Lukana Ngiwsara

Labdane diterpenes from the rhizomes of Hedychium coronarium

A new labdane diterpenoid, (E)-labda-8(17),12-dien-15,16-olide (1) together with eight known compounds, coronarin D (2), coronarin D methyl ether (3), coronarin D ethyl ether (4), isocoronarin D (5), coronarin B (6), labda-8(17),11,13-trien-15,16-olide (7), (E)-labda-8(17),12-diene-15,16-dial (8) and 16-hydroxylabda-8(17),11,13-trien-15,16-olide (9), are isolated from the rhizomes of Hedychium coronarium. Compounds 2–4, 5 and 9 are isolated as mixtures of C-15, C-14 and C-16 epimers, respectively. Their structures are determined on the basis of their spectroscopic data. The epimeric mixtures of 2 and 3 have not been reported before. Some of them were evaluated for their cytotoxicity.

Molecular analysis of the iduronate-2-sulfatase gene in Thai patients with Hunter syndrome

SummaryMolecular defects in the gene encoding the enzyme iduronate-2-sulfatase (IDS) result in Hunter disease (mucopolysaccharidosis type II, MPS II). To determine the molecular basis of MPS II in Thailand, the IDS gene was analysed in 20 Thai patients with Hunter syndrome from 18 unrelated families. A total of 19 different mutations, including 9 missense mutations, 3 nonsense mutations, 3 splice site alterations, 1 deletion, 2 indels, and 1 rearrangement were identified, 8 of which were novel (p.R101C, p.D148V, p.G224A, p.K227E, p.E254X, p.W337X, c.440_442delinsTT and c.720_731delinsTTTCAGATGTTCTCCCCAG). Evaluation of the IDS activity of two hemizygous variants identified in the same patient, p.R101C and p.R468Q, by expression of IDS with the individual mutations in COS 7 cells indicated that only the p.R468Q mutation affected IDS protein activity. Two exonic mutations, c.257C>T (p.P86L) and c.418G>A, were found to activate multiple cryptic splice sites, resulting in aberrantly spliced transcripts. Thus, MPS II in Thailand is caused by a diverse set of defects affecting both IDS protein production and activity.

Comparison of membrane-associated proteins in human cholangiocarcinoma and hepatocellular carcinoma cell lines

Cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) occur with relatively high incidence in Thailand. Cell line models, originating from Thai patients, are available for both diseases, including the human bile duct epithelial carcinoma cell line (HuCCA‐1) and the HCC cell line HCC‐S102. Here, we have prepared subproteomes enriched in membrane proteins or in cytosolic proteins from the HuCCA‐1 and the HCC‐S102 cell lines. Study of differential protein expression by 2‐DE and LC/MS/MS showed 195 proteins expressed in the two cell lines, including both membrane‐associated and cytosolic proteins. Eighteen proteins were found in both membrane and cytosolic fractions of HuCCA‐1, but not in HCC‐S102, while nine proteins were found in both membrane and cytosolic fractions of HCC‐S102, but not in HuCCA‐1. Ten membrane proteins were found in HuCCA‐1 but not in HCC‐S102, including integrin alpha‐6 precursor, ezrin, hippocalcin‐like protein 1, mitogen‐activated protein kinase kinase kinase 2 (MAPK/ERK kinase kinase 2), and calgizzarin. Proteins showing increased expression in the membrane fraction of HuCCA‐1 were mainly cytoskeletal proteins (40.9%), while proteins showing increased expression in the membrane fraction of HCC‐S102 were mainly metabolic proteins (39.4%). The subproteomic approach used here facilitates detection of potential biomarkers undetected by regular proteomic methods.

A synthetic 2,3-diarylindole induces cell death via apoptosis and autophagy in A549 lung cancer cells.

A series of 2,3-diarylindoles were synthesized via the Larock heteroannulation, and evaluated for their anticancer activity against A549 lung cancer cells. The most potent compound, PCNT13 with IC50=5.17 μM, caused the induction of two modes of programmed cell death, apoptosis and autophagy.

Hb Kurosaki [alpha7(A5)Lys -->Glu (AAG --> GAG)]: an alpha2-globin gene mutation found in Thailand.

Hb Kurosaki [α7(A5)Lys → Glu (AAG →GAG)], has been found for the first time in Thailand. The 30-year-old Thai male had a normal hematological profile at the steady state, but showed an abnormal hemoglobin (Hb) present at a level of 28%. Protein characterization was performed by automated sequencer analysis of the abnormal α-globin chain and amino acid analysis of the abnormal αT-1,2 peptide. Direct DNA sequence analysis of selectively amplified segments of the α1 and α2 genes showed that codon 7 of the α2-globin gene was heterozygous for AAG (Lys) and GAG (Glu). This was confirmed by restriction endonuclease digestion with Eco31I.

Aspirin suppresses components of lymphangiogenesis and lymphatic vessel remodeling by inhibiting the NF-κB/VCAM-1 pathway in human lymphatic endothelial cells:

Lymphangiogenesis is the process of new vessel formation from pre-existing lymphatic vessels. The process mainly involves cell adhesion, migration, and tubule formation of lymphatic endothelial cells. Tumor-induced lymphangiogenesis is an important factor contributing to promotion of tumor growth and cancer metastasis via the lymphatic system. Finding the non-toxic agents that can prevent or inhibit lymphangiogenesis may lead to blocking of lymphatic metastasis. Recently, aspirin, a non-steroidal anti-inflammatory drug (NSAID), has been reported to inhibit in vivo lymphangiogenesis in tumor and incision wound models, but the mechanisms of actions of aspirin on anti-lymphangiogenesis have been less explored. In this study, we aim to explore the mechanism underlying the anti-lymphangiogenic effects of aspirin in primary human dermal lymphatic microvascular endothelial (HMVEC-dLy) cells in vitro. Pretreatment of aspirin at non-toxic dose 0.3 mM significantly suppressed in vitro cord formation, adhesion, and the migration abilities of the HMVEC-dLy cells. Western blotting analysis indicated that aspirin decreased expression of vascular cell adhesion molecule-1 (VCAM-1), at both protein and mRNA levels, and these correlated with the reduction of NF-κB p65 phosphorylation. By using NF-κB inhibitor (BAY-11-7085) and VCAM-1 siRNA, we showed that VCAM-1 expression is downstream of NF-κB activation, and this NF-κB/VCAM-1 signaling pathway controls cord formation, adhesion, and the migration abilities of the HMVEC-dLy cells. In summary, we demonstrate the potential of aspirin as an anti-lymphangiogenic agent, and elucidate its mechanism of action.

p.X654R IDUA variant among Thai individuals with intermediate mucopolysaccharidosis type I and its residual activity as demonstrated in COS-7 cells

Mucopolysaccharidosis type I (MPS I) is a rare autosomal‐recessive disorder caused by defects in alpha‐L‐iduronidase (IDUA), a lysosomal enzyme encoded by the IDUA gene. Herein, we characterized IDUA mutations underlying mucopolysaccharidosis type I intermediate form (Hurler–Scheie syndrome) and its molecular pathogenic mechanisms.

Apigenin inhibits growth and induces apoptosis in human cholangiocarcinoma cells

A promising nutraceutical, apigenin, was recently revealed to exhibit biological activity in inhibiting several types of cancer. The effects of apigenin on the growth inhibition and apoptosis of the cholangiocarcinoma HuCCA-1 cell line were investigated. Protein alterations subsequent to apigenin treatment were studied using a proteomic approach. The values of 20, 50 and 90% inhibition of cell growth (IC20, IC50 and IC90) were determined by MTT cell viability assay. Apoptotic cell death was detected using two different methods, a flow cytometric analysis (Muse Cell Analyzer) and DNA fragmentation assay. A number of conditions including attached and detached cells were selected to perform two-dimensional gel electrophoresis (2-DE) to study the alterations in the expression levels of treated and untreated proteins and identified by liquid chromatography (LC)/tandem mass spectrometry (MS/MS). The IC20, IC50 and IC90 values of apigenin after 48 h treatment in HuCCA-1 cells were 25, 75 and 200 µM, respectively, indicating the cytotoxicity of this compound. Apigenin induced cell death in HuCCA-1 cells via apoptosis as detected by flow cytometric analysis and exhibited, as confirmed with DNA fragmentation, characteristics of apoptotic cells. A total of 67 proteins with altered expression were identified from the 2-DE analysis and LC/MS/MS. The cleavage of proteins involved in cytoskeletal, cytokeratin 8, 18 and 19, and high expression of S100-A6 and S100-A11 suggested that apoptosis was induced by apigenin via the caspase-dependent pathway. Notably, two proteins, heterogeneous nuclear ribonucleoprotein H and A2/B1, disappeared completely subsequent to treatment, suggesting the role of apigenin in inducing cell death. The present study indicated that apigenin demonstrates an induction of growth inhibition and apoptosis in cholangiocarcinoma cells and the apoptosis pathway was confirmed by proteomic analysis.

Molecular analysis of the novel IDS allele in a Thai family with mucopolysaccharidosis type II: The c.928C>T (p.Gln310*) transcript is sensitive to nonsense-mediated mRNA decay

Hunter syndrome (or mucopolysaccharidosis type II, MPS II) is an X-linked recessive disorder induced by a deficiency of the iduronate 2-sulfatase (IDS) enzyme, resulting in the accumulation of glycosaminoglycan substrates, heparan sulfate and dermatan sulfate, in the lysosomes. The progressive accumulation of undegraded metabolites induces cell and tissue dysfunction, leading to multi-systemic pathology. The heterogeneity of clinical phenotypes, ranging from mild to severe forms, results from different mutations in the IDS gene. To date, >550 MPS II causal mutations have been reported in the IDS gene, of which ~10% are nonsense mutations that lead to premature protein termination. In the present study, the IDS mutation causing MPS II in an extended Thai family was identified using IDS enzyme assay and IDS gene exon sequencing. Three family members were enzymatically confirmed to have MPS II and to carry the novel IDS nonsense allele c.928C>T (p.Gln310*). The IDS mRNA levels were evaluated by reverse transcription-quantitative polymerase chain reaction, which demonstrated that all patients exhibited a reduction of IDS mRNA, suggesting its degradation by nonsense-mediated mRNA decay. Expression of wild type and mutant IDS in COS-7 cells revealed that the IDS p.Gln310* mutant lacked IDS activity, consistent with production of a nonfunctional, prematurely truncated protein. Taken together, these results indicate that the IDS c.928C>T (p.Gln310*) mutation is a severe disease-causing mutation for MPS II.