammatory use and possible mechanism of Porphyra dentata in iNOS implicated diseases.

Cadmium(Cd)-induced oxidative stress down-regulates the gene expression of DNA mismatch recognition proteins MutS homolog 2 (MSH2) and MSH6 in zebrafish (Danio rerio) embryos.

DNA mismatch repair (MMR) of simple base mismatches and small insertion-deletion loops in eukaryotes is initiated by the binding of the MutS homolog 2 (MSH2)-MSH6 heterodimer to mismatched DNA. Cadmium (Cd) is a genotoxic heavy metal that has been recognized as a human carcinogen. Oxidant stress and inhibition of DNA repair have been proposed as major factors underlying Cd genotoxicity. Our previous studies indicated the ability of Cd to disturb the gene expression of MSH6 in zebrafish (Danio rerio) embryos. This study was undertaken to explore if Cd-induced oxidative stress down-regulated MSH gene activities. Following the exposure of zebrafish embryos at 1 h post fertilization (hpf) to sublethal concentrations of Cd at 3-5 μM for 4 or 9 h, a parallel down-regulation of MSH2, MSH6 and Cu/Zn superoxide dismutase (Cu/Zn-SOD) gene expression was detected by real-time RT-PCR and the expression levels were 40-50% of control after a 9-h exposure. Cd exposure also induced oxidative stress, yet no inhibition of catalase gene activity was observed. Whole mount in situ hybridization revealed a wide distribution of msh6 mRNA in the head regions of 10 hpf embryos and pretreatment of embryos with antioxidants butylhydroxytoluene (BHT), d-mannitol or N-acetylcysteine (NAC) at 1-10 μM restored Cd-suppressed msh6 expression. QPCR confirmed the protective effects of antioxidants on Cd-suppressed msh2/msh6 mRNA production. Down-regulated MSH gene activities reaching about 50% of control were also induced in embryos exposed to paraquat, a reactive oxygen species (ROS)-generating herbicide, or hydrogen peroxide at 200 μM. Hence, Cd at sublethal levels down-regulates msh2/msh6 expression primarily via ROS as signaling molecules. The transcriptional activation of human msh6 is known to be fully dependent on the specificity factor 1 (Sp1). Cd failed to inhibit the DNA binding activity of zebrafish Sp1 unless at lethal concentrations based on band shift assay, therefore excluding the involvement of Sp1 inactivation in Cd-induced MSH gene inhibition in zebrafish embryos.

Computed Tomography-Guided Core-Needle Biopsy Specimens Demonstrate Epidermal Growth Factor Receptor Mutations in Patients with Non-Small-Cell Lung Cancer

Background: Target therapy with a new class of epidermal growth factor receptor (EGFR) inhibitors shows improved clinical response in EGFR gene-mutated lung cancers. Purpose: To evaluate the use of computed tomography (CT)-guided core-needle biopsy specimens for the assessment of EGFR gene mutation in non-small-cell lung cancer (NSCLC). Material and Methods: Seventeen (nine males, eight females) patients with advanced NSCLC were enrolled in this study. All patients underwent CT-guided core-needle biopsy of the lung tumor prior to treatment with the EGFR inhibitor gefitinib. There were no life-threatening complications of biopsy. The specimens were sent fresh-frozen for EGFR mutation analysis and histopathological study. Results: There were 12 (70.6%) EGFR gene mutants and five (29.4%) nonmutants. The objective response rate to gefitinib therapy was 73.3% (11 of 15 patients), with 91.7% (11 of 12 mutants) for the mutant group and 0% for the nonmutant group. Conclusion: CT-guided core-needle biopsy of advanced NSCLC enables the acquisition of sufficient tissue for EGFR gene mutation analysis.

Sublethal levels of cadmium down-regulate the gene expression of DNA mismatch recognition protein MutS homolog 6 (MSH6) in zebrafish (Danio rerio) embryos.

MutS homolog 6 (MSH6) is the major mismatch contacting component of the MSH2-MSH6 heterodimeric complex (MutSα) that mediates DNA mismatch repair (MMR) of simple mispairs and small insertion-deletion loops in eukaryotes. This study examined the potential of cadmium (Cd) to disturb the gene expression of MSH6 in vertebrates using zebrafish (Danio rerio) embryo as a model organism. Semiquantitative RT-PCR indicated that msh2 and msh6 expressions were suppressed in embryos at 1h post fertilization (hpf), then drastically up-regulated in 2 hpf embryos and actively expressed in 3-25 hpf embryos. In the presence of a constitutive β-actin expression, exposure of 1 hpf embryos to sublethal concentrations of CdCl(2) at 0.5-3 μM for 4 or 9h caused a time and concentration-dependent down-regulation of msh6 transcription. Cd failed to inhibit msh2 transcription except at 3 μM, reflecting the higher sensitivity of msh6 than msh2 transcription to Cd. Whole mount in situ hybridization showed a wide distribution of msh6 transcripts in the front body portions of 10 hpf embryos and Cd-induced a general suppression of msh6 expression in zebrafish tissues. Cd-induced down-regulation of msh6 transcription paralleled with reduced levels of MSH6 protein synthesis and MSH6-mediated G-T mismatch binding activities identified by band shift assay using recombinant zebrafish MSH6 and an anti-human MSH6 antibody. Our results revealed the inhibition of Cd on MSH6 expression at both mRNA and protein levels and this mechanism may play a role in Cd genotoxicity.

Bisphosphonate zoledronic acid enhances the inhibitory effects of gefitinib on EGFR-mutated non-small cell lung carcinoma cells

Patients with non-small cell lung carcinoma (NSCLC) bearing epidermal growth factor receptor (EGFR) gene mutations are good responders to gefitinib (Iressa), an EGFR tyrosine kinase inhibitor (EGFR-TKI), yet these patients may eventually develop acquired resistance to all available EGFR-TKIs. Nitrogen-containing bisphosphonates (N-BPs) are inhibitors of farnesyl diphosphate (FPP) synthase as well as chelators of divalent cations. This study was undertaken to examine if the N-BP zoledronic acid (zoledronate) possessing antitumor activity could enhance the antitumor effect of gefitinib on the HCC827 NSCLC cell line expressing mutated EGFR. Both gefitinib and zoledronate were cytotoxic to HCC827 cells when treated alone. Combined treatment with gefitinib (0.025 microM) that induced G0/G1 arrest and zoledronate (50 microM) that caused S/G2/M accumulation generated an additive induction in cell cytotoxicity, sub-G1 cell population, and apoptosis. Gefitinib suppressed EGF-activated phosphorylation of ERK1/2 and Akt, while zoledronate seemed to impose its pharmacological effect independent of ERK1/2 and Akt phosphorylation. The volumes of xenografted tumors in nude mice co-administered with gefitinib (1 mg/kg/day, five days a week, p.o.) and zoledronate (10 microg/kg, twice weekly, i.p.) were significantly smaller than those of tumors in mice treated with gefitinib alone at the last stage of a 6-week in vivo study. Severe peri-tumoral fat loss frequently observed in gefitinib-treated mice disappeared in mice receiving the combined treatment. Hence, combined treatment of gefitinib with zoledronate may form a basis to develop a more effective and less toxic therapy for NSCLC with EGFR gene mutations.

A citrate-inducible gene, encoding a putative tricarboxylate transporter, is downregulated by the organic solvent DMSO in Agrobacterium tumefaciens

Aims:  To investigate the effects of the organic solvent dimethyl sulfoxide (DMSO) on the expression of a citrate‐inducible gene, encoding a putative tricarboxylate transporter, in Agrobacterium tumefaciens.

Expression of Estrogen Receptors Alfa and Beta mRNA and Alkaline Phosphatase in the Differentiation of Osteoblasts from Elderly Postmenopausal Women: Comparison with Osteoblasts from Osteosarcoma Cell Lines

OBJECTIVE To evaluate the expression of estrogen receptors (ER) alpha and beta, and activity of alkaline phosphatase during differentiation of primary osteoblast cells (hOB) from aged postmenopausal women and human osteosarcoma cell lines (HOS, MG63). MATERIALS AND METHODS Osteoblast cultures were prepared from the upper femur of postmenopausal patients (age, 60-74 years) and HOS. At the indicated times (days 5, 10, 15, 20, and 25), alkaline phosphatase activity and expression of ERalpha and ERbeta mRNA were evaluated. RESULTS In both cultures of primary hOB and HOS, alkaline phosphatase activity decreased at the osteoblast proliferation stage, whereas it subsequently increased at the matrix maturation stage. ER beta mRNA was strongly expressed in HOS on day 15 and remained at high levels of transcription through to day 25 (matrix maturation phase), whereas ERalpha mRNA was barely detectable during osteoblast differentiation. In hOB, transcription of ERalpha mRNA was much stronger than that of ERbeta mRNA. CONCLUSION The presence of ERalpha and ERbeta mRNA in osteoblasts supports the involvement of estrogen in human bone formation. The developmental expression of alkaline phosphatase was not correlated to ER mRNA expression during osteoblast differentiation. ER isoforms may have different functions or interact with each other during osteoblast differentiation. Since the expression of ER isoforms is different between postmenopausal women and osteosarcoma cell lines, characteristics of osteosarcoma cell lines may not be suitable as a model for the evaluation of estrogen effects on postmenopausal osteoporosis.

Response to Sorafenib in a Patient with Metastatic Xp11 Translocation Renal Cell Carcinoma

Sorafenib, a multikinase inhibitor of tumour-cell proliferation and angiogenesis, has been shown to have a role in the treatment of metastatic renal cell carcinoma (RCC). Xp11 translocation carcinoma is a rare subtype of RCC. We report an 18-year-old male patient with metastatic Xp11 translocation RCC who was responsive to sorafenib treatment. Six weeks after commencement of treatment with sorafenib, a CT scan of the patient showed increased central necrosis of the kidney mass and para-aortic lymph nodes as well as regression of the lung and pleural masses. The patient had a progression-free survival of 12 months, and overall survival of 15 months. The most severe adverse effects were grade 3 dermatitis and grade 3 anaemia. This case has demonstrated for the first time that sorafenib is active against Xp11 translocation RCC.

Studies on the susceptibility of various organs of zebrafish (Brachydanio rerio) to benzo(a)pyrene-induced DNA adduct formation

Abstract The susceptibility of various fish organs to polycylic aromatic hydrocarbons(PAHs)-induced DNA adduct formation was studied in zebrafish using benzo(a)pyrene as the representative carcinogenic PAH. Following exposure of fish to waterbome BaP at 0.2 mg/L for 3 days and at lmg/L for 4 days, 32P-postlabling analysis indicated that the adduct levels in intestine,liver, brain, and testis DNA were 13.3 ± l.2, 4.3 ± 2.5, 3.8 ± 0.5, and 0.2 0.l adducts per 108 nucleotides, respectively. When zebrafish were treated with BaP at 0.02 mg/L for 3 ±days and at 0.l mg/L for 4 days, a significant increase in the level of bulky adducts was detected only in intestine DNA (0.28 t 0.06 adduct / 108 nucleotides), and no adduct spots were observed for DNA isolated from other organs. Our data suggest that the fish intestine is a more sensitive target organ than the liver for biomonitoring the presence of carcinogenic PAHs in the aquatic enviroment, especially when PAHs are present at low levels.

Possible involvement of a 72-kDa polypeptide in nucleotide excision repair of Chlorella pyrenoidosa identified by affinity adsorption and repair synthesis assay

A DNA repair synthesis assay monitoring nucleotide excision repair (NER) was established in cell-free extracts of unicellular alga Chlorella pyrenoidosa using cisplatin- or mitomycin C-damaged plasmid DNA as the repair substrate. The algal extracts promoted a damage-dependent increase in 32P-dATP incorporation after normalization against an internal control. To identify the proteins responsible for NER, a biotin-labeled duplex 27 mer (2 µg) irradiated with or without UV (27 kJ m(-2)) was immobilized on streptavidin-conjugated agarose beads and incubated with C. pyrenoidosa extracts (50 µg) to pull down repair proteins. The extracts post incubation with beads carrying unirradiated 27 mer promoted an eightfold increase in repair synthesis in plasmid DNA (1 µg) damaged by 16.8 pmol of cisplatin. The extracts obtained after affinity adsorption with UV-damaged DNA ligand, however, failed to repair plasmid DNA treated with cisplatin, reflecting that some proteins crucial to NER had been sequestered by the damaged 27 mer. A polypeptide approximately 70-72 kDa in molecular mass was found to bind much more strongly to the damaged DNA than to the control DNA after analyzing the proteins bound to the beads by SDS-PAGE, and this polypeptide is believed to play a role in NER in C. pyrenoidosa.