Identification of differentially expressed, tumor-associated proteins in oral squamous cell carcinoma by proteomic analysis

Oral squamous cellular carcinoma is a malignant tumor with poor prognosis and therefore the discovery of early markers to discriminate malignant from normal cells would be of critical importance in clinical diagnosis. Subcellular fractions from oral squamous cell carcinoma (OSCC) and control samples, enriched in mitochondrial and cytosolic proteins, were analyzed by 2-DE, followed by MALDI-TOF-MS. Twenty proteins showed altered expression levels in OSCC; 14 were up- and 6 were down-regulated in comparison with the control samples. For 11 proteins, cofilin, C-reactive protein precursor, creatine kinase m-chain, fatty acid-binding protein, keratin type II, myosin light chain 2 and 3, nucleoside diphosphate kinase A, phosphoglycerate mutase 1, plakoglobulin, and retinoic acid-binding protein II, it is shown for the first time that they are differentially expressed in OSCC. Proteins with highly up-regulated levels may be of interest as potential diagnostic markers and consequently of clinical interest.     

Delimitation of squamous cell cervical carcinoma using infrared microspectroscopic imaging

Infrared (IR) spectroscopic imaging coupled with microscopy has been used to investigate thin sections of cervix uteri encompassing normal tissue, precancerous structures, and squamous cell carcinoma. Methods for unsupervised distinction of tissue types based on IR spectroscopy were developed. One-hundred and twenty-two images of cervical tissue were recorded by an FTIR spectrometer with a 64×64 focal plane array detector. The 499,712 IR spectra obtained were grouped by an approach which used fuzzy C-means clustering followed by hierarchical cluster analysis. The resulting false color maps were correlated with the morphological characteristics of an adjacent section of hematoxylin and eosin-stained tissue. In the first step, cervical stroma, epithelium, inflammation, blood vessels, and mucus could be distinguished in IR images by analysis of the spectral fingerprint region (950–1480 cm⁻¹). In the second step, analysis in the spectral window 1420–1480 cm⁻¹ enables, for the first time, IR spectroscopic distinction between the basal layer, dysplastic lesions and squamous cell carcinoma within a particular sample. The joint application of IR microspectroscopic imaging and multivariate spectral processing combines diffraction-limited lateral optical resolution on the single cell level with highly specific and sensitive spectral classification on the molecular level. Compared with previous reports our approach constitutes a significant progress in the development of optical molecular spectroscopic techniques toward an additional diagnostic tool for the early histopathological characterization of cervical cancer.     

Aberrant proteins in the saliva of patients with oral squamous cell carcinoma

Confirmation of oral squamous cell cancer (OSCC) currently relies on histological analysis, which does not provide clear indication of cancer development from precancerous lesions. In the present study, whole saliva proteins of patients with OSCC (n = 12) and healthy subjects (n = 12) were separated by 2DE to identify potential candidate biomarkers that are much needed to improve detection of the cancer. The OSCC patients’ 2DE saliva protein profiles appeared unique and different from those obtained from the healthy subjects. The patients’ saliva α1‐antitrypsin (AAT) and haptoglobin (HAP) β chains were resolved into polypeptide spots with increased microheterogeneity, although these were not apparent in their sera. Their 2DE protein profiles also showed presence of hemopexin and α‐1B glycoprotein, which were not detected in the profiles of the control saliva. When subjected to densitometry analysis, significant altered levels of AAT, complement C3, transferrin, transthyretin, and β chains of fibrinogen and HAP were detected. The increased levels of saliva AAT, HAP, complement C3, hemopexin, and transthyretin in the OSCC patients were validated by ELISA. The strong association of AAT and HAP with OSCC was further supported by immunohistochemical staining of cancer tissues. The differently expressed saliva proteins may be useful complementary biomarkers for the early detection and/or monitoring of OSCC, although this requires validation in clinically representative populations.     

Raman spectroscopic study on classification of cervical cell specimens

Cervix-cancer is the third most common female cancer worldwide. Papanicolaou (Pap) test, a well-recognized screening tool, is labor intensive, time consuming and prone to subjective interpretations. Optical spectroscopic methods, sensitive to molecular changes are being pursued as potential diagnostics tool. In this study we have explored Raman spectroscopic approach to differentiate exfoliated cell pellets using 94 cervical cell specimens (45-normal and 49-abnormal specimens). Study was carried out by two approaches. In the first approach, spectral data from 37 cell specimens were acquired and analyzed by Principal Component-Linear Discriminant Analysis (PC-LDA), which yielded classification efficiencies of 86% and 84% for normal and abnormal specimens, respectively. Mean and difference spectra suggest presence of blood in abnormal specimen as a major cause of discrimination. However, as tumor is vascular, bleeding was observed during abnormal sample collection. Hence, spectra of abnormal specimens show heme and fibrin features, and this can lead to false interpretations, as bleeding also occur in several non-cancerous conditions. Therefore, remaining 57 specimens were treated with Red Blood Corpuscles (RBC) lysis buffer in order to remove the RBC influence. PC-LDA resulted classification efficiency of about 79% and 78% for normal and abnormal smear, respectively – comparable to Pap test. Thus finding of the study suggests feasibility of Raman spectroscopic classification of normal and cancerous exfoliated cervical cell specimens.     

Evaluation of the accuracy of hTERT gene aberrant methylation using electrochemical hybridization assay and liquid-based cytology in screening for oral squamous cell carcinoma

In order to improve the survival rate of oral squamous cell carcinoma (OSCC) patients, a reliable diagnostic method for early OSCC detection is required that is minimally invasive, less burdensome to the patient, and has high sensitivity and specificity. Therefore, we performed the detection of abnormal methylation at three locations in the hTERT promoter region of oral exfoliated cells by employing the ferrocenylnaphthalene diimide (FND)-based electrochemical hybridization assay (FND-EHA) using three types of DNA probe-immobilized electrodes. We also performed liquid cytology using oral exfoliated cells and compared these obtained data to evaluate whether FND- EHA can be used as an OSCC screening system. The results showed a good correlation between this method and conventional OSCC screening, and cytology. In addition, FND-EHA was also able to determine samples that had been ambiguously determined by liquid cytology. This indicates that FND-EHA may be useful as an OSCC screening system.     

Discovery of lung squamous carcinoma biomarkers by profiling the plasma peptide with LC/MS/MS

Biomarkers can be used for the screening and clinical diagnosis of cancer, and peptidomics approach has been proven successful in the research of biomarkers. To develop better peptidomic technologies for fast, accurate, and reliable detection of peptides biomarkers for lung cancer, we have improved the procedures of blood collection to minimize the degradation of the blood proteins and optimize the extraction of peptidome peptides from plasma samples based on acetonitrile precipitation associated with size exclusion chromatography (SEC). Studies show that squamous cell carcinomas are found to express CAGE1, SPAT9 and TEX28 genes at significantly higher rates, and the results suggest that as tumors progress, the level of CAGE1, SPAT9 and TEX28 genes are likely to increase and lead to immunization. This suggests a potentially important therapeutic method for cancer testis-based cancer vaccines.     

In vitro evaluation of antiproliferative effect of ethyl gallate against human oral squamous carcinoma cell line KB

Although some polyphenols are known to possess anticancer activity against different cancer cell lines through induction of apoptosis, the mode of antiproliferative effect of ethyl gallate against human oral squamous carcinoma cell line KB was not studied until now. Therefore, the antiproliferative effect of ethyl gallate was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in comparison with the reference drug paclitaxel. Generation of reactive oxygen species, mitochondrial membrane potential loss, DNA damage and apoptosis were determined using 2,7-diacetyldichlorofluorescein fluorescence, uptake of rhodamine-123 by mitochondria, comet assay and acridine orange/ethidium bromide dual-dye staining method. Both ethyl gallate and paclitaxel exhibited cytotoxicity in a dose-dependent manner. The 50% inhibitory concentration for ethyl gallate was 30 and 20 μg/mL for paclitaxel. A volume of 50 μg/mL of ethyl gallate was found to be significantly effective (P < 0.05) in controlling the cancer cell proliferation leading to acute apoptosis.     

Electrophoretic analysis of the cleaved form of serpin,squamous cell carcinoma antigen-1 in normal and malignant squamous epithelial tissues

The aim of this study was to detect the cleaved form of serine proteinase inhibitor (serpin), squamous cell carcinoma (SCC) antigen-1 in normal and malignant squamous epithelial tissues, which implies the presence of its target proteinase. The cleaved SCC antigen-1 in normal squamous epithelium was identified as a single spot with pI 6.35 and M(r) 40,000 by two-dimensional electrophoresis (2-DE) combined with immunoblotting. Interestingly, the cleaved form showed different biochemical properties in heat stability or immunoreactivity with a monoclonal antibody for SCC antigen (Mab 426) compared to intact SCC antigen-1. Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of tissue extracts showed an abundant 40 kDa band of cleaved SCC antigen-1 in tumor tissue compared to normal tissue. Among the potential target proteinase of SCC antigen-1, immunoblotting analyses revealed that cathepsin L2 was remarkably overexpressed in tumor tissue, while cathepsin L was expressed in both normal and tumor tissues. These findings indicate that SCC antigen-1 interacts with specific endogenous proteinases such as cathepsins L and L2 in physiological and pathological states of squamous epithelium.     

Exosomal MicroRNA as Biomarkers for Diagnosing or Monitoring the Progression of Ovarian Clear Cell Carcinoma: A Pilot Study

Ovarian cancer is the most common cause of gynecological malignancy-related mortality since early-stage disease is difficult to diagnose. Advanced clear cell carcinoma of the ovary (CCCO) has dismal prognosis, and its incidence has been increasing in Japan, emphasizing the need for highly sensitive diagnostic and prognostic CCCO biomarkers. Exosomal microRNAs (miRNAs) secreted by tumor cells are known to play a role in carcinogenesis; however, their involvement in ovarian cancer is unclear. In this study, we performed expression profiling of miRNAs from exosomes released by five cell lines representing different histological types of ovarian cancer. Exosomes isolated from culture media of cancer and normal cells were compared for miRNA composition using human miRNA microarray. We detected 143 exosomal miRNAs, whose expression was ≥1.5-fold higher in ovarian cancer cells than in the control. Among them, 28 miRNAs were upregulated in cells of all histological ovarian cancer types compared to control, and three were upregulated in CCCO cells compared to other types. Functional analyses indicated that miR-21 overexpressed in CCCO cells targeted tumor suppressor genes PTEN, TPM1, PDCD4, and MASP1. The identified miRNAs could represent novel candidate biomarkers to diagnose or monitor progression of ovarian cancer, particularly CCCO.     

CoMFA and CoMSIA studies on a new series of xanthone derivatives against the oral human epidermoid carcinoma (KB) cancer cell line

Abstract  A new series of xanthone derivatives against the oral human epidermoid carcinoma (KB) cancer cell line is examined to determine the relationship between the structural properties and the biological activity of these compounds—the 3-D quantitative structure–activity relationship (3D-QSAR)—using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). The best CoMFA and CoMSIA models were obtained using the atom-based alignment of 33 compounds, 22 training compounds and 11 tested compounds, and these give desirable statistics; those for the CoMFA standard model were: r _cv² = 0.691, r ² = 0.998, S _press = 0.178, s = 0.014 and F = 1080.765, while CoMSIA combined steric, electrostatic, hydrophobic and hydrogen-bond acceptor fields: r _cv² = 0.600, r ² = 0.988, S _press = 0.206, s = 0.034 and F = 284.433. The 3D-QSAR models calculated satisfactory test set activities. The 3D-QSAR contour plots correlated strongly with the experimental data for the binding topology. For this reason, these results would be beneficial for predicting affinities with the compounds of interest, and they are advantageous for guiding the design and synthesis of new and more effective anticancer agents. Graphical abstract   A new and more effective anticancer agent of xanthone derivatives against the oral human epidermoid carcinoma (KB) cell line, as investigated by CoMFA and CoMSIA analysis