Motonari Nomura

Accumulation of cytosolic calcium induces necroptotic cell death in human neuroblastoma

Necrosis has been studied extensively since the early days of medicine, with some patterns of necrosis found to be programmed like apoptotic cell death. However, mechanisms of programmed necrosis (necroptosis) are yet to be fully elucidated. In this study, we investigated how the hemagglutinating virus of Japan-envelope (HVJ-E) induces necrosis in mouse xenografts of human neuroblastoma cells. HVJ-E-induced necrosis in this system was found to depend on phosphorylation of the death receptor kinase receptor interacting protein kinase 1 (RIP1) and on the production of reactive oxygen species. This process was interpreted as necroptosis, based on its suppression by the small molecule necrostatin-1, and it did not involve the TNF-α receptor pathway. We also demonstrated that increased concentrations of cytoplasmic calcium triggered necroptosis by activating calcium-calmodulin kinase (CaMK) II. Finally, we determined that RIP1 phosphorylation was mediated by CaMK II activation. Together, our results define an upstream pathway for the activation of necroptosis in neuroblastoma cells, with potential therapeutic implications.

TRAIL and Noxa are selectively upregulated in prostate cancer cells downstream of the RIG-I/MAVS signaling pathway by nonreplicating Sendai virus particles.

Purpose: The treatment of cancer with oncolytic viruses primarily depends on the selective viral replication in cancer cells. However, a replication-incompetent hemagglutinating virus of Japan (HVJ; Sendai virus) envelope (HVJ-E) suppresses the growth of human cancer cells as effectively as replication-competent live HVJ without producing toxic effects in nonmalignant cells. Here, we analyze the molecular mechanism of the oncolytic activity of HVJ-E. Experimental Design: The molecules responsible for HVJ-E–induced cancer cell death were elucidated in prostate cancer cell lines, and the effect of HVJ-E on orthotopic prostate cancers was evaluated in nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice. Results: The liposome-mediated transfer of viral RNA genome fragments from HVJ-E suppressed the viability of prostate cancer cells but not the viability of the noncancerous prostate epithelium. Knockdown experiments using siRNAs showed that the cancer cell–selective killing induced by HVJ-E was mediated by retinoic acid–inducible gene I (RIG-I) and mitochondrial antiviral signaling protein (MAVS). Downstream of the RIG-I/MAVS pathway, both TNF-related apoptosis-inducing ligand (TRAIL) and Noxa were upregulated by HVJ-E in the castration-resistant prostate cancer cell line PC3 but not in the noncancerous prostate epithelial cell line PNT2. TRAIL siRNA but not Noxa siRNA significantly inhibited HVJ-E–induced cell death in PC3 cells. However, Noxa siRNA effectively suppressed HVJ-E–induced cell death in DU145 cells, another castration-resistant prostate cancer cell line, in which Noxa but not TRAIL was upregulated by HVJ-E. Furthermore, the orthotopic prostate cancers were dramatically eradicated in immunodeficient mice injected with HVJ-E. Conclusion: The RIG-I/MAVS signaling pathway represents an attractive target for cancer therapy. Clin Cancer Res; 18(22); 6271–83. ©2012 AACR.

Influence of thoracoscopic esophageal atresia repair on esophageal motor function and gastroesophageal reflux.

BACKGROUND/PURPOSE Thoracoscopic repair has recently been attempted in newborns with esophageal atresia (EA), but it remains unclear whether thoracoscopic dissection reduces pathological gastroesophageal reflux. We investigated the influence of a thoracoscopic approach on esophageal motor function in patients with EA. METHODS Clinical and gastrointestinal data of 10 patients with EA with open repair (group A) and 7 with thoracoscopic repair (group B) were analyzed retrospectively. Videomanometry was conducted to investigate esophageal motor patterns. Esophageal acid exposure was evaluated with 24-hour esophageal pH monitoring. Data are expressed as medians and ranges. RESULTS Contractions in the distal esophagus were conspicuously absent in 1 and 3 patients in groups A and B, respectively (P = .26). There were no significant differences in esophageal acid exposure (5.5% [0.7%-24.6%] vs 3.7% [0.3%-56.8%]; P = .71) or mean esophageal acid reflux time (0.5 minutes [0.1-1.4 minutes] vs 0.5 minutes [0.1-1.3 minutes]; P = .87) between the 2 groups. Fundoplication was conducted in 2 patients in each group (P = .60), all of whom had conspicuously absent distal esophageal contractions. Those contractions were preserved in the remaining patients with the exception of 1 group B patient. CONCLUSION There are unlikely to be benefits from thoracoscopic repair of EA in terms of postoperative esophageal motor function.

Hedgehog signal inhibitors suppress the invasion of human rhabdomyosarcoma cells

PurposeIn the treatment of rhabdomyosarcoma (RMS), invasion and metastasis remain the most critical determinants of resectability and survival. The objective of this study was to determine whether Hedgehog (Hh) signaling plays a role in the invasion of RMS.MethodsTwo kinds of specific Hh signaling inhibitors, cyclopamine and forskolin, were used to suppress activated Hh signals in three RMS cell lines. The effects of the Hh signaling inhibitors on tumor cell invasion and motility were investigated using Matrigel invasion assays and wound closure assays, respectively.ResultsThe number of invaded cells counted in six random microscopic fields in the Matrigel chambers was significantly decreased by both cyclopamine and forskolin in every RMS cell line. Furthermore, the wound closure assays revealed that a blockade of the Hh signaling pathway by the Hh inhibitors strongly impairs RMS cell motility, as visualized by the delayed closure of the gaps generated in the cultured cell monolayers of the three RMS cell lines.ConclusionsBoth the invasive capacity and motility of RMS cells are significantly suppressed by Hh signaling inhibitors, demonstrating that the Hh pathway plays an important role in the invasion of RMS. Hh inhibitors may provide a new paradigm for the treatment of RMS.

13‐Cis retinoic acid can enhance the antitumor activity of non‐replicating Sendai virus particle against neuroblastoma

Hemagglutinating virus of Japan‐envelope (HVJ‐E) is a drug delivery vector based on inactivated Sendai virus. Recently, antitumor activities were found for HVJ‐E itself and clinical trials of HVJ‐E for some malignant tumors are now ongoing. We investigated the in vitro and in vivo antitumor effects of HVJ‐E against neuroblastoma, which is one of the most common malignant solid tumors in childhood. The sensitivity of human neuroblastoma cell lines to HVJ‐E correlated with the expression level of gangliosides, Sialylparagloboside (SPG) and GD1a, receptors for HVJ. Among the cell lines, SK‐N‐SH was the most sensitive to HVJ‐E in vitro and total SPG and GD1a expression was the highest. Complete eradication of subcutaneous tumors derived from SK‐N‐SH cells was achieved by intratumoral injection of HVJ‐E in SCID mice and no recurrence was observed for more than 300 days after HVJ‐E inoculation. In contrast, NB1 cells expressed the lowest amount of GD1a and SPG and were resistant to HVJ‐E in vitro. The expression of GD1a increased by 13‐cis retinoic acid (13cRA), which is a therapeutic drug for high risk neuroblastoma, thus leading to an improved sensitivity to HVJ‐E in vitro. Only growth inhibition of the subcutaneous tumors derived from NB1 cells was achieved by HVJ‐E in the SCID mice, but the combination of 13cRA and HVJ‐E could achieve partial eradication of the xenograft and also lead to an improved prognosis. In conclusion, HVJ‐E is a promising therapeutic modality for neuroblastoma and 13cRA can be used as an adjuvant to HVJ‐E.

Long-term outcomes of four patients with tracheal agenesis who underwent airway and esophageal reconstruction

PURPOSE The aim of this study was to evaluate the long-term outcomes of four patients with tracheal agenesis who underwent airway and esophageal/alimentary reconstruction. MATERIALS AND METHODS We reviewed the medical records of four long-term survivors of tracheal agenesis and collected the following data: age, sex, type of tracheal agenesis, method of reconstruction, nutritional management, and physical and neurological development. RESULTS The patients consisted of three boys and one girl, who ranged in age from 77 to 109months. The severity of their condition was classified as Floyds type I (n=2), II (n=1), or III (n=1). Mechanical respiratory support was not necessary in any of the cases. Esophageal/alimentary reconstruction was performed using the small intestine (n=2), a gastric tube (n=1), and the esophagus (n=1). The age at esophageal reconstruction ranged from 41 to 55months. All of the cases required enteral nutrition via gastrostomy. Three of the patients were able to swallow a small amount of liquid and one was able to take pureed food orally. The physical development of the subjects was moderately delayed-borderline in childhood. Neurological development was normal in two cases and slightly delayed in two cases. CONCLUSIONS None of the long-term survivors of tracheal agenesis required the use of an artificial respirator, and their development was close to normal. Future studies should aim to elucidate the optimal method for performing esophageal reconstruction to allow tracheal agenesis patients to achieve their full oral intake.

Abstract 4359: TRAIL and Noxa are selectively up-regulated in prostate cancer cells downstream of the RIG-I/MAVS signaling pathway by non-replicating Sendai virus particles.

PURPOSE: The treatment of cancer with oncolytic viruses primarily depends on the selective viral replication in cancer cells. However, we previously reported that a replication-incompetent hemagglutinating virus of Japan (HVJ; Sendai virus) envelope (HVJ-E) suppressed the growth of human cancer cells, including prostate cancer cells, as effectively as replication-competent live HVJ without producing toxic effects in nonmalignant cells. Here, we analyze the molecular mechanism of the oncolytic activity of HVJ-E and also analyze the treatment efficacy of HVJ-E using an orthotopic prostate cancer model. EXPERIMENTAL DESIGN: The molecules responsible for HVJ-E-induced cancer cell death were elucidated in human prostate cancer cell lines, PC3, DU145 and LNCap. The cell viability was examined using MTS assay after HVJ-E treatment or the transfer of viral RNA genome fragments from HVJ-E. The viral RNA genome fragments and siRNAs were transferred to cells using liposome transfection. The expression of TNF-related apoptosis-inducing ligand (TRAIL) and Noxa were examined using Western blot analysis. The orthotopic prostate cancers were established by the inoculation of PC3 cells into the prostate of nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice. After the tumors increased and microscopically visualized, HVJ-E was injected into the prostate of NOD-SCID mice. Tumor sections were prepared, and then, histological analysis and TUNEL assay were performed. RESULTS: The liposome-mediated transfer of viral RNA genome fragments from HVJ-E suppressed the viability of prostate cancer cells, PC3 and LNCaP, but not the viability of the noncancerous prostate epithelium, PNT2. Knockdown experiments using siRNAs showed that the cancer cell-selective killing induced by HVJ-E was mediated by retinoic acid-inducible gene I (RIG-I) and mitochondrial antiviral signaling protein (MAVS). Downstream of the RIG-I/MAVS pathway, both TRAIL and Noxa were up-regulated by either HVJ-E treatment or transfer of viral RNA genome fragments in PC3 cells but not in PNT2 cells. TRAIL siRNA and/or Noxa siRNA significantly inhibited HVJ-E-induced cell death in prostate cancer cells. Furthermore, the orthotopic prostate cancers were dramatically eradicated in NOD-SCID mice injected with HVJ-E. Although tubuloalveolar glands were histologically maintained in the prostate injected with HVJ-E, dramatic increase of TUNEL-positive apoptotic cells were detected exclusively in the tumors treated with HVJ-E. CONCLUSION: The oncolytic activity of HVJ-E was mediated by TRAIL and Noxa, downstream of the RIG-I/MAVS signaling pathway, selectively up-regulated in prostate cancer cells by HVJ-E treatment. The RIG-I/MAVS signaling pathway represents an attractive target for cancer therapy. Citation Format: Koji Hatano, Yasutomo Nakai, Taeko Matsushima-Miyagi, Motonari Nomura, Wataru Nakata, Takahiro Yoshida, Mototaka Sato, Atsunari Kawashima, Akira Nagahara, Kazutoshi Fujita, Motohide Uemura, Hitoshi Takayama, Yasufumi Kaneda, Norio Nonomura. TRAIL and Noxa are selectively up-regulated in prostate cancer cells downstream of the RIG-I/MAVS signaling pathway by non-replicating Sendai virus particles. [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 4359. doi:10.1158/1538-7445.AM2013-4359