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Supplemental Digital Content is available in the text., Objective: The ductus arteriosus (DA) is a fetal artery connecting the aorta and pulmonary arteries. Progressive matrix remodeling, that is, intimal thickening (IT), occurs in the subendothelial region of DA to bring anatomic DA closure. IT is comprised of multiple ECMs (extracellular matrices) and migrated smooth muscle cells (SMCs). Because glycoprotein fibulin-1 binds to multiple ECMs and regulates morphogenesis during development, we investigated the role of fibulin-1 in DA closure. Approach and Results: Fibulin-1–deficient (Fbln1−/−) mice exhibited patent DA with hypoplastic IT. An unbiased transcriptome analysis revealed that EP4 (prostaglandin E receptor 4) stimulation markedly increased fibulin-1 in DA-SMCs via phospholipase C-NFκB (nuclear factor κB) signaling pathways. Fluorescence-activated cell sorting (FACS) analysis demonstrated that fibulin-1 binding protein versican was derived from DA-endothelial cells (ECs). We examined the effect of fibulin-1 on directional migration toward ECs in association with versican by using cocultured DA-SMCs and ECs. EP4 stimulation promoted directional DA-SMC migration toward ECs, which was attenuated by either silencing fibulin-1 or versican. Immunofluorescence demonstrated that fibulin-1 and versican V0/V1 were coexpressed at the IT of wild-type DA, whereas 30% of versican-deleted mice lacking a hyaluronan binding site displayed patent DA. Fibulin-1 expression was attenuated in the EP4-deficient mouse (Ptger4−/−) DA, which exhibits patent DA with hypoplastic IT, and fibulin-1 protein administration restored IT formation. In human DA, fibulin-1 and versican were abundantly expressed in SMCs and ECs, respectively. Conclusions: Fibulin-1 contributes to DA closure by forming an environment favoring directional SMC migration toward the subendothelial region, at least, in part, in combination with EC-derived versican and its binding partner hyaluronan.

// Rina Nakakaji 1, 2, * , Masanari Umemura 1, * , Kenji Mitsudo 2 , Jeong-Hwan Kim 1 , Yujiro Hoshino 3 , Itaru Sato 1, 2 , Takatsugu Masuda 4 , Masahiro Yamamoto 5 , Mitomu Kioi 2 , Toshiyuki Koizumi 2 , Takayuki Fujita 1 , Utako Yokoyama 1 , Masaki Iida 2 , Motohiko Sato 6 , Hiroshi Sato 7 , Shoko Murofushi 7 , Sayaka Shibata 8 , Ichio Aoki 8 , Haruki Eguchi 7 , Iwai Tohnai 2 and Yoshihiro Ishikawa 1 1 Cardiovascular Research Institute, Yokohama City University Graduate School of Medicine, Yokohama, Japan 2 Department of Oral and Maxillofacial Surgery, Yokohama City University Graduate School of Medicine, Yokohama, Japan 3 Department of Environment and Natural Sciences, Yokohama National University Graduate School of Environment and Information Sciences, Yokohama, Japan 4 Tokyo Neutron Science Laboratory, Tokyo University Institute for Solid State Physics, Kashiwa, Japan 5 Department of Chemistry of Functional Molecules, Konan University Faculty of Science and Engineering, Kobe, Japan 6 Department of Physiology, Aichi Medical University, Nagakute, Japan 7 Advanced Applied Science Department, Research Laboratory, IHI Corporation, Yokohama, Japan 8 Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan * These authors contributed equally to this work Correspondence to: Masanari Umemura, email: umemurma@yokohama-cu.ac.jp Yoshihiro Ishikawa, email: yishikaw@med.yokohama-cu.ac.jp Keywords: iron-salen; taxol; oral cancer; paclitaxel; magnetism Received: July 14, 2017 Accepted: February 20, 2018 Epub: February 26, 2018 Published: March 20, 2018 ABSTRACT N,N’-Bis(salicylidene)ethylenediamine iron (Fe(Salen)) is an anti-cancer agent with intrinsic magnetic property. Here, we covalently linked Fe(Salen) to paclitaxel (PTX), a widely used anti-cancer drug, to obtain a magnetized paclitaxel conjugate (M-PTX), which exhibited magnetic characteristics for magnet-guided drug delivery and MRI visualization. M-PTX increased apoptosis and G2/M arrest of cultured human oral cancer cell lines in the same manner as PTX. Furthermore, marked contrast intensity was obtained in magnetic resonance imaging (MRI) of M-PTX. In a mouse oral cancer model, a permanent magnet placed on the body surface adjacent to the tumor resulted in distinct accumulation of M-PTX, and the anti-cancer effect was greater than that of M-PTX without the magnet. We believe that this strategy may improve future cancer chemotherapy by providing conventional anti-cancer drugs with novel functionalities such as magnet-guided drug delivery or MRI-based visualization/quantitation of drug distribution.

Sympathetic activation causes clinically important arrhythmias including atrial fibrillation (AF) and ventricular tachyarrhythmia. Although the usefulness of β-adrenergic receptor blockade therapy is widely accepted, its multiple critical side effects often prevent its initiation or continuation. The aim of this study is to determine the advantages of vidarabine, an adenylyl cyclase (AC)-targeted anti-sympathetic agent, as an alternative treatment for arrhythmia. We found that vidarabine, which we identified as a cardiac AC inhibitor, consistently shortens AF duration and reduces the incidence of sympathetic activation-induced ventricular arrhythmias. In atrial and ventricular myocytes, vidarabine inhibits adrenergic receptor stimulation-induced RyR2 phosphorylation, sarcoplasmic reticulum (SR) Ca2+ leakage, and spontaneous Ca2+ release from SR, the last of which has been considered as a potential arrhythmogenic trigger. Moreover, vidarabine also inhibits sympathetic activation-induced reactive oxygen species (ROS) production in cardiac myocytes. The pivotal role of vidarabine's inhibitory effect on ROS production with regard to its anti-arrhythmic property has also been implied in animal studies. In addition, as expected, vidarabine exerts an inhibitory effect on AC function, which is more potent in the heart than elsewhere. Indexes of cardiac function including ejection fraction and heart rate were not affected by a dosage of vidarabine sufficient to exert an anti-arrhythmic effect. These findings suggest that vidarabine inhibits catecholamine-induced AF or ventricular arrhythmia without deteriorating cardiac function in mice.

Pro-inflammatory cytokines are released in septic shock and impair cardiac function via the Jak-STAT pathway. It is well known that sympathetic stimulation leads to coupling of the β-adrenergic receptor/Gs/adenylyl cyclase, a membrane-bound enzyme that catalyzes the conversion of ATP to cAMP, thereby stimulating protein kinase A (PKA) and ultimately compensating for cardiac dysfunction. The mechanism of such compensation by catecholamine has been traditionally understood as PKA-mediated enforcement of cardiac contractility. We hypothesized that exchange protein activated by cyclic AMP (Epac), a new target of cAMP signaling that functions independently of protein kinase A, also plays a key role in protection against acute stresses or changes in hemodynamic overload. Lipopolysaccharide injection induced cytokine release and severe cardiac dysfunction in mouse. In mouse overexpressing Epac1 in the heart, however, the magnitude of such dysfunction was significantly smaller. Epac1 overexpression inhibited the Jak-STAT pathway, as indicated by decreased phosphorylation of STAT3 and increased SOCS3 expression, with subsequent inhibition of iNOS expression. In cultured cardiomyocytes treated with isoproterenol or forskolin, the increase of SOCS3 expression was blunted when Epac1 or PKCα was silenced with siRNA. Activation of the cAMP/Epac/PKCα pathway protected the heart against cytokine-induced cardiac dysfunction, suggesting a new role of catecholamine signaling in compensating for cardiac dysfunction in heart failure. Epac1 and its downstream pathways may be novel targets for treating cardiac dysfunction in endotoxemia.

Background It has been suggested that protein directly activated by cAMP (Epac), one of the downstream signaling molecules of β-adrenergic receptor (β-AR), may be an effective target for the treatment of arrhythmia. However, there have been no reports on the anti-arrhythmic effects or cardiac side-effects of Epac1 inhibitors in vivo. Methods and Results: In this study, the roles of Epac1 in the development of atrial and ventricular arrhythmias are examined. In addition, we examined the usefulness of CE3F4, an Epac1-selective inhibitor, in the treatment of the arrhythmias in mice. In Epac1 knockout (Epac1-KO) mice, the duration of atrial fibrillation (AF) was shorter than in wild-type mice. In calsequestrin2 knockout mice, Epac1 deficiency resulted in a reduction of ventricular arrhythmia. In both atrial and ventricular myocytes, sarcoplasmic reticulum (SR) Ca2+ leak, a major trigger of arrhythmias, and spontaneous SR Ca2+ release (SCR) were attenuated in Epac1-KO mice. Consistently, CE3F4 treatment significantly prevented AF and ventricular arrhythmia in mice. In addition, the SR Ca2+ leak and SCR were significantly inhibited by CE3F4 treatment in both atrial and ventricular myocytes. Importantly, cardiac function was not significantly affected by a dosage of CE3F4 sufficient to exert anti-arrhythmic effects. Conclusions These findings indicated that Epac1 is involved in the development of atrial and ventricular arrhythmias. CE3F4, an Epac1-selective inhibitor, prevented atrial and ventricular arrhythmias in mice.

Colorectal anastomotic leakage is one of the most feared and fatal complications of colorectal surgery. To date, no external coating material that can prevent anastomotic leakage has been developed. As myoblasts possess anti-inflammatory capacity and improve wound healing, we developed a multilayered human skeletal muscle myoblast (HSMM) sheet by periodic exposure to supraphysiological hydrostatic pressure during repeated cell seeding. We assessed whether the application of an HSMM sheet can promote the healing process after colonic anastomosis. Partial colectomy and insufficient suturing were employed to create a high-risk colo-colonic anastomosis model in 60 nude rats. Rats were divided into a control group ( n = 30) and an HSMM sheet group ( n = 30). Macroscopic findings, anastomotic bursting pressure, and histology at the colonic anastomotic site were evaluated on postoperative day (POD) 3, 5, 7, 14, and 28. The application of an HSMM sheet significantly suppressed abscess formation at the anastomotic site compared to the control group on POD3 and 5. The anastomotic bursting pressure in the HSMM sheet group was higher than that in the control group on POD3 and 5. Inflammatory cell infiltration in the HSMM sheet group was significantly suppressed compared to that in the control group throughout the time course. Collagen deposition in the HSMM sheet group on POD3 was significantly abundant compared to that in the control group. Regeneration of the mucosa at the colonic anastomotic site was promoted in the HSMM sheet group compared to that in the control group on POD14 and 28. Immunohistochemical analysis demonstrated that surviving cells in the HSMM sheet gradually decreased with postoperative time and none were detected on POD14. These results suggest that the application of a multilayered HSMM sheet may prevent postoperative colonic anastomotic leakage.

BACKGROUND Extremely preterm infants frequently have patent ductus arteriosus (PDA). Recent recommendations include immediately beginning amino acid supplementation in extremely preterm infants. However, the effect of amino acids on closure of the ductus arteriosus (DA) remains unknown.Methods and Results:Aminogram results in human neonates at day 2 revealed that the plasma glutamate concentration was significantly lower in extremely preterm infants (

Abdominal aortic aneurysm (AAA) is a progressive disease that has an increasing prevalence with aging, but no effective pharmacological therapy to attenuate AAA progression is currently available. We reported that the prostaglandin E receptor EP4 plays roles in AAA progression. Here, we show the effect of CJ-42794, a selective EP4 antagonist, on AAA using two mouse models (angiotensin II- and CaCl2 -induced AAAs) and human aortic smooth muscle cells isolated from AAA tissue. Oral administration of CJ-42794 (0.2 mg/kg per day) for 4 weeks significantly decreased AAA formation in ApoE-/- mice infused with angiotensin II (1 μg/kg per min), in which elastic fiber degradation and activations of matrix metalloproteinase (MMP)-2 and MMP-9 were attenuated. Interleukin-6 (IL-6) proteins were highly expressed in the medial layer of angiotensin II-induced mouse AAA tissues, whereas this expression was significantly decreased in mice treated with CJ-42794. AAA formation induced by periaortic CaCl2 application in wild-type mice was also reduced by oral administration of CJ-42794 for 4 weeks. After oral administration of CJ-42794 beginning 2 weeks after periaortic CaCl2 application and continuing for an additional 4 weeks, the aortic diameter and elastic fiber degradation grade were significantly smaller in CJ-42794-treated mice than in untreated mice. Additionally, in smooth muscle cells isolated from human AAA tissues, stimulation of CJ-42794 inhibited PGE2 -induced IL-6 secretion in a dose-dependent manner and decreased PGE2 -induced MMP-2 activity. These data suggest that inhibition of EP4 has the potential to be a pharmacological strategy for attenuation of AAA progression.

Aims At birth, dynamic changes occur in serum components and haemodynamics, such as closure of the ductus arteriosus (DA). A previous study demonstrated that, in full-term human neonates, serum osmolality decreased transiently after birth, but recovered over the next few days. However, the significance of this transient decrease in osmolality has never been addressed. The objective of the present study was to examine the role of changes in serum osmolality after birth in DA closure. Methods and results We found that rats exhibited a similar transient hypoosmolality after birth. Hypotonic stimulation induced constriction of DA rings and increased Ca2+ transient in DA smooth muscle cells, but not in the aorta. The hypoosmotic sensor transient receptor potential melastatin 3 (TRPM3) was highly expressed in the rat DA, and TRPM3 silencing abolished the Ca2+ response to hypoosmolality. Pregnenolone sulfate stimulation of TRPM3 induced rat DA constriction ex vivo and in vivo. Furthermore, hyper tonic fluid injection impaired rat DA closure. In humans, neonatal serum hypoosmolality was observed in relatively mature preterm infants (≥28 weeks). In extremely preterm infants (

Background In heart failure patients, chronic hyperactivation of sympathetic signaling is known to exacerbate cardiac dysfunction. In this study, the cardioprotective effect of vidarabine, an anti-herpes virus agent, which we identified as a cardiac adenylyl cyclase inhibitor, in dogs with pacing-induced dilated cardiomyopathy (DCM) was evaluated. In addition, the adverse effects of vidarabine on basal cardiac function was compared to those of the β-blocker, carvedilol.Methods and Results:Vidarabine and carvedilol attenuated the development of pacing-induced systolic dysfunction significantly and with equal effectiveness. Both agents also inhibited the development of cardiac apoptosis and fibrosis and reduced the Na+-Ca2+exchanger-1 protein level in the heart. Importantly, carvedilol significantly enlarged the left ventricle and atrium; vidarabine, in contrast, did not. Vidarabine-treated dogs maintained cardiac response to β-AR stimulation better than carvedilol-treated dogs did. Conclusions Vidarabine may protect against pacing-induced DCM with less suppression of basal cardiac function than carvedilol in a dog model. (Circ J 2016; 80: 2496-2505).

Principal mechanisms of doxorubicin-induced cardiotoxicity were considered as oxidative stress and apoptosis in cardiomyocytes. However the effect of doxorubicin on cardiac fibroblasts remains to be developed from the viewpoint of their function. The aim of this study is to investigate the direct

Pro-inflammatory cytokines are released in septic shock and impair cardiac function via the Jak-STAT pathway. It is well known that sympathetic and thus catecholamine signaling is activated thereafter to compensate for cardiac dysfunction. The mechanism of such compensation by catecholamine signaling has been traditionally understood to be cyclic AMP-dependent protein kinase (PKA)-mediated enforcement of cardiac contractility. We hypothesized that the exchange protein activated by cAMP (Epac), a newly identified target of cAMP signaling that functions independently of PKA, also plays a key role in this mechanism. In cultured cardiac myocytes, activation of Epac attenuated the inhibitory effect of interleukin-6 on the increase of intracellular Ca

The ubiquitin ligase, NEDD4L, participates in plasma volume and blood pressure regulation by controlling cell surface expression of kidney epithelial sodium channel (ENaC). Impairment of ENaC internalization with disrupting Nedd4L binding PY motif in the C-terminal of the protein showed rare Mendelian human hypertension disorder, Liddle syndrome. Therefore, possible involvements of ENaC-Nedd4L system abnormalities were suggested in development of human hypertension. Previously, we discovered a common variant, named v13(G/A), that affects the formation of an evolutionally new C2-encoding isoform of the Nedd4L gene in humans. The aim of this study was to test a hypothesis whether this variant is involved in essential hypertension; we performed the association study in a sample of the Japanese population. We found a gender-specific genotypic and allelic association with statistical significance achieved only in males. Our data suggest that this genetic variation may contribute to essential hypertension in Japanese male patients through the mechanism of disrupting the C2 encoding isoform in vivo. Furthermore, molecular and functional studies will be required to elucidate the mechanism by which genetic variation in NEDD4L contributes to the development of essential hypertension.

Hyperthermia is a promising anti-cancer treatment in which the tissue temperature is increased to 42-45 °C, and which is often used in combination with chemotherapy or radiation therapy. Our aim in the present work was to examine the feasibility of combination therapy for oral cancer with cisplatin and hyperthermia generated with ferucarbotran (Resovist(®); superparamagnetic iron oxide) in an alternating magnetic field (AMF). First, we established that administration of ferucarbotran at the approved dosage for magnetic resonance imaging provides an iron concentration sufficient to increase the temperature to 42.5 °C upon exposure to AMF. Then, we examined the effect of cisplatin combined with ferucarbotran/AMF-induced hyperthermia on cultured human oral cancer cells (HSC-3 and OSC-19). Cisplatin alone induced apoptosis of cancer cells in a dose-dependent manner, as is well known. However, the combination of cisplatin with ferucarbotran/AMF was significantly more effective than cisplatin alone. This result suggests that it might be possible to reduce the clinically effective dosage of cisplatin by administering it in combination with ferucarbotran/AMF-induced hyperthermia, thereby potentially reducing the incidence of serious cisplatin-related side effects. Further work seems justified to evaluate simultaneous thermo-chemotherapy as a new approach to anticancer therapy.

Multi-Detector Computed Tomography (MDCT) has been used to detect coronary lesions in a number of earlier studies. Here, we describe a rare case of congenital anomaly of the coronary artery, i.e., the R-II-B subtype (Lipton classification) of single coronary artery (SCA). A 61 year old man with angina was referred to our hospital. Assessment by coronary angiography (CAG) suggested SCA. The R-II-B subtype was confirmed by MDCT. Coronary artery bypass grafting (CABG) was then performed, and success of the grafting was evaluated by post- CABG MDCT. This subtype of SCA has been reported to be associated with high risk of cardiac sudden death because the aberrant vessel, which passes between the aorta and the main pulmonary artery, is readily compressed by these arteries. This subtype of SCA is also very rare: there is only one previously reported case in which MDCT was used for diagnosis. Ours is the first case of this subtype in which MDCT together with CAG was used for evaluation both pre- and post-CABG. Our results show that MDCT may be useful not only prior to CABG for risk stratification in patients with anomalous coronary arteries but also after CABG for evaluation.

There are few reports of acute myocardial infarction (AMI) relating to the occlusion of the conus branch, most of which are iatrogenic in nature. So far as we are concerned, this is the first case of spontaneous AMI with isolated conus branch occlusion. Electrocardiogram (ECG) showed mild elevation of ST segment in leads V(1) through V(3). Cardiac makers of myocardial infarction were positive. Right coronary angiography revealed an isolated occlusion of the conus branch. Penetration of the guidewire in the occluded lesion was attempted, and recanalization was successfully achieved. The patient was discharged without any adverse events.

Summary Background We report a rare case of acute pulmonary embolism (PE) induced by urinary retention and bladder distention with benign prostatic hyperplasia (BPH). Case report A 76-year-old male with BPH presented to the hospital with anuria of 24 h duration and abdominal distention. Physical examination revealed tenderness and distention of the lower abdomen and a swollen right leg. Echocardiography after urethral catheterization showed a large free-floating thrombus traversing back and forth through the tricuspid orifice. Computed tomographic angiography demonstrated filling defects at the level of the right inter lobar pulmonary artery and the segmental branches of both pulmonary arteries, indicating acute PE. The patient was treated with heparin and warfarin for three weeks to ensure the resolution of the pulmonary embolus. After the resolution of all symptoms, the patient was discharged without further complication. Conclusion This case suggested that a distended bladder is a potential risk factor for the development of deep vein thrombosis and PE.

Background: Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumor in humans. In spite of advances in treatment strategies, survival rate of GBM remains poor. We have recently reported a novel anti-cancer compound with intrinsic magnetism(EI236). In addition to its anti-cancer effect, EI236 generates a large amount of heat upon an alternating magnetic field (AMF) application because of its magnetism, i.e., hyperthermic effect. In this study, we have examined the effect of EI236 on human GBM in vitroand vivo. Materials and Methods: Human GBM cell lines, U251MG, YKG-1, and U87, were used in this study. Cell proliferation was assessed by methyl thiazolyl tetrazorium (MTT) assays in the presence of EI236, in comparison to temozolomide (TMZ) and calmustine (BCNU), i.e. the clinical standard drugs for GBM. Apoptosis was analyzed by fluorescence activated cells sorting. In addition, we have examined whether EI236 inhibited the tumor growth with/without AMF application in animal models. Tumors were induced by implantation of U251MG cells into the hip of female Balb-c mice. Seven days after the implantation of GBM cells, EI236, BCNU or normal saline (control) was injected respectively into the tumors and mice were exposed to an AMF application(330.8A, 280kHz) for 30 minutes. Therefore, we calculated the tumor volume and the regression rate of tumors twice a week. Result: EI236 inhibited the proliferation of all cell lines and increased apoptosis in a dose dependent manner. Moreover, we found that EI236 had much greater anti-cancer effect than the clinical standard drugs. Mice bearing GBM cancer cells in their hip were then subjected to a double combination therapy, consisting of local injection of EI236 and hyperthermic treatment by AMF application. Tumor sizes in both drug stimulation groups were reduced, especially that in EI236 with AMF application group was highly reduced, while that in control group was increased. Conclusion: EI236 has greater anti-cancer effect than TMZ and BCNU, i.e. the clinical standard drugs. Besides this strong anti-cancer effect, EI236 has hyperthermic effect upon an AMF application because of heat generation. Accordingly, EI236 may enable us to develop novel strategies in GBM treatment, i.e. hyperthermic-chemotherapy with a single-drug compound. Note: This abstract was not presented at the meeting. Citation Format: Makoto Ohtake, Masanari Umemura, Itaru Sato, Kayoko Oda, Akane Nagasako, Ayako Makino, Haruki Aoyama, Mayumi Katsumata, Haruki Eguchi, Nobutaka Kawahara, Yoshihiro Ishikawa. Simultaneous hyperthermic-chemotherapy for glioblastoma using a single anti-cancer compound with intrinsic magnetism. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4548. doi:10.1158/1538-7445.AM2015-4548

Cholesterol crystal embolism (CCE) is caused by the shedding of cholesterol crystals into the bloodstream, and it has been recently recognized as a serious complication after vascular procedures. Our case of CCE, which was diagnosed by skin and renal biopsies, occurred in a patient with hypertension and diabetes mellitus, 3 months after coronary angiography, with the development of renal failure and blue toes. After low-density lipoprotein apheresis (LDL-A), the skin lesions, including livedo reticularis and pain from the acrocyanotic toes, dramatically improved, with partial recovery of renal function. Following the administration of low-dose corticosteroid and candesartan--an angiotensin II type 1 receptor antagonist (ARB)--the eosinophilia disappeared and renal function improved gradually with a decrease in urinary protein excretion. Therefore, a combination therapy of LDL-A, low-dose corticosteroid, and an ARB is a possible treatment for CCE, although the possibility of spontaneous recovery of renal function cannot be eliminated for this patient.

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

Background: Hyperthermic therapy is a type of cancer treatment in which body tissue is exposed to high temperature (up to 113°F). Despite of its effectiveness, it has not been widely used. One reason is that it is technically difficult to increase high temperature only in a target cancer site. To address this issue, we have developed a novel nano-magnetic particle, i.e., EI236. EI236 exhibits not only anti-cancer effect, but also ferromagnetic property. Because of its magnetism, this drug can generate heat by itself when it is exposed to an alternating current magnetic field (ACMF). Method: We established a rabbit model of VX2 cells (rabbit osteosarcoma) grafted on legs to examine the anti-cancer and hyperthermal effects of EI236 in vivo. Rabbits were divided into five groups; 1) no treatment group 2) intra-venous (iv.) EI236 injection group 3) intra-arterial (ia.) EI236 group 4) ia. Methotrexate (MTX) injection group, and 5) ia. EI236+ACMF group. The volume of tumor was measured daily, and then the samples were harvested and evaluated histologically by HE, Ki67, and TUNEL staining. Results: The effect of EI236 was similar to that of MTX when EI236 was injected via intra-arterial infusion. Via intra-arterial infusion was more effective than iva intra-venous infusion. When EI236 was injected and the tumor was exposed to ACMF for simultaneous chemotherapeutic and hyperthermal effects, it showed the greatest regression of tumor among all the groups examined. EI236 significantly increased necrosis as determined by HE staining, and further increased it by ACMF. Similarly, EI236 decreased cell proliferation as determined by Ki67, and further decreased it by ACMF. Moreover EI236 increased apoptosis as determined by TUNEL staining, and further increased it by ACMF. These results demonstrated that the exposure of ACMF greatly enhanced the anti-cancer effect of EI236. Taken together, EI236 exhibited simultaneous effects of anti-cancer and thyperthermia. Conclusion: These results demonstrate that EI236 can provide us in developing a new strategy of simultaneous hyperthermo-chemotherapy in the future. Citation Format: Masanari Umemura, Hidenobu Fukumura, Itaru Sato, Xianfeng Feng, Haruki Eguchi, Yoshihiro Ishikawa. Development of a novel magnetic anti-cancer drug for hyperthermic therapy. [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 5567. doi:10.1158/1538-7445.AM2013-5567

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

Background: Fibroblast growth factor (FGF-2) regulates migration and angiogenesis in cancer. The binding of FGF-2 to its receptor requires N-sulfated glucosamine in heparan sulfate (HS), suggesting that a molecule that increases N-sulfation of glucosamine could enhance FGF-2 signaling. Recently, we demonstrated that Epac1, an exchange protein activated by cAMP, increases expression of N-deacetylase/N-sulfotransferase-1 (NDST-1) that increases N-sulfation of glucosamine. It is plausible that melanoma cells with abundant expression of Epac1 produce N-sulfation-rich glucosamine, which results in enhancement of intercellular FGF-2 signaling in melanoma microenvironment. The purpose of this study was to examine whether Epac1-rich melanoma cells can increase migration of surrounding Epac1-poor melanoma cells and endothelial cells. Methods: Immunohistochemistry was performed on human melanoma tissue microarrays to examine expressions of Epac1, NDST-1 and N-sulfated glucosamine. For in vitro studies, we used 2 human melanoma cell lines, WM1552C (radial growth phase melanoma) and C8161 (metastatic melanoma), and human umbilical vein endothelial cells (HUVEC). Since Epac1 expression was much lower in WM1552C than C8161 cells, WM1552C were used as Epac1-poor, and C8161 as Epac1-rich melanoma cells. We evaluated the migratory responses in WM1552C and HUVEC cells to conditioned medium (CM) from C8161 cells with or without Epac1 ablation (C8161/Epac1(−)) or overexpression (C8161/Epac1OE). Endothelial tube formation assay was performed in HUVEC with conditioned media. Results: The expression of Epac1 positively correlated with that of NDST-1, and N-sulfated glucosamine in human melanoma tissue microarray. Migration of WM1552C and HUVEC cells was increased by CM from C8161 cells, which was significantly enhanced by CM from C8161/Epac1OE, but reduced by CM from C8161/Epac1(−). The CM-induced migration was inhibited by a neutralizing antibody against FGF-2 (nFGF-2 ab) or heparitinase, a HS-degrading enzyme, suggesting involvement of FGF-2 and HS in the CM-induced migration. Binding of FGF-2 to FGF receptors in HUVEC cells was increased by CM from C8161 cells, which was significantly enhanced by CM from C8161/Epac1OE but reduced by CM from C8161/Epac1(−). Similar to migration, the CM-induced FGF-2 binding was inhibited by nFGF-2 ab or heparitinase. Since the expression of FGF-2 in C8161 cells was not changed by ablation of Epac1 or Epac1 overexpression, these data suggested that C8161 cells increase migration of WM1552C and HUVEC cells via modification of HS. Finally, tube formation of HUVEC was increased by CM from C8161 cells, but it was inhibited by Epac1 ablation on these cells. Conclusion: Epac1-rich melanoma cells increase migration of Epac1-poor melanoma cell, and endothelial cells via intercellular FGF-2-signaling, suggesting the role of Epac1 in melanoma progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3664. doi:10.1158/1538-7445.AM2011-3664