Identification of potential cervical cancer serum biomarkers in Thai patients.

Cervical cancer is one of the most common causes of cancer-associated mortality in females worldwide. Serum biomarkers are important tools for diagnosis, disease staging, monitoring treatment and detecting recurrence in different types of cancer. However, only a small number of established biomarkers have been used for clinical diagnosis of cervical cancer. Therefore, the identification of minimally invasive, sensitive and highly specific biomarkers for detection of cervical cancer may improve outcomes. In the present pilot study, changes in disease-relevant proteins in 31 patients with cervical cancer were compared with 16 healthy controls. The Human 14 Multiple Affinity Removal system was used to deplete the 14 most abundant serum proteins to decrease sample complexity and to enrich proteins that exhibited decreased levels of abundance in the serum samples. Immunoaffinity-depleted serum samples were analyzed by in-gel digestion, followed by liquid chromatography mass spectrometry analysis and data processing. Automated quantitative western blot assays and receiver operating characteristic (ROC) curves were used to evaluate the differential protein expression levels between the two groups. Capillary electrophoresis-based western blot analysis was performed to quantitatively determine serum levels of the candidate biomarkers. Significantly increased levels of α-1-antitrypsin (A1AT) and pyrroline-5-carboxylate reductase 2 (PYCR2) were detected, whereas the levels of transthyretin (TTR), apolipoprotein A-I (ApoA-I), vitamin D binding protein (VDBP) and multimerin-1 (MMRN1) were significantly decreased in patients with cervical cancer compared with the healthy controls. ROC curve analysis indicated that the sensitivity and specificity was improved through the combination of the 6 candidate biomarkers. In summary, the results demonstrated that 6 candidate biomarkers (A1AT, PYCR2, TTR, ApoA-I, VDBP and MMRN1) exhibited significantly different expression between serum samples from healthy controls and patients with cervical cancer. These proteins may represent potential biomarkers for distinguishing patients with cervical cancer from healthy controls and for differentiation of patient subgroups.

Perfluorooctanoic Acid Enhances Invasion of Follicular Thyroid Carcinoma Cells Through NF-κB and Matrix Metalloproteinase-2 Activation.

BACKGROUND/AIM: Perfluorooctanoic acid (PFOA) is one of the most common perfluorinated compounds widely used in several applications. Due to its persistence in the environment, PFOA has been associated with various diseases, including cancer. This study explored the effects of PFOA on follicular thyroid carcinoma cells (FTC133). MATERIALS AND METHODS: Cell invasion, migration, adhesion and activity of matrix metalloproteinase-2 (MMP-2) were investigated using Transwell assays, adhesion assay and gelatin zymography, respectively. The underlying mechanism involved in the effects observed was evaluated by immunoblot analyses. RESULTS: Treatment with PFOA did not affect cell migration, but enhanced cell invasion, adhesion and activity of MMP-2 in FTC133 cells. PFOA selectively enhanced the phosphorylation of nuclear factor kappa B (NF-κB) p65, as well as induced NF-κB nuclear translocation. Treatment with a NF-κB inhibitor (BAY 11-7085) was able to reverse PFOA-induced cell invasiveness. CONCLUSION: PFOA promotes invasiveness of FTC133 cells mediated through the activation of NF-κB signaling.

Tumor Susceptibility Gene 101 Mediates Anoikis Resistance of Metastatic Thyroid Cancer Cells.

BACKGROUND/AIM: Resistance to anoikis is a pre-requisite step in metastasis, a major cause of death in patients with cancer, including thyroid cancer. Impairing anoikis resistance is a possible strategy for therapy of metastatic cancer. We, therefore, we aimed to investigate the key players of anoikis resistance. MATERIALS AND METHODS: Papillary-type (BCPAP), follicular-type (FTC133), and anaplastic-type (ARO) thyroid carcinoma cells, cultured in poly(2-hydroxyethyl methacrylate)-coated plates to mimic circulating cells, were used as model systems in this study. Flow cytometry and soft-agar assays were used to determine cells exhibiting anoikis resistance. Proteomics was used to identify candidate proteins and validated using western blot and siRNA knockdown. RESULTS: Only ARO cells showed both anoikis resistance potential and anchorage-independent growth ability. Tumor susceptibility gene 101 protein (TSG101) was identified to be potentially important in anoikis resistance, which was confirmed by an increase in anoikis and expression of a pro-apoptotic protein (BCL-2 like protein 4) and an apoptotic marker (cleaved poly-ADP ribose polymerase) in floating siTSG101-knockdown cells. CONCLUSION: To our knowledge, this is the first study that implicates the importance of TSG101 in anoikis resistance of thyroid cancer.

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.

Involvement of vimentin in neurite outgrowth damage induced by fipronil in SH-SY5Y cells.

Fipronil, a phenylpyrazole insecticide, is more selective in its potency towards insects than humans and is thus commonly used. In this study, we demonstrated that exposure to fipronil may pose a human health risk. We observed in vitro the shortening of neurite outgrowths of SH-SY5Y neuroblastoma cells upon treatment with fipronil, even at a non-cytotoxic concentration. Fipronil induced apoptosis involving caspase-6, which is an apoptotic effector highly implicated in neurodegenerative diseases. Moreover, at a concentration that did not induce apoptosis, mitochondrial dysfunction and autophagic vacuole formation were detected. Interestingly using proteomics, we identified vimentin to be dramatically expressed by SH-SY5Y cells as a response to fipronil treatment. Not only did the expression of total vimentin increase, different isoforms were observed, indicating alterations in post-translational modifications. Vimentin was localized at the neurite outgrowth, possibly to repair the damage in cellular structure. However at high concentrations of fipronil, vimentin was found in less defined fibrils, in bridge-like formation, and dense surrounding vacuoles. In all, our results indicate that vimentin plays an important role in fipronil-induced neurotoxicity in SH-SY5Y cells.

Unveiling a novel biomarker panel for diagnosis and classification of well-differentiated thyroid carcinomas.

Thyroid cancer is the most common human endocrine malignancy with increasing global incidence. Papillary thyroid carcinomas (PTC) and follicular thyroid carcinomas (FTC) are well-differentiated thyroid cancers (WDTC) accounting for 95% of all thyroid cancer cases, with survival rates of almost 100% when diagnosed early. Since PTC and FTC have different modes of metastasis, they require different treatment strategies. Standard diagnosis by fine needle aspiration with cytopathological examination can be inaccurate in approximately 10-30% of all cases and difficult to definitively classify as WDTC. Currently, there is no single or panel of biomarkers available for thyroid cancer diagnosis and classification. This study identified novel biomarkers for thyroid cancer diagnosis and classification using proteomics, which may be translated into a biomarker panel for clinical application. Two-dimensional SDS-PAGE and mass spectrometry were used to identify potential biomarkers in papillary and follicular thyroid carcinoma cell lines, and the biomarkers were validated in five PTC and five FTC tissues, with their adjacent normal tissues from Thai patients. Eight biomarkers could distinguish PTC from normal tissues, namely enolase 1, triose phosphate isomerase, cathepsin D, annexin A2, cofilin 1, proliferating cell nuclear antigen (PCNA), copine 1 and heat shock protein 27 kDa (HSP27). These biomarkers can also discriminate FTC from normal tissues, except for annexin A2. On the contrary, annexin A2, cofilin 1, PCNA and HSP27 can be used to classify the types of WDTC. These findings have potential for use as a novel multi-marker panel for more accurate diagnosis and classification to better guide physicians on thyroid cancer treatment. Moreover, our results suggest the involvement of proteins in cell growth and proliferation, and the p53 pathway in the carcinogenesis of WDTC, which may lead to targeted therapy for thyroid cancer.

Kinetic and structural characterization of phosphofructokinase from Lactobacillus bulgaricus.

Phosphofructokinase from Lactobacillus delbrueckii subspecies bulgaricus (LbPFK) has been reported to be a nonallosteric analogue of phosphofructokinase from Escherichia coli at pH 8.2 [Le Bras et al. (1991) Eur. J. Biochem. 198, 683-687]. A reexamination of the kinetics of this enzyme shows LbPFK to have limited binding affinity toward the allosteric ligands, MgADP and PEP, with dissociation constants of approximately 20 mM for both. Their allosteric effects are observed only at high concentrations of these ligands, with both exhibiting inhibitory effects on substrate binding. No pH dependence was observed for the binding and the influence of MgADP and PEP on the enzyme. To attempt to explain these results, the crystal structure of LbPFK was solved using molecular replacement to 1.86 A resolution. A comparative study of the LbPFK structure with that of phosphofructokinases from E. coli (EcPFK) and Bacillus stearothermophilus (BsPFK) reveals a structure with conserved fold and substrate binding site. The effector binding site, however, shows many differences that could explain the observed decreases in binding affinity for MgADP and PEP in LbPFK as compared to the other two enzymes.

Disentangling the web of allosteric communication in a homotetramer: heterotropic activation in phosphofructokinase from Escherichia coli.

Phosphofructokinase from Escherichia coli (EcPFK) is a homotetramer with four active sites and four allosteric sites. Understanding the allosteric activation of EcPFK by MgADP has been complicated by the complex web of possible interactions, including active site homotropic interactions, allosteric site homotropic interactions, and heterotropic interactions between active and allosteric sites. The current work has simplified this web of possible interactions to a series of single heterotropic interactions by forming and isolating hybrid tetramers. Each of the four unique heterotropic interactions have independently been isolated and compared to a control that has all four of the unique heterotropic interactions. If the interactions are labeled with the distances between interacting ligands, the 45-A interaction contributes 20% +/- 1%, the 33-A interaction contributes 34% +/- 1%, the 30-A interaction contributes 21% +/- 1%, and the 23-A interaction contributes 25% +/- 1% with respect to the total free energy of MgADP/fructose 6-phosphate (Fru-6-P) activation in the control. The free energies of the isolated interactions sum to 100% +/- 2% of the total. Therefore, the four unique interactions are all contributors to activation, are nonequivalent, and are additive.

Identification of substrate contact residues important for the allosteric regulation of phosphofructokinase from Eschericia coli.

The side chains of Escherichia coli phosphofructokinase (EcPFK) that interact with bound substrate, fructose 6-phosphate (Fru-6-P), are examined for their potential roles in allosteric regulation. Mutations that severely decrease Fru-6-P affinity and/or k(cat)/K(m) were created at each contact residue, with the exception of the catalytic base, D127. Even though Fru-6-P affinity was greatly decreased for R162E, M169A, E222A/H223A, and R243E, the mutated proteins retained the ability to be activated by MgADP and inhibited by phosphoenolpyruvate (PEP). R252E did not show an allosteric response to either MgADP or PEP. The H249E mutation retained MgADP activation but did not respond to PEP. R72E, T125A, and R171E maintained allosteric inhibition by PEP. Both R72E and T125A displayed a MgADP-dependent decrease in k(cat) but no MgADP-dependent K-type effects. R171E maintained MgADP-dependent K-type activation but also displayed a MgADP-dependent decrease in k(cat). Localization of mutations that alter MgADP activation near the transferred phosphate group indicates the importance of the 1-methoxy region of Fru-6-P in allosteric regulation by MgADP. A region near the 6'-phosphate may be similarly important for PEP inhibition. R252 is uniquely positioned between the 1'- and 6'-phosphates of bound Fru-1,6-BP, and the mutation at this position may alter both allosterically responsive regions. The differential functions of specific regions in the Fru-6-P contact residues support different mechanisms for allosteric activation and inhibition. In addition, the lack of correlation between mutations that decrease Fru-6-P affinity and those that abolish allosteric communications supports the independence of affinity and allosteric coupling.