Kazuhiko Okumura

Enhancing Effects of Epidermal Growth Factor on Human Squamous Cell Carcinoma Motility and Matrix Degradation But Not Growth

In order to ascertain the effects of epidermal growth factor (EGF) on human cancer invasion abilities, three cell lines of human oral squamous cell carcinoma were studied using a phagokinetic track assay and zymography. EGF (1-100 ng/ml) was found to inhibit the growth but enhance the random motility of all three cell lines in a concentration-dependent fashion. Exposure to EGF, dose-dependently, led to an increased production of urokinase-type plasminogen activator and M(r) 92 kD matrix metalloproteinase by the same cells. These results strongly suggest that EGF may promote human squamous cell carcinoma invasion and metastasis.

Ceragenin CSA‐13 exhibits antimicrobial activity against cariogenic and periodontopathic bacteria

INTRODUCTION Ceragenin CSA-13 is a bile-acid-based mimic of endogenous antimicrobial peptides and shares a mechanism of action with many of these antimicrobial agents. Because CSA-13 is not peptide based, it is not a substrate for the proteases that are found in the oral cavity, which are capable of degrading antimicrobial peptides. Furthermore, the simplicity of the ceragenins makes them easier to prepare and purify than antimicrobial peptides. In this study, we examined the antimicrobial activities of CSA-13 against oral pathogens and found that this compound was bactericidal against all of the strains tested. METHODS The strains used were isolates of Streptococcus mutans and Porphyromonas species. Minimum inhibitory concentrations (MIC) were determined using agar dilution methods. In susceptibility testing, viable counts were determined after incubation with CSA-13. RESULTS CSA-13 was potent against all 23 strains tested with MICs of 1-8 microg/ml for S. mutans and 1-16 microg/ml for 24 strains of the genus Porphyromonas. The MIC(50) was 2 and the MIC(90) was 8 mug/ml for S. mutans. MIC ranges for protease-positive P. gingivalis and P. cangingivalis were 2-16 microg/ml, and 1-2 microg/ml for protease-negative P. circumdentaria. CSA-13 interacted with lipopolysaccharide-sensitized erythrocytes at a concentration of 5.0-20.0 microg/ml. CONCLUSION CSA-13 displays broad-spectrum activity against cariogenic and periodontopathic bacteria. CSA-13 was effective against protease-positive Porphyromonas. It was shown to bind to erythrocytes coated with lipopolysaccharide and lipoteichoic acid from diverse bacterial strains. These results suggest that CSA-13 may be useful for the prevention and treatment of oral microbial diseases.

Expression of MIP-3α/CCL20, a macrophage inflammatory protein in oral squamous cell carcinoma

Abstract We have examined the expression of MIP-3α/CCL20 in oral squamous cell carcinoma (SCC) in vivo and in vitro. In addition, we have investigated whether the expression of MIP-3α/CCL20 is regulated by bacterial infection and inflammatory cytokines. In order to determine the mRNA level of MIP-3α, quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed with LightCycler™ using the double-stranded DNA dye, SYBR Green I. Oral epithelial cells and six SCC cell lines (SCC-9, SAS, BSC-OF, HSC-4, HSC, Ca9-22) were found to express MIP-3α mRNA. The expression of MIP-3α was upregulated by infection with Actinobacillus actinomycetemcomitans and by stimulation with lipopolysaccharide and TNF-α. By in situ hybridization, the detectable MIP-3α expression in SCC was localized primarily at the epithelial pearls corresponding to the spinous layer. These results suggest that MIP-3α contributes to the oral immunoresponse to bacterial infection, and may be involved in the growth of SCC.

Establishment of high- and low-invasion clones derived for a human tongue squamous-cell carcinoma cell line SAS

Distant-organ metastasis and regional lymph node metastasis are still the major cause of mortality of oral-cavity squamous-cell cancer (SCC). However, only a few studies have been undertaken to elucidate the mechanism of invasion and metastasis of oral SCC. In this study, we attempted to establish human oral SCC clones with different invasiveness, defined by endothelial cell monolayer assay, which can be used for the study of invasion and metastasis of oral SCC. We established five clones from the human oral SCC cell line SAS by a limiting-dilution method. Two distinct clones, SAS-L1 with very low invasive potential and SAS-H1 with very high invasive potential, were picked out by rat lung endothelial cell monolayer assay. The number of SAS-H1 that penetrated the rat lung endothelial cell monolayer was six fold higher than the number of SAS-L1. There were no differences of metalloproteinase production and cell adhesiveness to Matrigel of SAS-L1 and SAS-H1. However, SAS-H1 exhibited a higher migration ability than SAS-L1. This pair of clones would be a useful experimental model to help in the study of the invasiveness of human oral SCC.

The Human Cathelicidin Antimicrobial Peptide LL-37 and Mimics are Potential Anticancer Drugs

Antimicrobial peptides (AMPs) play a critical role in innate host defense against microbial pathogens in many organisms. The human cathelicidin, LL-37, has a net positive charge and is amphiphilic, and can eliminate pathogenic microbes directly via electrostatic attraction toward negatively charged bacterial membranes. A number of studies have shown that LL-37 participates in various host immune systems, such as inflammatory responses and tissue repair, in addition to its antibacterial properties. Moreover, recent evidence suggests that it is also involved in the regulation of cancer. Indeed, previous studies have suggested that human LL-37 is involved in carcinogenesis via multiple reporters, such as FPR2 (FPRL1), epidermal growth factor receptor, and ERBb2, although LL-37 and its fragments and analogs also show anticancer effects in various cancer cell lines. This discrepancy can be attributed to peptide-based factors, host membrane-based factors, and signal regulation. Here, we describe the association between AMPs and cancer with a focus on anticancer peptide functions and selectivity in an effort to understand potential therapeutic implications.

Differential diagnosis of cervical lymph nodes in head and neck cancer by ultrasonography

OBJECTIVE Determination of whether an enlarged cervical lymph node is metastatic or not is clinically important in head and neck oncology. Differential diagnosis of the lymph node, however, is still a diagnostic problem. The purpose of this study is to clarify the ultrasonographic findings of the metastatic lymph nodes of head and neck squamous cell carcinoma and to establish the criteria. METHODS We investigated 36 metastatic lymph nodes in head and neck squamous cell carcinoma and 24 non-metastatic nodes in benign disease with a 10-MHz transducer. We examined the size, shape, and internal echo (echo level, punctate bright echogenic spots, hilus echogenic line, cystic pattern) of these nodes. Based on this investigation, we evaluated 70 lymph nodes from 25 other patients by ultrasonography. RESULTS The short axis diameter and shape of metastatic nodes were larger and rounder than those of non-metastatic ones. Of the metastatic nodes, 69% showed hypoechoic and 31% isoechoic levels, and 78% exhibited punctate bright echogenic spots. Of the non-metastatic nodes, 92% showed hypoechoic and 8% isoechoic levels, and none of them showed the spots. The hilus echogenic line was not present in any metastatic node, but it was seen in 58% of non-metastatic ones. Of the metastatic nodes, 19% exhibited a cystic pattern; none of the non-metastatic nodes showed the pattern. According to our criteria based on these results, the accuracy rate was 98.6% (69/70). The sensitivity and specificity were 97.2% (35/36) and 100% (34/34), respectively. The false positive rate and the false negative rate were 0% (0/70) and 1.4% (1/70), respectively. CONCLUSION Internal echo findings and shape of lymph nodes can be an important diagnostic tool, and our ultrasonographical criteria of the lymph nodes are very useful for the differential diagnosis of the cervical lymph nodes.

Antimicrobial activity of three tick defensins and four mammalian cathelicidin-derived synthetic peptides against lyme disease spirochetes and bacteria isolated from the midgut.

In this study, chemically synthesized tick defensins and cathelicidin-derived mammalian peptides were used to investigate the activity spectrum against Borrelia garinii and symbiotic Stenotrophomonas maltophila. Synthetic tick defensins showed antimicrobial activity against Staphylococcus aureus but not B. garinii and S. maltophila. Mammalian peptides which have cationic property similar to tick defensins, showed antimicrobial activity similar to tick defensins. The antimicrobial peptides in ticks and mammalian hosts have common characteristics against microbial invasion in the innate immune system.

Basaloid-squamous cell carcinoma of the oral mucosa: Report of two cases and study of the proliferative activity

Two cases of basalold‐squamous cell carcinoma (BSC) of the oral mucosa are described. The first case occurred at the floor of the mouth in a 58‐year‐old man, and the second case occurred at the mandibular gingiva in a 79‐year‐old woman. The laboratory data of the first case showed a positive response to hepatitis C virus antibody. in the first case, the tumor mass measured 4 times 4 cm in size, and was i‐texl at the lingual side of the median mandible beside the sublingual gland. In the second case, the tumor mass measured 25 times 15 mm In size, and was located in the alveolar mucosa of the right mandible. Histologically, both tumors showed a neoplastic epithelium arranged in a solid pattern with evidence of peripheral palisading, central necrosis, and some squamous differentiation. The pro‐ilferathfe activities of the BSC were compared with conventional squamous cell carcinomas (SCC) in the oral floor and gingiva, respectively, by employing a sensitive argy‐rophillc nuclear organizer region (AgNOR) staining method. The number of AgNOR per nucleus of the BSC was higher than that of any other SCC cases. The results support the opinion that BSC of the oral mucosa has a worse prognosis than conventional SCC.

Protein phosphatase inhibitor calyculin A induces hyperphosphorylation of cytokeratins and inhibits amylase exocytosis in the rat parotid acini

Calyculin A, a protein phosphatase inhibitor with a chemical structure completely different from that of okadaic acid, reproduced the inhibitory effect of okadaic acid on cyclic AMP‐mediated amylase release from rat parotid acinar cells. Calyculin A markedly enhanced phosphorylation of cytokeratins in the cytoskeletal fraction of the cells, whereas cAMP had apparently no effect on the phosphorylation. Microscopic observations showed that parotid acini incubated with 100 nM calyculin A for 15 min had large vacuoles in the cytoplasm and conspicuous blebs on the basal plasma membrane. K252a, a nonselective protein kinase inhibitor, clearly reduced calyclin A‐induced phosphorylation of cytokeratins, and it markedly blocked the inhibition of amylase release and morphological changes evoked by calyculin A. These results suggest that hyperphosphorylation of cytokeratins profoundly affects the morphology and secretory activity of parotid acinar cells.

Effect of cytochalasin D on acinar cell structure and secretion in rat parotid salivary glands in vitro

Cytochalasin D, a microfilament disrupting agent, considerably inhibited isoproterenol-stimulated amylase release from enzymatically dispersed parotid acini. Histologically cytochalasin D caused a loss of microvilli lining acinar lumina and luminal enlargement. Nearly empty vacuoles appeared near the luminal and lateral surface, and the membrane bordering on the vacuoles was often continuous with the plasma membrane. Therefore, the vacuolization probably resulted from an elongation of the membrane lining the lumen. Fluorescence staining with rhodamine-phalloidin showed that cytochalasin D caused disruption of microfilaments. When stimulating the cytochalasin D-treated cells with isoproterenol, the number of secretory granules in the cytoplasm diminished markedly and secretory material was observed in the vacuoles, indicating that inhibition of amylase release by cytochalasin D is not due to blocking of exocytosis but to the retention of amylase discharged into vacuoles. These findings suggest that microfilaments are essential in maintaining the parotid acinar structure but do not play a direct part in the movement of secretory granules and their fusion with the luminal membrane.