Hideyuki Nakashima

Overexpression of caveolin-1 in a human melanoma cell line results in dispersion of ganglioside GD3 from lipid rafts and alteration of leading edges, leading to attenuation of malignant properties.

Caveolin‐1 is a component of lipid rafts, and is considered to be a tumor suppressor molecule. However, the mechanisms by which caveolin‐1 functions in cancer cells are not well understood. We generated caveolin‐1 transfectant cells (Cav‐1+ cells) using a human melanoma cell line (SK‐MEL‐28) and investigated the effects of caveolin‐1 overexpression on the GD3‐mediated malignant properties of melanomas. Cav‐1+ cells had decreased cell growth and motility, and reduced phosphorylation levels of p130Cas and paxillin relative to controls. In floatation analysis, although GD3 was mainly localized in glycolipid‐enriched microdomain (GEM)/rafts in control cells, it was dispersed from GEM/rafts in Cav‐1+ cells. Correspondingly, GD3 in Cav‐1+ cells stained uniformly throughout the membrane, whereas control cells showed partial staining of the membrane, probably at the leading edge. p130Cas and paxillin were stained in the leading edges and colocalized with GD3 in the control cells. In contrast, these molecules were diffusely stained and no definite leading edges were detected in Cav‐1+ cells. These results suggest that caveolin‐1 regulates GD3‐mediated malignant signals by altering GD3 distribution and leading edge formation. These results reveal one of the mechanisms by which caveolin‐1 curtails the malignant properties of tumor cells. (Cancer Sci 2007; 98: 512–520)

Retrograde superselective intra-arterial chemotherapy and daily concurrent radiotherapy for stage III and IV oral cancer: Analysis of therapeutic results in 112 cases

PURPOSE To evaluate the therapeutic results and rate of organ preservation in patients with stage III or IV oral cancer treated with retrograde superselective intra-arterial chemotherapy and daily concurrent radiotherapy. MATERIALS AND METHODS One hundred and twelve patients with stage III and IV oral squamous cell carcinoma underwent intra-arterial chemoradiotherapy. Catheterization from the superficial temporal and occipital arteries was performed. Treatment consisted of superselective intra-arterial chemotherapy (docetaxel, total 60mg/m(2), cisplatin, total 150mg/m(2)) and daily concurrent radiotherapy (total of 60Gy) for 6 weeks. RESULTS The median follow-up for all patients was 46.2 months (range, 10-76 months). After intra-arterial chemoradiotherapy, primary site complete response was achieved in 98 (87.5%) of 112 cases. Five-year survival and local control rates were 71.3% and 79.3%, respectively. Grade 3 or 4 toxicities included mucositis in 92.0%, neutropenia in 30.4%, dermatitis in 28.6%, anemia in 26.8%, and thrombocytopenia in 7.1% of patients. Grade 3 toxicities included dysphagia in 72.3%, nausea/vomiting in 21.4%, fever in 8.0%, and renal failure in 0.9% of patients. CONCLUSION Retrograde superselective intra-arterial chemotherapy and daily concurrent radiotherapy for stage III and IV oral cancer provided good overall survival and local control.

Functional Activation of Src Family Kinase Yes Protein Is Essential for the Enhanced Malignant Properties of Human Melanoma Cells Expressing Ganglioside GD3

The possible roles of Src family kinases in the enhanced malignant properties of melanomas related to GD3 expression were analyzed. Among Src family kinases only Yes, not Fyn or Src, was functionally involved in the increased cell proliferation and invasion of GD3-expressing transfectant cells (GD3+). Yes was located upstream of p130Cas and paxillin and at an equivalent level to focal adhesion kinase. Yes underwent autophosphorylation even before serum treatment and showed stronger kinase activity in GD3+ cells than in GD3− cells following serum treatment. Coimmunoprecipitation experiments revealed that Yes bound to focal adhesion kinase or p130Cas more strongly in GD3+ cells than in GD3− cells. As a possible mechanism for the enhancing effects of GD3 on cellular phenotypes, it was shown that majority of Yes was localized in glycolipid-enriched microdomain/rafts in GD3+ cells even before serum treatment, whereas it was scarcely detected in glycolipid-enriched microdomain/rafts in GD3− cells. An in vitro kinase assay of Yes revealed that coexistence of GD3 with Yes in membranous environments enhances the kinase activity of GD3− cell-derived Yes toward enolase, p125, and Yes itself. Knockdown of GD3 synthase resulted in the alleviation of tumor phenotypes and reduced activation levels of Yes. Taken together, these results suggest a role of GD3 in the regulation of Src family kinases.

Molecules in the signaling pathway activated by gangliosides can be targets of therapeutics for malignant melanomas

There have been a number of studies on tumor‐specific glycolipid antigens. In particular, neuroectoderm‐derived cancers express characteristic ganglioside antigens, some of them have been used as tumor markers and/or target of immunotherapy. Molecules in the signaling pathway activated by gangliosides have been analyzed. Here, we reported results on the functions of molecules involved in the signaling pathway to enhance malignant properties of human melanomas under GD3 expression, and emphasized that those molecules including tumor‐associated antigens can be targets of therapeutics for malignant melanomas.

M2-polarized macrophages contribute to neovasculogenesis, leading to relapse of oral cancer following radiation

Despite the fact that radiation is one of the standard therapies in the treatment of patients with oral cancer, tumours can recur even in the early stages of the disease, negatively impacting prognosis and quality of life. We previously found that CD11b+ bone marrow-derived cells (BMDCs) were recruited into human glioblastoma multiforme (GBM), leading to re-organization of the vasculature and tumour regrowth. However, it is not yet known how these cells contribute to tumour vascularization. In the present study, we investigated the role of infiltrating CD11b+ myeloid cells in the vascularization and recurrence of oral squamous cell carcinoma (OSCC). In a xenograft mouse model, local irradiation caused vascular damage and hypoxia in the tumour and increased infiltration of CD11b+ myeloid cells. These infiltrating cells showed characteristics of M2 macrophages (M2Mφs) and are associated with the promotion of vascularization. M2Mφs promoted tumour progression in recurrence after irradiation compared to non-irradiated tumours. In addition, we found that CD11b+ myeloid cells, as well as CD206+ M2Mφs, are increased during recurrence after radiotherapy in human OSCC specimens. Our findings may lead to the development of potential clinical biomarkers or treatment targets in irradiated OSCC patients.

Simultaneous hyperthermia-chemotherapy with controlled drug delivery using single-drug nanoparticles

We previously investigated the utility of μ-oxo N,N′- bis(salicylidene)ethylenediamine iron (Fe(Salen)) nanoparticles as a new anti-cancer agent for magnet-guided delivery with anti-cancer activity. Fe(Salen) nanoparticles should rapidly heat up in an alternating magnetic field (AMF), and we hypothesized that these single-drug nanoparticles would be effective for combined hyperthermia-chemotherapy. Conventional hyperthermic particles are usually made of iron oxide, and thus cannot exhibit anti-cancer activity in the absence of an AMF. We found that Fe(Salen) nanoparticles induced apoptosis in cultured cancer cells, and that AMF exposure enhanced the apoptotic effect. Therefore, we evaluated the combined three-fold strategy, i.e., chemotherapy with Fe(Salen) nanoparticles, magnetically guided delivery of the nanoparticles to the tumor, and AMF-induced heating of the nanoparticles to induce local hyperthermia, in a rabbit model of tongue cancer. Intravenous administration of Fe(Salen) nanoparticles per se inhibited tumor growth before the other two modalities were applied. This inhibition was enhanced when a magnet was used to accumulate Fe(Salen) nanoparticles at the tongue. When an AMF was further applied (magnet-guided chemotherapy plus hyperthermia), the tumor masses were dramatically reduced. These results indicate that our strategy of combined hyperthermia-chemotherapy using Fe(Salen) nanoparticles specifically delivered with magnetic guidance represents a powerful new approach for cancer treatment.

IL-13 receptor-directed cancer vaccines and immunotherapy

Many immunotherapy approaches including therapeutic cancer vaccines targeting specific tumor-associated antigens are at various stages of development. Although the significance of overexpression of (IL-13Rα2) in cancer is being actively investigated, we have reported that IL-13Rα2 is a novel tumor-associated antigen. The IL-13Rα2-directed cancer vaccine is one of the most promising approaches to tumor immunotherapy, because of the selective expression of IL-13Rα2 in various solid tumor types but not in normal tissues. In this article, we will summarize its present status and potential strategies to improve IL-13Rα2-directed cancer vaccines for an optimal therapy of cancer.

Abstract 4576: Development of thermochemotherapy using cisplatin and ferucarbotran (Resovist®) in head and neck cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Radical surgery for patients with advanced head and neck cancer causes dysfunctions as well as decreases quality of life. To overcome this issue, we developed a new combination therapy of cisplatin and inductive hyperthermia using ferucarbotran (Resovist®). Ferucarbotran, which is made of superparamagnetic iron oxide, generates heat when exposed to an alternating magnetic fields (AMF). Herein, we explored whether ferucarbotran could be used as a heat source for hyperthermia upon exposure to AMF in the presence of cisplatin. Our aim is to evaluate the simultaneous therapeutic efficacy of chemotherapy and inductive hyperthermia for head and neck cancer. Materials and Methods: OSC-19 and HSC-3, human oral cancer cell lines, were used in this study. Cell proliferation was assessed by methyl thiazolyl tetrazorium (MTT) assay. The intracellular level of reactive oxygen species (ROS) was measured using fluorescent dye 2′, 7′-dichlorodihydrofluorescein diacetate. Apoptotic cells were stained with Annexin V, allophycocyanin conjugate and 7-amino-actinomycin D, and measured by fluorescence activated cell sorting (FACS), to evaluate early and late apoptosis. Thermal images and temperature were obtained by thermograpy and thermometer. Alternating magnetic fields were generated by a transistor-driven vertical coil at a frequency of 308 KHz and electric current (EC) 250 A. Results: Ferucarbotran generated heat in a dose- and time-dependent manner when exposed to an AMF, suggesting that ferucarbotran could be used as a heat source for hyperthermia. As we expected, Cisplatin suppressed proliferation of OSC-19 and HSC-3 cells in a dose-dependent manner, not only furucarbtran. First, we performed MTT assay and ROS generation assay to evaluate whether hyperthermia effect enhanced anti-cancer effect in the presence of cisplatin. Simply incubation at 42 °C for one hour enhanced the anti-cancer effect and ROS generation in the presence of cisplatin. Cisplatin induced apoptosis of OSC-19 and HSC-3 cells in a dose-dependent manner. Ferucarbotran further promoted cisplatin-induced apoptosis compared to cisplatin alone, when exposed to an AMF for an hour. Thus, the combination of cisplatin with ferucarbotran /AMF was more effective than cisplatin alone, suggesting that we could reduce the amount of cisplatin in clinical usage. Conclusion: Our findings suggest that combination therapy of cisplatin and ferucarbotran in an AMF may be used to develop a new combination therapy for head and neck cancer. Citation Format: Itaru Sato, Masanari Umemura, Kenji Mitsudo, Xianfeng Feng, Hideyuki Nakashima, Mitomu Kioi, Akiyoshi Miyajima, Haruki Eguchi, Iwai Tohnai, Yoshihiro Ishikawa. Development of thermochemotherapy using cisplatin and ferucarbotran (Resovist®) in head and neck cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4576. doi:10.1158/1538-7445.AM2014-4576

Communication Between the Transverse Cervical Nerve and the Marginal Mandibular Branch of the Facial Nerve: A Rare Anatomical Variant

Although anatomical knowledge of the facial nerve (FN) is very important to avoid iatrogenic injury during surgery, surgeons are concerned less with the cervical branch, which plays a definitive role in lower lip esthetic and function compared with other branches of the FN. Although communication between branches of the cervical plexus and FN is not well documented, the transverse cervical nerve commonly communicates with the cervical branch of the FN. There are few reports describing direct communication with the marginal mandibular branch. As a rare anatomical variant, the authors report communication between the transverse cervical nerve and the marginal mandibular branch of the FN.

Abstract 5568: Thermochemotherapy with controlled drug delivery using a novel magnetic anti-cancer drug .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Radical surgery for patients with oral cancer may cause dysfunction, such as dysphagia, dysarthria, or mastication disorders, leading to decreased quality of life in cancer patients. Here, we report the development of a novel magnetic anti-cancer drug, EI236, which may enable us to reduce such problems. EI236 possesses three features; anti-cancer effect alike cisplatin, controlled drug delivery using a magnet, and hyperthermic effect upon exposure to an alternating current magnetic field (ACMF). We thus examined the efficacy of EI236 in treating oral cancer in a rabbit cancer model. Methods: Cell proliferation was measured by MTT assays. We established the rabbit tongue cancer model using VX2 cells, which are readily transplantable in vivo. Electromagnet was used to attract EI236 to the site of tongue cancer by controlled drug delivery (CDR), and ACMF to generate heat from EI236 (HT). Rabbits were divided into 4 groups: control group, intravenous EI236 injection group (5mg/kg×7days) (i.v.), intravenous EI236 injection + electromagnet group (i.v. + CDR), intravenous EI236 injection + electromagnet + ACMF group (i.v. + CDR + HT). The size of tumors was measured daily for 7 days, followed by histopathological evaluation. Of note, EI236 was injected at the same dose in the three groups. Results: EI236 showed anti-cancer effects in a dose-dependent manner in VX2 cells. The IC50 value of EI236 was 7.5μM in cultured cells. The resultant tumor volume after EI236 i.v. injection treatment in rabbits was as follows; 319.3±40.0% for control, 228.5±94.3% for i.v., 91.5±27.4X% for i.v. + CDR, 24±8.2% for i.v. + CDR + HT, indicating that the i.v. + CDR + HT group showed the greatest decrease. It also showed the largest necrotic area in H-E staining analysis. The tumor cell nucleus, as determined by Ki67 staining, were mostly disappeared. Conclusion: Anti-cancer effect of EI236 was synergistically increased in the presence of CDR and/or HT. EI236 may serve as a novel anti-cancer therapy that enables CDR and HT at the same time. Citation Format: Itaru Sato, Kenji Mitsudo, Masanari Umemura, Xianfeng Feng, Hidenobu Fukumura, Haruki Eguchi, Hideyuki Nakashima, Mitomu Kioi, Iwai Tohnai, Yoshihiro Ishikawa. Thermochemotherapy with controlled drug delivery using a novel magnetic anti-cancer drug . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5568. doi:10.1158/1538-7445.AM2013-5568