Yasuaki Yamakawa

Dual Programmed Cell Death Pathways Induced by p53 Transactivation Overcome Resistance to Oncolytic Adenovirus in Human Osteosarcoma Cells

Tumor suppressor p53 is a multifunctional transcription factor that regulates diverse cell fates, including apoptosis and autophagy in tumor biology. p53 overexpression enhances the antitumor activity of oncolytic adenoviruses; however, the molecular mechanism of this occurrence remains unclear. We previously developed a tumor-specific replication-competent oncolytic adenovirus, OBP-301, that kills human osteosarcoma cells, but some human osteosarcoma cells were OBP-301–resistant. In this study, we investigated the antitumor activity of a p53-expressing oncolytic adenovirus, OBP-702, and the molecular mechanism of the p53-mediated cell death pathway in OBP-301–resistant human osteosarcoma cells. The cytopathic activity of OBP-702 was examined in OBP-301–sensitive (U2OS and HOS) and OBP-301–resistant (SaOS-2 and MNNG/HOS) human osteosarcoma cells. The molecular mechanism in the OBP-702–mediated induction of two cell death pathways, apoptosis and autophagy, was investigated in OBP-301–resistant osteosarcoma cells. The antitumor effect of OBP-702 was further assessed using an orthotopic OBP-301–resistant MNNG/HOS osteosarcoma xenograft tumor model. OBP-702 suppressed the viability of OBP-301–sensitive and -resistant osteosarcoma cells more efficiently than OBP-301 or a replication-deficient p53-expressing adenovirus (Ad-p53). OBP-702 induced more profound apoptosis and autophagy when compared with OBP-301 or Ad-p53. E1A-mediated miR-93/106b upregulation induced p21 suppression, leading to p53-mediated apoptosis and autophagy in OBP-702–infected cells. p53 overexpression enhanced adenovirus-mediated autophagy through activation of damage-regulated autophagy modulator (DRAM). Moreover, OBP-702 suppressed tumor growth in an orthotopic OBP-301–resistant MNNG/HOS xenograft tumor model. These results suggest that OBP-702–mediated p53 transactivation is a promising antitumor strategy to induce dual apoptotic and autophagic cell death pathways via regulation of miRNA and DRAM in human osteosarcoma cells. Mol Cancer Ther; 12(3); 314–25. ©2012 AACR.

Ablation of MCL1 expression by virally induced microRNA-29 reverses chemoresistance in human osteosarcomas

Osteosarcoma is a rare disease diagnosed as malignant bone tumor. It is generally refractory to chemotherapy, which contributes to its poor prognosis. The reversal of chemoresistance is a major clinical challenge to improve the prognostic outcome of osteosarcoma patients. We developed a tumor-specific replication-competent oncolytic adenovirus, OBP-301 (telomelysin) and assessed its synergistic effects with chemotherapeutic agents (cisplatin and doxorubicin) using human osteosarcoma cell lines and a xenograft tumor model. The molecular mechanism underlying the chemosensitizing effect of OBP-301 was evaluated in aspects of apoptosis induction. OBP-301 inhibits anti-apoptotic myeloid cell leukemia 1 (MCL1) expression, which in turn leads to chemosensitization in human osteosarcoma cells. The siRNA-mediated knockdown of MCL1 expression sensitized human osteosarcoma cells to common chemotherapeutic agents. We also found that upregulation of microRNA-29 targeting MCL1 via virally induced transcriptional factor E2F-1 activation was critical for the enhancement of chemotherapy-induced apoptosis in osteosarcoma cells. Telomerase-specific oncolytic adenovirus synergistically suppressed the viability of human osteosarcoma cells in combination with chemotherapeutic agents. The combination treatment also significantly inhibited tumor growth, as compared to monotherapy, in an osteosarcoma xenograft tumor model. Our data suggest that replicative virus-mediated tumor-specific MCL1 ablation may be a promising strategy to attenuate chemoresistance in osteosarcoma patients.

Favorable outcome after complete resection in elderly soft tissue sarcoma patients: Japanese Musculoskeletal Oncology Group study.

BACKGROUND The surgical management of soft tissue sarcoma (STS) in elderly patients has only been addressed in a few studies. The objective of the current study was to assess surgical outcomes in patients with STS aged 70 years and older and the association of older age with the survival after complete resection. METHODS A retrospective analysis was conducted in 158 elderly patients with localized STS who visited 11 institutions participating in Japanese Musculoskeletal Oncology Group between 1995 and 2006 and were treated by surgical resection. Univariate and multivariate analyses were performed to identify prognostic factors. RESULTS Median follow-up period was 38 months. Histologically high-grade tumors were detected in 71% of the patients. Wide resection with adequate margins was performed in 66% of the cases. Systemic chemotherapy was performed in only 5 patients. Univariate analysis identified histological grade and gender as statistically significant prognostic factors for sarcoma-specific survival. Multivariate analysis did not identify significant prognostic factors for sarcoma-specific survival, although high grade sarcoma emerged as a potentially significant prognostic factor (P = 0.050). Local recurrence was detected in 19% of the patients. Multivariate analysis of local recurrence-free survival showed that tumor site and surgical margins were statistically significant prognostic factors. CONCLUSIONS Older age was not identified as a prognostic factor for sarcoma-specific survival, which is not consistent with the findings of previous studies showing that older age was associated with decreased sarcoma-specific survival. Complete resection should be indicated and can lead to optimal treatment outcome for properly selected elderly patients.

Role of zoledronic acid in oncolytic virotherapy: promotion of antitumor effect and prevention of bone destruction

Osteosarcoma is an aggressive malignant bone tumor that causes bone destruction. Although tumor‐specific replicating oncolytic adenovirus OBP‐301 induces an antitumor effect in an osteosarcoma tumor, it cannot prevent bone destruction. Zoledronic acid (ZOL) is a clinically available agent that inhibits bone destruction. In this study, we investigated the potential of combination therapy with OBP‐301 and ZOL against osteosarcomas with bone destruction. The antitumor activity of OBP‐301 and ZOL in monotherapy or combination therapy was assessed using three human osteosarcoma cell lines (143B, MNNG/HOS, SaOS‐2). The cytotoxic effect of OBP‐301 and/or ZOL was measured by assay of cell apoptosis. The effect of OBP‐301 and ZOL on osteoclast activation was investigated. The potential of combination therapy against tumor growth and bone destruction was analyzed using an orthotopic 143B osteosarcoma xenograft tumor model. OBP‐301 and ZOL decreased the viability of human osteosarcoma cells. Combination therapy with OBP‐301 and ZOL displayed a synergistic antitumor effect, in which OBP‐301 promoted apoptosis through suppression of anti‐apoptotic myeloid cell leukemia 1 (MCL1). Combination therapy significantly inhibited tumor‐mediated osteoclast activation, tumor growth and bone destruction compared to monotherapy. These results suggest that combination therapy of OBP‐301 and ZOL suppresses osteosarcoma progression via suppression of MCL1 and osteoclast activation.

Venous thromboembolism in major trauma patients: a single-center retrospective cohort study of the epidemiology and utility of D-dimer for screening

Venous thromboembolism (VTE) can be a life‐threatening complication after major trauma. The aim of this study was to investigate the epidemiology of VTE and to assess the usefulness of D‐dimer for screening for VTE in major trauma cases among the Japanese population.

Intraocular Silicone Oil Masquerading as Eye Hemorrhage

An 87-year-old man presented to the emergency department complaining of a frontal head injury. Brain computed tomography scan revealed hyperdensity in the vitreous cavity of the right eye, suggesting intraocular hemorrhage. The patient’s family reported that he had a history of retinal detachment, which was presumably treated by tamponade with silicone oil. Eye examination by an ophthalmologist indicated no vitreous or retinal hemorrhage. Since intraocular silicone oil is being increasingly used for treatment of retinal detachment, emergency physicians must become familiar with its unique characteristic of mimicking hemorrhage in medical imaging.

A case of traumatic cardiopulmonary arrest with good neurological outcome predicted by amplitude-integrated electroencephalogram

Highlights • Traumatic cardiopulmonary arrest requires unique therapeutic approaches, although angiography and percutaneous coronary intervention are often required in other forms of arythmogenic cardiopulmonary arrest.• Amplitude-integrated electroencephalogram was useful for traumatic cardiopulmonary arrest to predict the neurological outcome in the early stage of clinical course.• Amplitude-integrated electroencephalogram may help clinicians to start invasive and intensive treatments in a case of traumatic cardiopulmonary arrest.

Abstract 5188: Molecular radiosensitization in soft tissue sarcomas by telomerase-specific oncolytic adenovirus

INTRODUCTION:Treatment options for soft tissue sarcoma (STS) include surgical resection and adjuvant chemotherapy and radiotherapy. Despite the development of combined modality treatments in recent years, a significant proportion of patients with sarcomas respond poorly to adjuvant therapy, leading to local recurrence or distant metastasis. Therefore, there is an urgent need to develop novel therapeutic strategies for improvement of patient prognoses. We previously reported the therapeutic potential of OBP-301, a telomerase-specific oncolytic adenoviruses, and OBP-702, an armed OBP-301 expressing the wild-type p53 tumor suppressor gene against bone and soft tissue sarcoma cells. Furthermore, the synergistic anti-tumor effect of OBP-301 in combination with radiation has been observed. While OBP-702 suppressed the viability of OBP-301-sensitive and -resistant osteosarcoma cells more efficiently, the combination effect of OBP-702 and radiotherapy has been unknown. In this study, we investigated the radiosensitizing effect of OBP-702 against human STS cells. METHODS:We used four human STS cell lines, HT1080 (fibrosarcoma), NMS-2 (malignant peripheral nerve sheath tumor), and SYO-1 (synovial sarcoma). Cells were irradiated 24 h after infection with OBP-301 and OBP-702, and cell viability was assessed by XTT assay 4 days after irradiation. Combined effect of radiation with OBP-301 and OBP-702 was analyzed with the CalcuSyn software (BioSoft). These cells were also analyzed for apoptosis and DNA damage using Western blot analysis. RESULTS:While OBP-301 and OBP-702 showed anti-tumor effect for STS cell lines respectively, HT1080 and NMS-2 were highly resistant to radiation. When combined with radiation, not only OBP-301 but also OBP-702 enhanced the inhibitory anti-tumor effect in all STS cell lines. The calculation of combination index demonstrated additive or synergistic anti-tumor effect in combination therapy. Further analysis revealed that OBP-301 and OBP-702 increased radiation-induced apoptosis in STS cells. Notably, the radio-sensitizing effect of OBP-702 was associated with increase of p53 expression and interruption of anti-apoptotic myeloid cell leukemia 1 (MCL1) expression. DISCUSSION:Our study demonstrated that OBP-702 had much stronger anti-tumor effect compared to OBP-301, and sensitized radiotherapy to various types of osteosarcoma cell lines. Currently, the clinical trial of OBP-301 is performed, and preclinically OBP-702 accumulates good therapeutic results in various tumors. Thus, OBP-702 may provide a novel treatment strategy for STSs and wide application of radiotherapy for localized as well as advanced STSs. Citation Format: Tadashi Komatsubara, Toshinori Omori, Hiroshi Tazawa, Kazuhisa Sugiu, Yusuke Mochizuki, Yasuaki Yamakawa, Shuhei Osaki, Joe Hasei, Tomohiro Fujiwara, Toshiyuki Kunisada, Yasuo Urata, Tsoshifumi Ozaki, Toshiyoshi Fujiwara. Molecular radiosensitization in soft tissue sarcomas by telomerase-specific oncolytic adenovirus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5188. doi:10.1158/1538-7445.AM2017-5188

Abstract 4677: Tumor suppressor p53 reactivation by oncolytic adenovirus reverses chemoresistance in human osteosarcomas

Background: Osteosarcoma is a primary malignant bone tumor. Despite recent advances in multi-agent chemotherapy and aggressive surgical resection, the poor response to chemotherapy often contributes to poor prognosis in osteosarcoma patients. Therefore, the development of novel strategies for reversing the chemoresistance is a pivotal approach to improve the clinical outcome for osteosarcoma patients. We recently developed a tumor suppressor p53-expressing oncolytic adenovirus, OBP-702, which drives the adenoviral E1 gene under the control of the human telomerase reverse transcriptase promoter for tumor-specific virus replication and induces profound p53 expression for tumor-specific cell death. We recently found that OBP-702 effectively kills human osteosarcoma cells. In this study, we investigated the therapeutic potential of OBP-702 as a chemosensitizing reagent in human osteosarcoma cells with different p53 status. Methods: We used 4 human osteosarcoma cell lines with different p53 status, including U2OS (p53 wild-type), MNNG/HOS (p53 mutant), 143B (p53 mutant), SaOS2 (p53 null). We also used the doxorubicin (DOX)-resistant U2OS cells, which were established by sequential exposure to DOX over 3 months. We performed the XTT assay to examine the antitumor effects of DOX and OBP-702. Combination efficacy between DOX and OBP-702 was assessed by calculating the combination index using CalcuSyn software (BioSoft, Inc.). We further investigated the DOX- and OBP-702-mediated apoptosis in parental and DOX-resistant U2OS cells using Western blot analysis. Results: OBP-702 improved the sensitivity to DOX in a dose-dependent manner in all 4 osteosarcoma cell lines. The calculation of combination index revealed the synergistic effect in all 4 osteosarcoma cell lines. Combination with DOX and OBP-702 induced more profound apoptosis than monotherapy in all 4 osteosarcoma cell lines. Moreover, in DOX-resistant U2OS cells, OBP-702 induced the cytopathic effect as well as parental U2OS cells. Synergistic effect was also observed in DOX-resistant U2OS cells when we treated with DOX and OBP-702. Although DOX-resistant U2OS cells was more resistant to the DOX-mediated apoptosis than parental cells, OBP-702 enhanced the DOX-mediated apoptosis in DOX-resistant U2OS cells as well as parental cells. Conclusions: These results suggest that OBP-702-mediated p53 reactivation reverses the chemoresistance in human osteosarcomas. Citation Format: Kazuhisa Sugiu, Hiroshi Tazawa, Joe Hasei, Shuhei Osaki, Yasuaki Yamakawa, Toshinori Omori, Tadashi Komatsubara, Kouji Uotani, Tomohiro Fujiwara, Toshiyuki Kunisada, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Tumor suppressor p53 reactivation by oncolytic adenovirus reverses chemoresistance in human osteosarcomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4677.

Abstract 1794: Preclinical evaluation of radiotherapy in combination with radio-sensitizing telomerase-specific oncolytic virus for human bone and soft tissue sarcomas

Background: Despite major advances in the treatment of bone and soft tissue sarcomas, including surgery, chemotherapy, and radiation, some sarcoma patients show poor prognosis due to resistance to conventional therapy. Therefore, the development of a novel therapeutic strategy for sarcoma patients is needed. We recently revealed that a telomerase-specific, replication-competent oncolytic adenovirus OBP-301 efficiently killed human sarcoma cells. Moreover, combination therapy with OBP-301 and radiation has been confirmed to show synergistic antitumor effect in epithelial malignant tumor cells; however, its effect in human sarcoma cells remains elusive. In this study, we investigated the antitumor effect of OBP-301 in combination with radiation against human sarcoma cells. Methods: We used five human sarcoma cell lines, SK-ES-1 (Ewing sarcoma), RD-ES (Ewing sarcoma) SYO-1 (synovial sarcoma), U2OS (osteosarcoma) and HOS (osteosarcoma). Cells were irradiated 24 h after infection with OBP-301, and cell viability was assessed by XTT assay 4 days after irradiation. Combined effect of radiation with OBP-301 was analyzed with the CalcuSyn software (BioSoft). These cells were analyzed for apoptosis using western blot analysis. To analyze the effect of OBP-301 in DNA repair process, immunofluorescence staining was performed after treatment of irradiation with or without OBP-301 infection on SK-ES-1 cell. We further investigated the in vivo combined effect of OBP-301 and radiation. The SK-ES-1 tumor-bearing mice were irradiated at a dosage of 1 Gy/tumor once per week for three cycles, and OBP-301 (1 × 108 plaque-forming units per tumor), or PBS was injected into the tumor 3 times per week for three cycles. Results: Combination treatment with OBP-301 and radiation showed synergistic or additive antitumor effect in all human sarcoma cells. Western blot analysis showed that combination treatment increased the expression of cleaved-PARP, and prolonged the level of γH2AX protein than radiation treatment. Combination therapy of OBP-301 with radiation showed antitumor effect more significantly than monotherapy in SK-ES-1 xenograft tumor model. Conclusions: These results suggest that combination therapy of OBP-301 with radiation is a promising antitumor strategy for bone and soft tissue sarcomas. Citation Format: Toshinori Omori, Yasuaki Yamakawa, Joe Hasei, Hiroshi Tazawa, Shuhei Osaki, Tusyoshi Sasaki, Kazuhisa Sugiu, Tomohiro Fujiwara, Toshiyuki Kunisada, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Preclinical evaluation of radiotherapy in combination with radio-sensitizing telomerase-specific oncolytic virus for human bone and soft tissue sarcomas. [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 1794. doi:10.1158/1538-7445.AM2015-1794