Minoru Ikuta

Expression of heparanase in oral cancer cell lines and oral cancer tissues

In the process of metastasis, cancer cells secrete several enzymes which degrade extracellular matrices (ECMs) and basement membranes (BMs) of blood vessels. One of them, heparanase, has been reported to be an important enzyme when metastatic cancer cells invade blood vessels. The enzyme cleaves heparan sulfate (HS), a main component of ECM and BM. In the present study, HS-degrading ability of several human oral cancer cell lines (HSC2, HSC3, HSC4, Ca9-22, NA, ACC3 and Ab-J) and tissues derived from human oral squamous cell carcinomas (both metastatic and non-metastatic) were investigated by measuring heparanase activities and levels of heparanase mRNA by a quantitative reverse transcriptase-polymerase chain reaction. The catalytic activities and the mRNA levels of heparanase showed a good agreement. Clinical demonstration of cancer metastasis generally correlated with high levels of heparanase activity and its mRNA. The results suggest that heparanase activity and its mRNA level are good diagnostic parameters for evaluating the metastatic properties of human oral cancer cells.

Heparanase and tumor invasion patterns in human oral squamous cell carcinoma xenografts

The role of heparanase, an endo‐β‐glucuronidase specifically degrading heparan sulfate (HS) glycosaminoglycans, in the mechanism of cancer cell invasion was investigated. Three human oral squamous cell carcinoma (SCC) cell lines (i.e., HSC‐2, HSC‐3 and LMF4), exhibiting various degrees of invasiveness to their surrounding tissues, were xenografted to the tongue of SCID mice in order to establish experimental cancer foci. Cancer cells and their surrounding tissues were examined for the expression of heparanase mRNA by an in situ hybridization technique, and for various basement membrane (BM)‐associated molecules (i.e., perlecan, laminins and type IV collagen) by immunohistochemical procedures. BM structures surrounding cancer tissues were also examined by electron microscopy. Increasing levels of heparanase mRNA expression were observed with the progression of cancer invasiveness, as manifested by the destruction of BM structures. Enhanced heparanase enzyme activities in cancer tissues with more invasive properties were demonstrated by the disappearance of HS glycosaminoglycans in the face of retained HS pro‐teoglycan core proteins. These results demonstrated a positive correlation between the heparanase enzyme activities and the invasiveness of human oral SCC. The roles of heparanase in cancer cell invasion were not precisely clarified by the present morphological study, but the enhanced heparanase activity in an early phase of BM destruction by cancer cells suggested the participation of this enzyme from the early phase of cancer invasion. (Cancer Sci 2003; 94: 277–285)

Characterization of Heparanase from a Rat Parathyroid Cell Line

Cell surface heparan sulfate proteoglycans undergo unique intracellular degradation pathways after they are endocytosed from the cell surface. Heparanase, an endo-β-glucuronidase capable of cleaving heparan sulfate, has been demonstrated to contribute to the physiological degradation of heparan sulfate proteoglycans and therefore regulation of their biological functions. A rat parathyroid cell line was found to produce heparanase with an optimal activity at neutral and slightly acidic conditions suggesting that the enzyme participates in heparan sulfate proteoglycan metabolism in extralysosomal compartments. To elucidate the detailed properties of the purified enzyme, the substrate specificity against naturally occurring heparan sulfates and chemically modified heparins was studied. Cleavage sites of rat heparanase were present in heparan sulfate chains obtained from a variety of animal organs, but their occurrence was infrequent (average, 1–2 sites per chain) requiring recognition of both undersulfated and sulfated regions of heparan sulfate. On the other hand intact and chemically modified heparins were not cleaved by heparanase. The carbohydrate structure of the newly generated reducing end region of heparan sulfate cleaved by the enzyme was determined, and it represented relatively undersulfated structures. O-Sulfation of heparan sulfate chains also played important roles in substrate recognition, implying that rat parathyroid heparanase acts near the boundary of highly sulfated and undersulfated domains of heparan sulfate proteoglycans. Further elucidation of the roles of heparanase in normal physiological processes would provide an important tool for analyzing the regulation of heparan sulfate-dependent cell functions.

Clinical characteristics of multiple primary carcinomas of the oral cavity

OBJECTIVES This study aimed to clarify the clinical characteristics of multiple primary carcinomas of the oral cavity. MATERIALS AND METHODS We retrospectively reviewed the cases of 1015 patients who were treated during follow up for oral cancer at Tokyo Medical and Dental University between March 2001 and December 2012. We compared the clinical characteristics of 961 patients who developed single primary oral squamous cell carcinoma (SCC) during follow up and 54 patients who subsequently developed multiple primary carcinomas in the oral cavity. RESULTS Mean age at first diagnosis was significantly higher in patients with multiple primary carcinomas than single primary carcinoma. Multiple primary carcinomas showed a female predilection, were most prevalent in the gingiva, and tended to show earlier tumor and nodal stages than single primary carcinoma. The local recurrence rate was higher for multiple primary carcinomas than single primary carcinoma, and it increased with the number of multiple primary occurrences. The disease-specific survival rate at 10 years for patients with single primary carcinoma was 85.3% and that for patients with multiple primary carcinomas was 79.6%. The cumulative incidence rate for metachronous second multiple primary carcinomas after the onset of first carcinoma at 10 years was 8.0%. The recurrence of multiple primary carcinomas did not decrease the survival rate. CONCLUSION Differences were found in the clinical characteristics between patients with single oral SCC and those with multiple primary oral carcinomas. Early diagnosis and treatment as well as close long-term follow up are needed for patients with multiple primary oral carcinomas.