Pantipa Subhasitanont

Detection of cathepsin B up-regulation in neoplastic thyroid tissues by proteomic analysis

Nodular or multinodular goiter is the most common non‐neoplastic thyroid disease and may be difficult to distinguish from true neoplastic thyroid diseases using microscopic criteria. We have used two‐dimensional gel electrophoresis to study the protein patterns of thyroid tissues including normal thyroid, multinodular goiter, diffuse hyperplasia, follicular adenoma, follicular carcinoma and papillary carcinoma. Specific proteins, in the region of molecular mass 15–30 kDa and isoelectric point 4.5–6.5, were identified by electrospray tandem mass spectrometry and protein sequencing. The most distinctive protein found is cathepsin B, which could be detected as four spots, with differential expression in different thyroid diseases. In particular, two of these cathepsin B spots CB2 and CB3 are strongly up‐regulated in neoplastic diseases, compared to non‐neoplastic diseases. In addition, overexpression of ATP synthase D chain and prohibitin were observed in papillary carcinoma, which should allow it to be differentiated from follicular carcinoma. Changes in expression of other proteins were also observed in disease states compared to normal tissues, namely translationally controlled tumor protein, thioredoxin peroxidase 1, glutathione‐S‐transferase P, DJ‐1 protein, superoxide dismutase (Cu, Zn), and heat shock protein 27, but these changes are less characteristic, so they do not allow the differentiation between neoplastic and non‐neoplastic tissues. Thus, the proteomic approach is a useful diagnostic tool for studying diseases involving the thyroid nodule.

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.

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.

Role of curcuminoids in ameliorating oxidative modification in β-thalassemia/Hb E plasma proteome

Thalassemic patients often exhibit high levels of oxidative stress and iron overload, which can lead to hazardous complications. Curcuminoids, extracted from the spice turmeric, are known to have antioxidant and iron-chelating properties and have been proposed as a potential upstream therapy of thalassemia. Here we have applied proteomic techniques to study the protein profile and oxidative damage in the plasma of β-thalassemia/Hb E patients before and after treatment with curcuminoids. In this study, 10 β-thalassemia/Hb E patients were treated with 500 mg curcuminoids daily for 12 months. The plasma protein profile and protein carbonyl content were determined at baseline, 6 and 12 months using two-dimensional fluorescence difference gel electrophoresis and carbonyl immunoblotting, respectively. Other hematological, clinical, and biochemical parameters were also analyzed. Twenty-six spots, identified as coagulation factors and proteins involved in iron homeostasis, showed significantly decreased intensity in thalassemic plasma, compared to those of normal subjects. Treatment with curcuminoids up-regulated the plasma levels of these proteins and reduced their oxidative damage. Serum non-transferrin bound iron, platelet factor-3 like activity, oxidative stress parameters and antioxidant enzymes were also improved after curcuminoids treatment. This study is the first proteomic study of plasma in the thalassemic state and also shows the ameliorating role of curcuminoids towards oxidative stress and iron overload in the plasma proteome.

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.

Effective enrichment of cholangiocarcinoma secretomes using the hollow fiber bioreactor culture system.

The Northeastern region of Thailand is well known to have high incidence of bile duct cancer known as cholangiocarcinoma. So there is a continued need to improve diagnosis and treatment, and discovery of biomarkers for early detection of bile duct cancer should greatly improve treatment outcome for these patients. The secretome, a collection of proteins secreted from cells, is a useful source for identifying circulating biomarkers in blood secreted from cancer cells. Here a Hollow Fiber Bioreactor culture system was used for enrichment of cholangiocarcinoma secretomes, since this culture system mimics the dense three-dimensional microenvironment of the tumor found in vivo. Two-dimensional fluorescence difference gel electrophoresis using a sensitive Fluor saturation dye staining, followed by LC/MS/MS, was used to compare protein expression in the secretomes of cells cultured in the Hollow Fiber system and cells cultured in the monolayer culture system. For the first time, the 2D-patterns of cholangiocarcinoma secretomes from the two culture systems could be compared. The Hollow Fiber system improved the quality and quantity of cholangiocarcinoma secreted proteins compared to conventional monolayer system, showing less interference by cytoplasmic proteins and yielding more secreted proteins. Overall, 75 spots were analyzed by LC/MS/MS and 106 secreted proteins were identified. Two novel secreted proteins (C19orf10 and cystatin B) were found only in the Hollow Fiber system and were absent from the traditional monolayer culture system. Among the highly expressed proteins, 22 secreted soluble proteins were enriched by 5 fold in Hollow Fiber system compared to monolayer culture system. The Hollow Fiber system is therefore useful for preparing a wide range of proteins from low-abundance cell secretomes.

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.