Toshio Shigetomi

Selective hyperthermia using magnetoliposomes to target cervical lymph node metastasis in a rabbit tongue tumor model

The effect of hyperthermia on cervical lymph node metastasis of VX7 tongue cancer in female Japanese white rabbits was investigated. Magnetoliposomes (MLs) with a neutral surface charge and a size of 94.1 nm were used as heating mediators. MLs were injected into the tongue 20 days after tumor transplantation, and we examined whether they reached the metastatic deep cervical lymph node. The highest magnetite concentration 24 h after ML injection was detected in the lymph node, followed by tongue, spleen, blood, and liver. Rabbits were separated into three groups: group I as the control; group II with ML injection alone; and group III with ML injection and hyperthermia 24 h after ML injection, generated by applying an alternating magnetic field (118 kHz, 384 Oe) to the neck region. The hyperthermic effect was evaluated in terms of the percentage of necrosis in proportion to the metastatic tumor and the apoptotic index (AI), defined as the ratio of TUNEL‐positive cells. The temperature of lymph nodes in group III reached over 44°C. The mean area of necrosis in group III was 58.0%, which was significantly higher than that in group I (19.6%) or group II (20.4%). The AI in group III was 22.9%, significantly higher than in group I (1.67%) or II (1.42%). The difference between group I and II was not statistically significant. Group III tumor sites around MLs showed necrosis or apoptosis‐positive cells induced by hyperthermia. These results indicate that MLs injected into the tongue can target cervical lymph node metastases and accumulate there at concentrations sufficient to generate therapeutically effective temperatures.

Up-regulation of CD109 expression is associated with carcinogenesis of the squamous epithelium of the oral cavity

CD109 is a glycosylphosphatidylinositol (GPI)–anchored glycoprotein whose expression is up‐regulated in squamous cell carcinomas (SCCs) of the lung, esophagus, and uterus. The purpose of this study was to evaluate CD109 expression in oral tumors, including premalignant lesions, and to assess the clinical application of CD109 in oral cancer. CD109 expression in oral normal and tumor tissues from 124 patients was examined by immunohistochemical staining with anti‐CD109 antibody, and significant relations between clinical features and CD109 expression were statistically assessed. We found that high levels of CD109 expression were frequently detected in SCCs and premalignant lesions of the oral cavity, but not in normal squamous epithelia. The CD109 expression level was higher in well‐differentiated SCCs than in poorly differentiated SCCs. Furthermore, premalignant lesions highly expressing CD109 showed higher risk to progress to SCCs. Oral SCC cell lines overexpressing CD109 exhibited accelerated cell growth in vitro compared with control cell lines. In addition, overexpression of CD109 impaired the transforming growth factor (TGF)–β1‐mediated suppression of cell growth. These findings suggest that CD109 plays a role in the development of oral cancers, and is a useful prognostic marker to predict malignant transformation of premalignant lesions. (Cancer Sci 2008; 99: 1916–1923)

Processing of CD109 by furin and its role in the regulation of TGF-|[beta]| signaling

CD109 is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein, whose expression is upregulated in squamous cell carcinomas of the lung, esophagus, uterus and oral cavity. CD109 negatively regulates transforming growth factor (TGF)-β signaling in keratinocytes by directly modulating receptor activity. In this study, we further characterized CD109 regulation of TGF-β signaling and cell proliferation. We found that CD109 is produced as a 205 kDa glycoprotein, which is then processed in the Golgi apparatus into 180 kDa and 25 kDa proteins by furin (furinase). 180 kDa CD109 associated with GPI-anchored 25 kDa CD109 on the cell surface and was also secreted into the culture medium. To investigate whether furinase cleavage of CD109 is necessary for its biological activity, we mutated arginine 1273 in the CD109 furinase cleavage motif (amino acid 1270-RRRR-1273) to serine (R1273S). Interestingly, CD109 R1273S neither significantly impaired TGF-β signaling nor affected TGF-β-mediated suppression of cell growth, although it was expressed on the cell surface as a 205 kDa protein. Consistent with this finding, the 180 kDa and 25 kDa CD109 complex, but not CD109 R1273S, associated with the type I TGF-β receptor. These findings indicate that processing of CD109 into 180 kDa and 25 kDa proteins by furin, followed by complex formation with the type I TGF-β receptor is required for the regulation of TGF-β signaling in cancer cells and keratinocytes.

Selected salivary-gland cell culture and the effects of isoproterenol, vasoactive intestinal polypeptide and substance P

To establish a selected salivary-gland cell culture and determine the effect of neuropeptides, monolayers were cultured using 3T3 cells as a feeder layer. To confirm the origin of these cultured cells, amylase production was examined by electron microscopy and periodic acid-Schiff staining, together with immunocytochemical analysis of myosin, anti-cytokeratin (CK-1, CK 10/13, CK MNF116, CK LMW, CK HMW and CK-19) and amylase antibody. The cultured cells demonstrated secretion granules containing amylase and presented features characteristic of acinar cells, which they retained until passage two. By using a feeder layer in conjunction with a newly formulated culture medium, the selectability of these cells was improved. Changes in proliferation of cultured salivary-gland cells in the presence of selected neurotransmitters were also examined. Isoproterenol enhanced cellular proliferation. On the other hand, vasoactive intestinal polypeptide and substance P, which increase the weight of salivary glands in vivo, showed no significant enhancement of proliferation.

Acceleration of rat salivary gland tissue repair by basic fibroblast growth factor.

A model of atrophic rat submandibular gland was used to examine the ability of basic fibroblast growth factor (bFGF) to accelerate tissue repair. The gland duct was separated carefully from associated blood vessels and nerve, and ligated with a 8-0 suture under a surgical microscope. Two weeks after ligation, the glandular tissue showed severe atrophy and weight loss (to 26% of that in a sham-operated group). Thereafter, the ligature was removed and various amounts of bFGF, isoproterenol or saline were instilled retrogradely through the duct. Both isoproterenol and bFGF increased cell proliferation significantly. bFGF accelerated the proliferation of various cell types, including both acinar and ductal. The proliferative effects of bFGF peaked at a dose of 1 ng/gland. When bFGF (1 ng/gland) was administered to the atrophic gland, its weight increased to 125% of the glands in saline-treated control animals after 2 weeks. The effects of bFGF were also examined in normal submandibular glands: bFGF stimulated cell proliferation, but the effective concentration was at least 50 times higher than that required in the atrophic gland. The results from immunohistochemical tests against anti-FGF receptor-type 1 antibody demonstrated increased immunoreactivity in the damaged gland, which might be involved in the difference in the response to bFGF between damaged and normal glands. Overall, the results indicate that bFGF can accelerate tissue repair in salivary gland.

Prognostic Evaluation of Preoperative Thermochemoradiotherapy for N3 Cervical Lymph Node Metastases of Oral Cancer

Objective: The purpose of this study was to evaluate the clinical efficacy, histopathological efficacy, and response to preoperative thermochemoradiotherapy for N3 cervical lymph node metastases of oral cancer. Methods: Preoperative thermochemoradiotherapy was performed in 8 patients with oral cancer and N3 cervical lymph node metastasis. These patients underwent four-weekly sessions of hyperthermia, combined with radiotherapy (40 Gy) as well as chemotherapy with cisplatin (CDDP; 100 mg/m2), all prior to surgery. Radical neck dissection was performed 4 weeks after completion of preoperative thermochemoradiotherapy. Results: The preoperative treatment of cervical lymph node metastases yielded a partial response in 6 patients, while 2 patients demonstrated no change. Histopathologically, grade III was detected in 1, grade IIb in 4 and grade IIa in 3 patients after surgery, according to the criteria of Shimosato. The follow-up period ranged from 13 to 64 months (mean 34). Of the 8 patients, 2 died (1 of lymph node metastasis and 1 had metastasis to a distant site), and 6 patients were alive at the last follow-up, with the longest postoperative disease-free survival being 63 months. The 5-year cumulative survival rate was 70.0%. Conclusion: These results indicate that preoperative thermochemoradiotherapy is a promising modality for patients with N3 cervical lymph node metastasis of oral cancer.

Effects of basic fibroblast growth factor on cultured rat and human submandibular salivary gland cells.

Basic fibroblast growth factor (bFGF) is a strong mitogen for most mesoderm- and ectoderm-derived cells. Although bFGF exists in rat and human salivary glands, its physiological role in those glands is unknown. In this study, the effects of bFGF were investigated in monolayer culture of normal rat and human submandibular gland cells. Epithelial cells from rat and human submandibular glands were cultivated with the aid of 3T3 cells as a feeder layer. The effects of different concentrations of bFGF on the second passage of these cultured cells were examined. In both the rat and human cells, the percentage of bromodeoxyuridine (BrdU)-positive cells gradually increased up to 50 ng/ml, and then increased sharply at 100 ng/ml. However, at concentrations higher than 100 ng/ml, the percentages of BrdU-positive cells reached a plateau. In both rat and human cells, total cell numbers at 100 ng/ml bFGF were significantly higher than those of the control group from culture day 4. On the other hand, the morphology of the cultured cells showed no difference either with or without bFGF. These results indicate that a major effect of bFGF on salivary gland epithelial cells is to act as a mitogenic stimulus.

Chemoradiotherapy for maxillary sinus adenoid cystic carcinoma using superselective intra-arterial infusion via a superficial temporal artery

The efficacy of concurrent intra‐arterial infusion chemoradiotherapy for adenoid cystic carcinoma (ACC) has been described in only a few reports. Herein, we report on 2 patients with unresectable ACC of the maxillary sinus treated with this approach.

Effects of Ca2+ removal and of tetraethylammonium on membrane currents induced by carbachol in isolated cells from the rat parotid gland

In freshly dispersed rat parotid acinar cells, 10 μM carbachol increased outward currents at 0 mV and also inward currents at −70 mV recorded with the whole-cell clamp method using patch pipettes containing 1 mM EGTA. When EGTA in the pipette was increased to 2.4 mM, carbachol increased only outward currents and a further increase of EGTA to 4 mM blocked the carbachol response. Effects of changes in external K+ and Cl− concentrations suggested that outward currents were carried by K+ and inward by Cl−. Effects of Ca2+ removal from the medium differed between experiments with 0 and 5 mM ATP in the patch pipettes. When pipettes contained no ATP, responses evoked by repeated applications of 10 μM carbachol (0.5–1 min) at 1.5–4 min intervals decreased only slowly after Ca2+ removal, outward currents being reduced to 90±6% and inward currents to 47±11% (n=6) in 10 min. On the other hand, when 5 mM ATP was included in the electrodes, Ca2+ removal abolished the carbachol responses in about 5 min (n=4). It was also found that tetraethylammonium (5 mM) strongly reduced both currents, by blocking muscarinic receptors, while Ba2+ (2.4 mM) inhibited only the outward K+ current.

Optimization of hyperthermia and dendritic cell immunotherapy for squamous cell carcinoma.

The aims of this study were to explore the feasibility of a novel combination therapy comprising hyperthermia (HT) and dendritic cell (DC) application for squamous cell carcinoma (SCC), and to optimize the conditions of this therapy. In vitro, the correlation between maturation of DCs by co-culture with SCCVII cells and HT was investigated. DCs did not mature in simple HT (43 °C for 30 min) with SCCVII cells. On the other hand, DC maturation occurred in additional mild HT (mHT: 41 °C for 30 min) with simple HT. To assess whether additional mHT was effective, in vivo combined treatment was performed using tumor-bearing C3H/HeJ mice. A more suppressive effect of tumor growth was observed, and cytotoxic T cell infiltration was significantly increased by adding mHT compared to conventional only simple HT with DCs. These phenomena also occurred in non-treated contralateral tumors as well as treated ones. Our data suggest that the combination of 43 °C preheated simple HT SCCVII tumors and additional 41 °C heat mHT promotes DC maturation, resulting in suppression of tumor growth systemically and lifetime prolongation in mice. A three-step process of additional mHT after local HT and intratumoral immature DC (iDC) injection could be a more effective and novel method for the treatment of SCC.