The clinical evaluation of the serum SCC antigen levels with "SCC RIABEAD" by immunoradiometric assay

The clinical significance of serum SCC antigen level was evaluated by the monoclonal antibody method (SCC.RIABEAD Dinabot Co. Ltd). Patients with squamous cell carcinoma showed a high positive SCC antigen level and positive rate elevated with the advance of the clinical stage. The serum SCC antigen level was decreased by treatment, and it increased again before obvious clinical recurrence was recognized. The results suggest that measurement of serum SCC antigen level is useful as a follow up of cancer treatment.     

Fundamental and clinical evaluation of an immunoradiometric assay procedure "Amerwell AFP" for estimation of serum alpha-fetoprotein

The efficacy of assay procedure for estimation of serum alpha-fetoprotein (AFP) was examined on "Amerwell AFP" immunoradiometric assay (IRMA) using monoclonal AFP antibody. The condition of incubation was most satisfactory for 2 hours at 37 degrees C. Coefficients of variance for intraassay and interassay were 5.1-10.0% and 8.4-9.9% respectively. Dilution test gave satisfactory results. The binding capacity of microplate-well tagged monoclonal AFP antibody with AFP antigen was satisfactory for assay reactions. This method showed a good correlation to the AFP RIA bead (Dinabot Co.) method. The normal range (reference value) was within the level of 5.0 IU/ml due to Hoffmann's method in the examination of 860 subjects. Estimation of AFP with "Amerwell AFP" IRMA kit was a feasible routine method of clinical application for tumor marker.     

Electrophoretic characterization of heat-stable squamous cell carcinoma antigen.

The aim of this study was to investigate the heat stability of squamous cell carcinoma (SCC) antigen, a tumor-associated serine proteinase inhibitor (serpin), in tumor tissue extract by electrophoretic methods. After heat treatment at 70 degrees C for 2 h, the tumor tissue extract showed a single main protein band of 45 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) which reacted with a monoclonal antibody specific for SCC antigen. The heat-stable SCC antigen was separated by two-dimensional electrophoresis (2-DE) into four spots with pI 6.4-5.9 and Mr 44500-45 000 of SCC antigen-1. Furthermore, the SCC antigen-1 still showed its inhibitory activity against a cysteine proteinase, papain, by gelatin zymography. These results suggest that heat treatment of protein sample at 70 degrees C for 2 h may be a useful method for a partial purification of SCC antigen-1 which can inhibit lysosomal cysteine proteinases such as cathepsin L, S, and K.     

Enzyme-conjugated hybridization chain reaction for magneto-controlled immunoassay of squamous cell carcinoma antigen with pH meter

A nanoparticle-based potentiometric immunoassay was designed for the sensitive detection of squamous cell carcinoma antigen (SCCA; cervical carcinoma marker) on a portable pH meter coupling enzyme-labeled hybridization chain reaction (HCR) with two alternating hairpin DNA probes for the signal amplification. Initially, a sandwich-type immunoreaction was carried out between anti-SCCA capture antibody-conjugated magnetic bead and detection antibody/initiator strand-coated gold nanoparticle (AuNP). Then, the HCR reaction was readily executed between two glucose oxidase (GOx)-labeled hairpins through the initiator strand to form numerous GOx concatamers on the AuNP via the long nicked double-helix. The concatenated GOx oxidized glucose into gluconic acid and hydrogen peroxide, thus resulting in the pH change of the detection solution on a handheld pH meter. Several labeling protocols including GOx-antibody, GOx-AuNP-antibody and GOx-HCR-AuNP-antibody were investigated for detection of target SCCA, and improved analytical features were obtained with the immune-HCR assay. Under optimum conditions, the immune-HCR assay exhibited good pH responses for the determination of SCCA at a concentration as low as 5.7 pg/mL. Additionally, the immune-HCR assay had good precision and reproducibility, high specificity, and acceptable accuracy for analyzing human serum specimens.     

Enzyme-conjugated hybridization chain reaction for magneto-controlled immunoassay of squamous cell carcinoma antigen with pH meter

A nanoparticle-based potentiometric immunoassay was designed for sensitive detection of squamous cell carcinoma antigen on a portable pH meter by coupling enzyme-labeled hybridization chain reaction with two alternating hairpin DNA probes for the signal amplification.     

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.     

A new screening procedure for the estimation of oxidizable organic compounds in water samples

Summary Immobilized lead dioxide (supported on SiO₂) has been used as the packing material in a solid phase reactor for the oxidation of organic compounds in water samples by flow injection analysis (FIA). On-line oxidation takes place in a FIA-system; this allows the detection of mobilized Pb²⁺ either photometrically, after complex formation with 4-(2-pyridylazo)-recorcinol (PAR), or directly with flame-AAS. The oxidation yield is quite different (0–100%) for a variety of organic compounds; however, calibration was possible in all cases investigated. Thus the systems can be used for the screening of polluted waters and as a post-column chemical-reaction detector (e.g. after HPLC-separation of organic compounds). After modification the FIA determination of COD equivalent values should be possible.     

Development and validation of a stability-indicating reversed-phase high performance liquid chromatography method for assay of betamethylepoxide and estimation of its related compounds

Betamethylepoxide (16beta-methyl-Delta(1,4)-pregnadiene-9beta-11beta-oxide-17alpha,21-diol-3,20-dione) is a key intermediate for the synthesis of various active pharmaceutical ingredients (APIs) of steroid compounds. A stability-indicating reversed-phase HPLC method for assay of betamethylepoxide and estimation of its related compounds has been developed and validated. This method can accurately quantitate betamethylepoxide in the presence of numerous structurally related compounds (including the alpha-epimer, known as alphamethylepoxide). This method can also adequately separate most of the impurities from each other and estimate their quantities in betamethylepoxide samples. The stability-indicating capability of this method has been demonstrated by adequate separation of the degradation products from betamethylepoxide in stress degraded and aged stability samples. The HPLC column used in the method was a 5 cm YMC Hydrosphere C(18) column (4.6 mm I.D.) and the mobile phase consisted of (A) water and (B) acetonitrile:methanol (8:25, v/v).     

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.     

Shell click-crosslinked (SCC) nanoparticles: a new methodology for synthesis and orthogonal functionalization

A new methodology for the preparation of well-defined core-shell nanoparticles was developed, based upon the employment of a multifunctional crosslinker to coincidently stabilize supramolecular polymer assemblies and imbed into the shell unique chemical functionalities. Amphiphilic diblock copolymers of poly(acrylic acid)(80)-b-poly(styrene)(90) that had been assembled into micelles and partially functionalized throughout the corona with alkynyl groups were utilized as Click-readied nanoscaffolds for the formation of shell Click-crosslinked nanoparticles (SCCs). Divergently grown dendrimers of the zero, first, second, and third generations having increasing numbers of azide terminating groups ((N(3))(2)-[G-0], (N(3))(4)-[G-1], (N(3))(8)-[G-2], and (N(3))(16)-[G-3], respectively) were investigated as crosslinkers via Click reactions with the alkynyl groups to form covalent linkages throughout the block copolymer micelle corona, thus forming a crosslinked shell. The crosslinking reactions were characterized by (1)H NMR and IR spectroscopies, differential scanning calorimetry (DSC), and dynamic light scattering (DLS) measurements. Only the first generation dendrimer ((N(3))(4)-[G-1]) possessed a sufficient balance of polyvalency and water solubility to achieve crosslinking and establish a robust nanostructure. The resulting SCC was further characterized with atomic force microscopy (AFM), transmission electron microscopy (TEM), and analytical ultracentrifugation (AU). The dendritic crosslinker is important as it also allows for the incorporation of excess functionality that can undergo complementary reactions. Within the shell of this SCC the remaining azide termini of the dendrimer crosslinker were then consumed in a secondary Click reaction with an alkynyl-functionalized fluorescein to yield a fluorescently labeled SCC that was characterized with DLS, AFM, TEM, AU, UV-vis, and fluorescent measurements as a function of pH.     

Evidence for a stem-cell lineage in corneal squamous cell carcinoma using synchrotron-based Fourier-transform infrared microspectroscopy and multivariate analysis

The cornea is one of the few human tissues where the in situ locations of stem cells (SCs), transient-amplifying (TA) cells and terminally-differentiated (TD) cells have been relatively well localised and characterised. Mid-infrared (IR) (4000-400 cm(-1)) is absorbed by biological molecules and facilitates the acquisition in the biochemical-cell fingerprint region (1800-900 cm(-1)) of spectra representative of structure and function. Human cornea derived from normal or squamous cell carcinoma (SCC) samples were acquired, cryosectioned (10 μm), floated onto BaF(2) windows and interrogated using synchrotron-based radiation (SRS) Fourier-transform IR (FTIR) microspectroscopy. Spectra were analysed using principal component analysis (PCA) with or without linear discriminant analysis (LDA) to allow cluster analysis of the cell categories. A clear cell lineage emanating from SCs to TA cells to TD cells was noted in normal samples. Within the SCC samples, a small sub-population of the cell-derived spectra pointed to a SC-like phenotype with the vast majority pointing to a TA cell-like character; these cells would tend to be the most proliferative within a tissue. Our findings suggest that SRS FTIR microspectroscopy has the potential to identify and characterise cancer SCs.     

Characterization of newly established oral cancer cell lines derived from six squamous cell carcinoma and two mucoepidermoid carcinoma cells

Since genetic abnormalities of human cancer are greatly geographically dependent, cultural and environmental backgrounds are thought to be closely related to the carcinogenic process. In the present study, eight human cell lines were established by culture from untreated carcinomas of the oral cancer, of which five were from primary oral squamous cell carcinomas (OSC), one from a mucoepidermoid carcinoma (MEC) and one each originating from metastatic OSC and MEC. All the studied tumor lines grew as monolayers, and showed: i) an epithelial origin by the presence of cytokeratin, and ii) tumorigenic potential in nude mice. Western blot analysis revealed i) over expression of EGFR in six of the cell lines ii) decreased expression of E-cadherin in six cell lines compared to normal human oral mucosa. A mutational analysis showed: point mutations of p53 at exon 7, with transversion, and at exon 8, with transition. These well-characterized human YD cell lines should serve as useful tools in the study of the molecular pathogenesis and biological characteristics of head and neck cancer cells, and in the future testing of new therapeutic reagents for oral cancer.