Daranee Chokchaichamnankit

Proteomic studies of cholangiocarcinoma and hepatocellular carcinoma cell secretomes.

Cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) occur with relatively high incidence in Thailand. The secretome, proteins secreted from cancer cells, are potentially useful as biomarkers of the diseases. Proteomic analysis was performed on the secreted proteins of cholangiocarcinoma (HuCCA-1) and hepatocellular carcinoma (HCC-S102, HepG2, SK-Hep-1, and Alexander) cell lines. The secretomes of the five cancer cell lines were analyzed by SDS-PAGE combined with LC/MS/MS. Sixty-eight proteins were found to be expressed only in HuCCA-1. Examples include neutrophil gelatinase-associated lipocalin (lipocalin 2), laminin 5 beta 3, cathepsin D precursor, desmoplakin, annexin IV variant, and annexin A5. Immunoblotting was used to confirm the presence of lipocalin 2 in conditioned media and cell lysate of 5 cell lines. The results showed that lipocalin 2 was a secreted protein which is expressed only in the conditioned media of the cholangiocarcinoma cell line. Study of lipocalin 2 expression in different types of cancer and normal tissues from cholangiocarcinoma patients showed that lipocalin 2 was expressed only in the cancer tissues. We suggest that lipocalin 2 may be a potential biomarker for cholangiocarcinoma.

Identification of novel allergen in edible insect, Gryllus bimaculatus and its cross-reactivity with Macrobrachium spp. allergens

Edible insects have recently been promoted as a source of protein and have a high nutrition value. Identification of allergens and cross-reactivity between Macrobrachium spp. and the field cricket (Gryllus bimaculatus) is necessary for food safety control and to assist in the diagnosis and therapy of allergy symptoms. Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate proteins. Allergens were determined and identified by IgE-immunoblotting with pooled sera from prawn-allergic patients (n=16) and LC-MS/MS. Arginine kinase (AK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were determined as the important allergens in muscle of Macrobrachium rosenbergii whereas, hemocyanin (HC) was identified as an allergen in Macrobrachium spp. The allergens in Macrobrachium lanchesteri were identified as AK and HC. In addition, hexamerin1B (HEX1B) was identified as a novel and specific allergen in G. bimaculatus. The important allergen in G. bimaculatus and Macrobrachium spp. is AK and was found to cross-react between both species.

Proteomic analysis and abrogated expression of O-GlcNAcylated proteins associated with primary breast cancer

O‐GlcNAcylation is a dynamic PTM of nuclear and cytoplasmic proteins, regulated by O‐GlcNAc transferase (OGT) and O‐GlcNAcase, which catalyze the addition and removal of O‐GlcNAc, respectively. This modification is associated with glucose metabolism, which plays important roles in many diseases including cancer. Although emerging evidence reveals that some tumor‐associated proteins are O‐GlcNAc modified, the total O‐GlcNAcylation in cancer is still largely unexplored. Here, we demonstrate that O‐GlcNAcylation was increased in primary breast malignant tumors, not in benign tumors and that this augmentation was associated with increased expression of OGT level. Using 2D O‐GlcNAc immnoblotting and LC‐MS/MS analysis, we successfully identified 29 proteins, with seven being uniquely O‐GlcNAcylated or associated with O‐GlcNAcylation in cancer. Of these identified proteins, some were related to the Warburg effect, including metabolic enzymes, proteins involved in stress responses and biosynthesis. In addition, proteins associated with RNA metabolism, gene expression, and cytoskeleton were highly O‐GlcNAcylated or associated with O‐GlcNAcylation. Moreover, OGT knockdown showed that decreasing O‐GlcNAcylation was related to inhibition of the anchorage‐independent growth in vitro. These data indicate that aberrant protein O‐GlcNAcylation is associated with breast cancer. Abnormal modification of these O‐GlcNAc‐modified proteins might be one of the vital malignant characteristics of cancer.

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.

Aberrant O-GlcNAc-modified proteins expressed in primary colorectal cancer

O-GlcNAcylation is a post-translational modification of serine and threonine residues which is dynamically regulated by 2 enzymes; O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) that catalyze the addition and removal of a single N-acetylglucosamine (GlcNAc) molecule, respectively. This modification is thought to be a nutrient sensor in highly proliferating cells via the hexosamine biosynthesis pathway, a minor branch of glycolysis. Although emerging evidence suggests that O-GlcNAc modification is associated with many types of cancer, identification of O-GlcNAc-modified proteins and their role in cancer remain unexplored. In the present study, we demonstrated that O-GlcNAcylation is increased in primary colorectal cancer tissues, and that this augmentation is associated with an increased expression of OGT levels. Using 2-dimensional O-GlcNAc immunoblotting and LC-MS/MS analysis, 16 proteins were successfully identified and 8 proteins showed an increase in O-GlcNAcylation, including cytokeratin 18, heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/B1), hnRNP H, annexin A2, annexin A7, laminin-binding protein, α-tubulin and protein DJ-1. Among these identified proteins, annexin A2 was further confirmed to show overexpression of O-GlcNAc in all cancer samples. The results, therefore, indicate that aberrant O-GlcNAcylation of proteins is associated with colorectal cancer and that identification of O-GlcNAc-modified proteins may provide novel biomarkers of cancer.

Increased oxidative metabolism is associated with erythroid precursor expansion in β0-thalassaemia/Hb E disease

Erythropoiesis in β0-thalassaemia/Hb E patients, the most common variant form of β-thalassaemia in Southeast Asia, is characterized by accelerated differentiation and over-expansion of erythroid precursor cells. The mechanism driving this accelerated expansion and differentiation remain unknown. To address this issue a proteomic analysis was undertaken to firstly identify proteins differentially expressed during erythroblast differentiation and a second analysis was undertaken to identify proteins differentially expressed between β0-thalassaemia/Hb E erythroblasts and control erythroblasts. The majority of proteins identified as being differentially expressed between β0-thalassaemia/Hb E and control erythroblasts were constituents of the glycolysis/TCA pathway and levels of oxidative stress correlated with the degree of erythroid expansion. A model was constructed linking these observations with previous studies showing increased phosphorylation of ERK1/2 in thalassemic erythroblasts which predicted the increased activation of PKA, PKB and PKC which Western analysis confirmed. Inhibition of PKA or PKC reduced β0-thalassaemia/Hb E erythroblast differentiation and/or expansion. We propose that increased expansion and differentiation of β0-thalassaemia/Hb E erythroblasts occur as a result of feedback loops acting through increased oxidative metabolism.

Metabolic alteration of HepG2 in scaffold‐based 3‐D culture: Proteomic approach

3‐D cell culture models are important in cancer biology since they provide improved understanding of tumor microenvironment. We have established a 3‐D culture model using HepG2 in natural collagen‐based scaffold to mimic the development of small avascular tumor in vivo. Morphological characterization showed that HepG2 colonies grew within the interior of the scaffold and showed enhanced extracellular matrix deposition. High levels of cell proliferation in the outermost regions of the scaffold created a hypoxic microenvironment in the 3‐D culture system, as indicated by hypoxia‐inducible factor‐1α stabilization, detectable by Western blotting and immunohistochemistry. Proteomic studies showed decreased expression of several mitochondrial proteins and increased expression of proteins in anaerobic glycolysis under 3‐D culture compared to monolayer culture. Creatine kinase was also upregulated in 3‐D culture, indicating its possible role as an important energy buffer system under hypoxic microenvironment. Increased levels of proteins in nucleotide metabolism may relate to cellular energy. Thus, our results suggest that HepG2 cells under 3‐D culture adapt their energy metabolism in response to hypoxic conditions. Metabolic alterations in the 3‐D culture model may relate to physiological changes relevant to development of small avascular tumor in vivo and their study may improve future therapeutic strategies.

Identification of a novel allergen from muscle and various organs in banana shrimp (Fenneropenaeus merguiensis)

BACKGROUND The increasing consumption of shellfish can cause an increase in allergic symptoms. Shrimp allergy can be species specific, but specific allergies in different organs have not been studied. Identification of allergens in muscle and others organs of banana shrimp is necessary for improved diagnostics of allergies for shrimp and food safety control. OBJECTIVE To identify the IgE-binding proteins in various organs of Fenneropenaeus merguiensis by immunoblotting and tandem mass spectrometry. METHODS Proteomic methods were used to investigate the allergenic proteins from banana shrimp. Proteins from muscle and various organs were separated by denaturing polyacrylamide gel electrophoresis. Allergens were analyzed by immunoblotting with pooled sera from shrimp allergic patients (n = 21) and tandem mass spectrometry. RESULTS The important allergens in banana shrimp are arginine kinase, sarcoplasmic calcium-binding protein, myosin heavy chain, hemocyanin, enolase, and glyceraldehyde-3-phosphate dehydrogenase, which can be demonstrated by immunoblotting in muscle and shell. Moreover, vitellogenin, ovarian peritrophin 1 precursor, β-actin, and 14-3-3 protein were suggested as allergens in the ovary at different stages of ovarian development. CONCLUSION Ten allergens were identified as allergens in various organs, and they are suggested as novel allergens in banana shrimp. The major allergen in muscle and shell from this shrimp is arginine kinase, whereas the major allergen in the ovary is vitellogenin.

A proteomic analysis of Curcuma comosa Roxb. rhizomes

BackgroundThe similarly in plant physiology and the difficulty of plant classification, in some medicinal plant species, especially plants of the Zingiberaceae family, are a major problem for pharmacologists, leading to mistaken use. To overcome this problem, the proteomic base method was used to study protein profiles of the plant model, Curcuma comosa Roxb., which is a member of the Zingiberaceae and has been used in traditional Thai medicine as an anti-inflammatory agent for the treatment of postpartum uterine bleeding.ResultsDue to the complexity of protein extraction from this plant, microscale solution-phase isoelectric focusing (MicroSol-IEF) was used to enrich and improve the separation of Curcuma comosa rhizomes phenol-soluble proteins, prior to resolving and analyzing by two-dimensional polyacrylamide gel electrophoresis and identification by tandem mass spectrometry. The protein patterns showed a high abundance of protein spots in the acidic range, including three lectin proteins. The metabolic and defense enzymes, such as superoxide dismutase (SOD) and ascorbate peroxidase, that are associated with antioxidant activity, were mainly found in the basic region. Furthermore, cysteine protease was found in this plant, as had been previously reported in other Zingiberaceae plants.ConclusionThis report presents the protein profiles of the ginger plant, Curcuma comosa. Several interesting proteins were identified in this plant that may be used as a protein marker and aid in identifying plants of the Zingiberaceae family.

Comparative proteomic analysis of oral squamous cell carcinoma and adjacent non-tumour tissue from Thailand

OBJECTIVE The study was aimed at analysing and identifying the proteins that are differentially expressed in oral squamous cell carcinoma (OSCC) compared to adjacent non-tumour tissue. MATERIALS AND METHODS Two-dimensional (2D) sodium dodecyl sulphate-polyacrylamide gel electrophoresis accompanied by mass spectrometry (matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry and liquid chromatography-tandem mass spectrometry) was used to analyse and identify the differentially expressed proteins in 10 pairs of tumours and adjacent non-tumour tissues from five cases of early-stage and five cases of late-stage OSCC. The statistical differences of the protein spots were analysed by the Wilcoxon signed-rank test. A validation study using immunohistochemistry and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) was performed. RESULTS A total of 68 proteins (63 up-regulated, five down-regulated) were differentially expressed in early-stage disease, and 39 proteins (37 up-regulated, two down-regulated) were significantly altered in late-stage disease. Among these, 14 proteins were altered in both groups. A total of 44 proteins were identified, including heat shock proteins (HSPs: Hsp90, HSPA5 and HSPA8), keratins (K1, K6A and K17), tubulin, cofilin 1, 14-3-3σ and metabolic enzymes. These proteins are involved in various cellular processes essential for cell growth, survival and cell migration. The validation study on α-tubulin and 14-3-3σ using immunohistochemistry and KIAA1199 expression using real-time RT-PCR confirmed the results in proteomics analysis. CONCLUSIONS The study identified many proteins, both known and unknown, for cancer cell processes. At least two proteins, KIAA1199 and Horf6, are novel for oral cancer.