Shinri Koshika

Inostamycin, an inhibitor of cytidine 5′-diphosphate 1,2-diacyl-sn-glycerol (CDP-DG): Inositol transferase, suppresses invasion ability by reducing productions of matrix metalloproteinase-2 and -9 and cell motility in HSC-4 tongue carcinoma cell line

Inostamycin is an inhibitor of cytidine 5′-diphosphate 1,2-diacyl-sn-glycerol (CDP-DG): inositol transferase. It significantly reduced epidermal growth factor (EGF)-induced in vitro invasion of the tongue carcinoma cell line, HSC-4, through reconstituted basement membrane Matrigel®. Since phosphatidylinositol (PI) 4,5-biphosphate is important for signal transduction through protein kinase C and actin reorganisation, we further examined the effect of inostamycin on production of two matrix metalloproteinases (MMPs), MMP-2 and -9, and on cell motility. Zymographic analysis showed that inostamycin suppressed pro-MMP-2 and pro-MMP-9 levels at a dose-dependent fashion, while MMP-2 activity was not significantly affected. By reverse transcription-polymerase chain reaction, it was found that inostamycin diminished steady state levels of MMP-2 and -9 but not membrane type 1-MMP mRNA expressions. Inostamycin partially blocked both EGF- and phorbol 12-myristate 13-acetate-stimulated pro-MMP- 9 production. A cytoplasmic calcium chelator (BAPTA-AM) dramatically elevated pro- MMP-9 and slightly elevated pro-MMP-2 secretions. EGF-stimulated motility of HSC-4 cells was suppressed by inostamycin treatment along with reduction of actin cytoskeletal reorganisation, filopodia formation and cdc42 expression. These results suggested that inostamycin would be useful for an anti-invasive agent in tongue cancer.

Stimulation of Motility of Human Renal Cell Carcinoma by SPARC/Osteonectin/BM-40 Associated with Type IV Collagen

SPARC is known to be important in development and tissue remodelling. Here, we examined the effects of SPARC (secreted protein, acidic and rich in cysteine; osteonectin) derived from a rat osteosarcoma cell line on migration of renal cell carcinoma (RCC) by a Boyden chamber assay. YCR RCC cells migrated through type IV collagen-coated filters without stimuli (basal level). SPARC in the lower compartment stimulated chemotactic activity to 120% of the basal level, whereas premixing of YCR with purified SPARC before inoculation reduced their migration to 72% of the basal level. Furthermore, SPARC mixed with type IV collagen more efficiently stimulated their migration in a concentration-dependent manner (up to 170% of the basal level). This suggests that SPARC bound to type IV collagen plays a role in tumor invasion.

High production of SPARC/osteonectin/BM-40 in mouse metastatic B16 melanoma cell lines

Production of SPARC/osteonectin/BM-40 was determined in mouse B16 melanoma clones BL6 and F10 (high metastatic) and Fl (low metastatic). SPARC was produced greater amount in BL6 and F10 than in Fl cells, showing a good agreement with their metastatic potentials. Moreover, SPARC production was not influenced by culture pH, even in the acidic conditions (≈pH 5.9). Although tumor tissues show often low pH due to excessive amount of acidic metabolites such as lactate, most studies have been done in neutral pH. High SPARC production in the acidic medium, therefore, is thought to be an important potential for tumor invasive behaviour.

Cytostatic effect of inostamycin, an inhibitor of cytidine 5'-diphosphate 1,2-diacyl-sn-glycerol (CDP-DG): inositol transferase, on oral squamous cell carcinoma cell lines.

Inostamycin, which was recently isolated from Streptomyces sp. MH816‐AF15 as an inhibitor of cytidine 5′‐diphosphate 1,2‐diacyl‐sn‐glycerol (CDP‐DG): inositol transferase, caused a G1‐phase accumulation in the cell cycle of small cell lung carcinomas. To investigate whether the cytostatic effect of inostamycin is restricted to lung carcinoma cell lines or applicable to other type of cells, we tested five oral squamous cell carcinoma (SCC) cell lines. Cell growth was suppressed in 62.5–125ng/ml inostamycin in the culture medium in all oral cancer cell lines tested, with non‐viable cells being <1%, indicating inostamycin is cytostatic on SCC cell lines. Decrease in cyclin D1 mRNA and protein expression due to the inostamycin treatment was accompanied by suppression of phosphorylated retinoblastoma susceptibility gene product (pRB‐P) levels. Moreover, flow cytometric analysis showed that inostamycin induced an increase in G1/G0 cells (1.2–3.2 fold) over 24h. These results suggest that inostamycin is a useful agent for tumour dormant cytostatic therapy for oral SCC.

Characterization of packing columns for the liquid chromatographic determination of corticosteroids in human plasma

Abstract Commercially available columns were characterized by examining the retentions of steroid hormones in liquid chromatography. The phase systems used were dichloromethane-ethanol for silica gel, acetonitrile-water for ODS and CN bonded columns. Solvent compositions were optimized so as to make the retention times of standard steroids less than sixty minutes. Retention data were obtained and reproducibility of the retention times was examined by repetitive injections of the standard samples. These chromatographic phase systems were applied to determine steroid hormones in human plasma following extraction by dichloromethane. CN columns showed the best reproducibility and resolution. Changes in the standing of corticosteroids in plasma under general anesthesia were investigated using CN column systems.

Testosterone metabolism in new squamous cell carcinoma cell line (RSS18) from 7,12-dimethylbenz[a]anthracene-induced submandibular gland of female rat

We established a new squamous cell carcinoma cell line, designated RSS18, from a 7,12-dimethyl-benz[a]anthracene (DMBA)-induced submandibular gland of the female rat, and investigated a testosterone metabolism in the cells. During 6 h incubation of RSS18 cells with testosterone as a substrate, the cells produced a significant amount of 5alpha-dihydrotestosterone (DHT) and three kinds of minor metabolites, and their percentages metabolized against total metabolites were in descending order of DHT (89 %) > 5alpha-androstane-3alpha,17beta-diol (9.0 %) > 5alpha-androstanedione(1.6%) > 4-androstene-3,17-dione (0.69%). Therefore, testosterone in RSS18 cells was predominantly converted to DHT by 5alpha-reductase. Growth of RSS18 cells was stimulated by DHT (10(-11)-10(-9) M) to around 170%. By reverse transcription-polymerase chain reaction, the androgen receptor mRNA was significantly detected in RSS18 cells. As a result of these findings, DHT production from testosterone and expression of androgen receptor mRNA, we concluded that RSS18 proliferation may be stimulated by DHT through 5alpha-reductase from testosterone.

Evidence of gelatinase secretion by the submandibular gland in prepubescent rats.

The gelatin cleaving activities in secretions of cultured fragments of male rat submandibular glands were studied using zymography. Gelatinolytic activities of 88-, 64-, and 57-kDa proteins detected in the tissues from 22-28-day old animals were undetectable in 31-70-day old rats. The traces of gelatinolytic activity associated with 28-kDa protein were detectable from 22-day old rats in serum-free media, and this activity of the enzyme markedly increased with aging from 38-days old. At 52-days and the subsequent stages, in addition to 28-kDa, activities associated with 60-, 32-, and 29-kDa proteins were strong. When the conditioned media were treated with 1,10-phenanthroline and diisopropyl fluorophosphate (DFP), both products inhibited activity of 88-kDa enzyme, indicating that this enzyme is Cls-like enzyme. The 64- and 57-kDa activities were inhibited by 1,10-phemanthroline, but not by DFP; when the conditioned medium of the tissue from 24-day old rats was treated with p-aminophenylmercuric acetate, gelatinolytic activity associated with 64-kDa converted to 57-kDa. Therefore, 64- and 57-kDa activities were concluded to be progelatinase A and gelatinase A, respectively. On the other hand, the gelatinolytic activities associated with 60-, 32-, 29- and 28-kDa proteins were inhibited by DFP but not by 1,10-phenanthroline, indicating that these enzymes belong to the family of serine proteinase, most probably kallikrein-related enzymes. From these findings, it was suggested that gelatinase A, along with Cls-like enzyme, participates in the maturation of the submandibular gland before it becomes active as an exocrine organ.

Trypsin-like protease and glucose-6-phosphate dehydrogenase in the human submandibular salivary gland.

The proteolytic activity of N-benzoyl-D,L-arginine-p-nitroanilide hydrochloride, a trypsin-like protease, was weak, whereas strong glucose-6-phosphate dehydrogenase activity was found in all subjects. The enzyme activities of males and females were similar.

Inhibitory effects by a submandibular gland extract on luteinizing hormone‐stimulated testosterone production by testicular cells

In order to evaluate the role of the submandibular gland (SMG) on testosterone (T) production by the testis, primary cultured testicular cells were prepared from rats that had the submandibular gland surgically ablated (G‐) and control (sham operated) rats (S.O.) respectively. The cells were incubated with or without 100 ng/ml luteinizing hormone (LH) and/or SMG extract. The same linear increase in T secretion was shown by both S.O.G‐ cells on multi‐stimulation with LH for up to 96 hrs. However, while an equivalent response was shown for S.O. cells after a single LH stimulation at 96 hrs, T secretion by the G‐ cells reached a plateau after 24 hrs. The level at 96 hrs was thus approximate 30% and 33% of those of S.O. cells with and without multi‐stimulus by LH for 96 hrs, respectively. When S.O. cells were cultured with SMG extract, LH‐stimulated T secretion was dose‐dependently inhibited and there was no effect on basal T secretion. The inhibitory effect was abolished by treatment at 95 °C for 5 min. Ultra‐filtration indicated that the molecular size of the inhibitory agent was greater than 30,000. It is proposed that SMG may contain a high molecular weight, heat labile soluble factor(s) which affects T secretion by inhibiting LH action in testicular cells.

Inhibition of estradiol‐17β secretion in ovarian granulosa cells by an extract from the submandibular gland of the rat

The submandibular salivary gland (SMG) in the rodent is a rich source of growth factors. The estradiol‐17β (E2) secretions were studied in the primary cultures of ovarian granulosa cells from sialoadenectomized (surgical removal of SMG, SDX) immature rats. The secretions of luteinizing hormone (LH) and follicle‐stimulating hormone (FSH) were also studied in the primary cultures of anterior pituitary cells from SDX rats. E2 secretion from the ovarian granulosa cells of SDX rats was not affected by male and female SMG extracts. FSH‐stimulated E2 secretion, however, was inhibited by the SMG extracts. Substances in the SMG extract, which inhibited FSH‐stimulated E2 secretion in the cells, were heat and urea stable, and were not stripped by charcoal. SMG extracts did not affect the secretions of LH and FSH in the anterior pituitary cells, with or without luteinizing hormone‐releasing hormone. The present findings suggest that male and female rat SMG contain the E2 modulating factor which affect ovarian granulosa cells.