Human Soft Tissue Sarcoma Cell Research Articles

P53-Armed Oncolytic Virotherapy Improves Radiosensitivity in Soft-Tissue Sarcoma by Suppressing BCL-xL Expression.

Soft-tissue sarcoma (STS) is a heterogeneous group of rare tumors originating predominantly from the embryonic mesoderm. Despite the development of combined modalities including radiotherapy, STSs are often refractory to antitumor modalities, and novel strategies that improve the prognosis of STS patients are needed. We previously demonstrated the therapeutic potential of two telomerase-specific replication-competent oncolytic adenoviruses, OBP-301 and tumor suppressor p53-armed OBP-702, in human STS cells. Here, we demonstrate in vitro and in vivo antitumor effects of OBP-702 in combination with ionizing radiation against human STS cells (HT1080, NMS-2, SYO-1). OBP-702 synergistically promoted the antitumor effect of ionizing radiation in the STS cells by suppressing the expression of B-cell lymphoma-X large (BCL-xL) and enhancing ionizing radiation-induced apoptosis. The in vivo experiments demonstrated that this combination therapy significantly suppressed STS tumors' growth. Our results suggest that OBP-702 is a promising antitumor reagent for promoting the radiosensitivity of STS tumors.

X-ray radio-enhancement by Ti3C2Tx MXenes in soft tissue sarcoma.

Radiotherapy is a cornerstone of cancer treatment. However, due to the low tissue specificity of ionizing radiation, damage to the surrounding healthy tissue of the tumor remains a significant challenge. In recent years, radio-enhancers based on inorganic nanomaterials have gained considerable interest. Beyond the widely explored metal and metal oxide nanoparticles, 2D materials, such as MXenes, could present potential benefits because of their inherently large specific surface area. In this study, we highlight the promising radio-enhancement properties of Ti3C2Tx MXenes. We demonstrate that atomically thin layers of titanium carbides (Ti3C2Tx MXenes) are efficiently internalized and well-tolerated by mammalian cells. Contrary to MXenes suspended in aqueous buffers, which fully oxidize within days, yielding rice-grain shaped rutile nanoparticles, the MXenes internalized by cells oxidize at a slower rate. This is consistent with cell-free experiments that have shown slower oxidation rates in cell media and lysosomal buffers compared to dispersants without antioxidants. Importantly, the MXenes exhibit robust radio-enhancement properties, with dose enhancement factors reaching up to 2.5 in human soft tissue sarcoma cells, while showing no toxicity to healthy human fibroblasts. When compared to oxidized MXenes and commercial titanium dioxide nanoparticles, the intact 2D titanium carbide flakes display superior radio-enhancement properties. In summary, our findings offer evidence for the potent radio-enhancement capabilities of Ti3C2Tx MXenes, marking them as a promising candidate for enhancing radiotherapy.

Radiosensitizing Effect of Trabectedin on Human Soft Tissue Sarcoma Cells.

Trabectedin is used for the treatment of advanced soft tissue sarcomas (STSs). In this study, we evaluated if trabectedin could enhance the efficacy of irradiation (IR) by increasing the intrinsic cell radiosensitivity and modulating tumor micro-environment in fibrosarcoma (HS 93.T), leiomyosarcoma (HS5.T), liposarcoma (SW872), and rhabdomyosarcoma (RD) cell lines. A significant reduction in cell surviving fraction (SF) following trabectedin + IR compared to IR alone was observed in liposarcoma and leiomyosarcoma (enhancement ratio at 50%, ER50: 1.45 and 2.35, respectively), whereas an additive effect was shown in rhabdomyosarcoma and fibrosarcoma. Invasive cells' fraction significantly decreased following trabectedin ± IR compared to IR alone. Differences in cell cycle distribution were observed in leiomyosarcoma and rhabdomyosarcoma treated with trabectedin + IR. In all STS lines, trabectedin + IR resulted in a significantly higher number of γ-H2AX (histone H2AX) foci 30 min compared to the control, trabectedin, or IR alone. Expression of ATM, RAD50, Ang-2, VEGF, and PD-L1 was not significantly altered following trabectedin + IR. In conclusion, trabectedin radiosensitizes STS cells by affecting SF (particularly in leiomyosarcoma and liposarcoma), invasiveness, cell cycle distribution, and γ-H2AX foci formation. Conversely, no synergistic effect was observed on DNA damage repair, neoangiogenesis, and immune system.

PO-1921 Radiosensitizing effect of Trabectidin on human soft tissue sarcoma cells

PO-1921 Radiosensitizing effect of Trabectidin on human soft tissue sarcoma cells

Oncolytic virotherapy promotes radiosensitivity in soft tissue sarcoma by suppressing anti-apoptotic MCL1 expression.

Soft tissue sarcoma (STS) is a rare cancer that develops from soft tissues in any part of the body. Despite major advances in the treatment of STS, patients are often refractory to conventional radiotherapy, leading to poor prognosis. Enhancement of sensitivity to radiotherapy would therefore improve the clinical outcome of STS patients. We previously revealed that the tumor-specific, replication-competent oncolytic adenovirus OBP-301 kills human sarcoma cells. In this study, we investigated the radiosensitizing effect of OBP-301 in human STS cells. The in vitro antitumor effect of OBP-301 and ionizing radiation in monotherapy or combination therapy was assessed using highly radiosensitive (RD-ES and SK-ES-1) and moderately radiosensitive (HT1080 and NMS-2) STS cell lines. The expression of markers for apoptosis and DNA damage were evaluated in STS cells after treatment. The therapeutic potential of combination therapy was further analyzed using SK-ES-1 and HT1080 cells in subcutaneous xenograft tumor models. The combination of OBP-301 and ionizing radiation showed a synergistic antitumor effect in all human STS cell lines tested, including those that show different radiosensitivity. OBP-301 was found to enhance irradiation-induced apoptosis and DNA damage via suppression of anti-apoptotic myeloid cell leukemia 1 (MCL1), which was expressed at higher levels in moderately radiosensitive cell lines. The combination of OBP-301 and ionizing radiation showed a more profound antitumor effect compared to monotherapy in SK-ES-1 (highly radiosensitive) and HT1080 (moderately radiosensitive) subcutaneous xenograft tumors. OBP-301 is a promising antitumor reagent to improve the therapeutic potential of radiotherapy by increasing radiation-induced apoptosis in STS.

The Oncolytic Activity of Myxoma Virus against Soft Tissue Sarcoma Is Mediated by the Overexpression of Ribonucleotide Reductase.

Background:Myxoma virus (MYXV) is an oncolytic poxvirus that lacks the gene for 1 of the subunits of ribonucleotide reductase (RR), a crucial DNA synthesis and repair enzyme. The overexpression of RR has been implicated in the invasiveness of several cancers, including soft tissue sarcomas (STS). The purpose of the study was to investigate the oncolytic efficacy of MYXV in STS with different levels of RR expression.Methods:The oncolytic effect of recombinant MYXV was evaluated in 4 human STS cell lines, LS141 (a dedifferentiated liposarcoma), DDLS8817 (a dedifferentiated liposarcoma), RDD2213 (recurrent dedifferentiated liposarcoma), and HSSYII (a synovial sarcoma) using infectivity and cytotoxicity assays. Following the overexpression of RRM2 by cDNA transfection and silencing of RRM2 by siRRM2 in these STS cell lines, the RRM2 expression levels were analyzed by Western blot.Results:We observed a direct correlation between viral oncolysis and RRM2 mRNA levels (R = 0.96) in STS. Higher RRM2 expression was associated with a more robust cell kill. Silencing the RRM2 gene led to significantly greater cell survival (80%) compared with the control group (P = .003), whereas overexpression of the RRM2 increased viral oncolysis by 33% (P < .001).Conclusions:Our results show that the oncolytic effects of MYXV correlate directly with RR expression levels and are enhanced in STS cell lines with naturally occurring or artificially induced high expression levels of RR. Myxoma virus holds promise in the treatment of advanced soft tissue cancer, especially in tumors overexpressing RR.

The application of aluminium phthalocyanine AlPs-4-mediated photodynamic therapy against human soft tissue sarcoma (RMS) cell line

The main challenge in the cancer treatment is the on-target drug delivery to the affected cells. Various therapies have been designed to target the affected cells efficiently but still the success is awaited. An iron and cobalt nanocomposite for the effective drug delivery to target cells was designed. The photodynamic effect of anticancer drugs loaded with iron oxide and cobalt ferrite nanomaterials coated with polyvinyl alcohol (PVA) was studied. The iron oxide nanoparticles (IONPs) and cobalt ferrite (CF) NPs without the loaded drugs were characterized by UV, XRD, FTIR, SEM and EDX techniques. The photodynamic effect of the photosensitizer, doxorubicin, and dacarbazine loaded nanomaterials were screened against human rhabdomyosarcoma (RMS) cells after incubation for 3 h, 24 h, and 48 h using MTT assay. The combination of photodynamic therapy (PDT) with chemo drugs is studied over different doses. When RMS cells were exposed to nanomaterials loaded with chemo drugs and PDT alone, it resulted in less cell killing compared to chemo drugs followed by PDT. These results revealed that in the case of combined treatment (combination therapy) the cell viability decreases as compared to individual treatment (monotherapy). The in vitro studies showed positive results which give a new pathway for the in vivo studies.

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

Abstract 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 2426: Oncopig soft-tissue sarcomas recapitulate key transcriptional features of human sarcomas

Abstract Human soft-tissue sarcomas (STS) are rare, aggressive mesenchymal tumors with a late stage 5-year survival rate (50-60%) that has for decades remained unchanged. Research into STS treatment is hampered by the limited human STS cell line availability and the large number of STS subtypes. Therefore, there is a need to develop STS cell lines and animal models representative of diverse human STS subtypes. Pigs represent ideal human disease models due to their similar size, anatomy, metabolism, genetics, and epigenetics compared to humans. In this regard, porcine cancer models provide the opportunity to produce STS cell lines and in vivo tumors similar to those clinically observed in humans. The Oncopig encodes Cre recombinase inducible porcine transgenes encoding KRASG12D and TP53R167H, allowing the Oncopig to model a number of human sarcomas in an inducible and temporal manner. However, comparative analysis is required to determine to what extend the Oncopig STS model mimics human STS on a molecular level. The purpose of this study was to identify similarities between Oncopig and human STS transcriptional profiles to validate the Oncopig model as a viable model for human STS. Towards this end, Oncopig fibroblasts were isolated from ear notches of 4 Oncopigs and cultured in vitro. Following confirmation of their mesenchymal origin (positive vimentin immunostaining), Oncopig fibroblasts were transformed via Cre recombinase exposure, resulting in the formation of Oncopig STS cell lines. Oncopig STS tumors were produced in vivo through intramuscular injection of adenovirus encoding Cre in 2 Oncopigs (2 sites/Oncopig), resulting in the formation of 4 tumors detectable by 10 days post injection. The mesenchymal origin of the resulting tumors was confirmed through histological characterization. Genome-wide expression of Oncopig STS cell lines and tumors was profiled via RNA-seq. Reproducible Oncopig STS cell line and tumor expression profiles were observed, and Oncopig STS cell lines also displayed high temporal reproducibility. Differential expression analysis was performed by comparing Oncopig STS cell lines and tumors to untransformed fibroblasts and skeletal muscle, respectively. A total of 3,360 and 7,652 differentially expressed genes were identified in the Oncopig STS cell lines and tumors, respectively. Commonly identified alterations in human STS gene expression and pathway regulation were identified in Oncopig STS, including altered TP53 signaling, activation of Wnt signaling, and evidence of epigenetic reprogramming. Furthermore, master regulators of Oncopig STS gene expression were identified, including FOSL1, which was previously identified as a potential therapeutic target for human STS. These results demonstrate the Oncopig STS model’s ability to mimic human STS on a transcriptomic level, making the Oncopig a valuable resource for sarcoma research and cell line development. Citation Format: Kyle M. Schachtschneider, Yingkai Liu, Suvi Makelainen, Ole Madsen, Laurie A. Rund, Martien A. Groenen, Regina M. Schwind, Ron C. Gaba, Schook B. Lawrence. Oncopig soft-tissue sarcomas recapitulate key transcriptional features of human sarcomas [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 2426. doi:10.1158/1538-7445.AM2017-2426

Curcumin and Viscum album Extract Decrease Proliferation and Cell Viability of Soft-Tissue Sarcoma Cells: An In Vitro Analysis of Eight Cell Lines Using Real-Time Monitoring and Colorimetric Assays

ABSTRACTBackground: The cytostatic effects of the polyphenol curcumin and Viscum album extract (VAE) were assessed in soft-tissue sarcoma (STS) cells. Methods: Eight human STS cell lines were used: fibrosarcoma (HT1080), liposarcoma (SW872, T778, MLS-402), synovial sarcoma (SW982, SYO1, 1273), and malignant fibrous histiocytoma (U2197). Primary human fibroblasts served as control cells. Cell proliferation, viability, and cell index (CI) were analyzed by BrdU assay, MTT assay, and real-time cell analysis (RTCA). Results: As indicated by BrdU and MTT, curcumin significantly decreased the cell proliferation of five cell lines (HT1080, SW872, SYO1, 1273, and U2197) and the viability of two cell lines (SW872 and SW982). VAE led to significant decreases of proliferation in eight cell lines (HT1080, SW872, T778, MLS-402, SW982, SYO1, 1293, and U2197) and reduced viability in seven STS lines (HT1080, SW872, T778, MLS-402, SW982, SYO1, and 1273). As indicated by RTCA for 160 h, curcumin decreased the CI of all synovial sarcoma cell lines as well as T778 and HT1080. VAE diminished the CI in most of the synovial sarcoma (SW982, SYO1) and liposarcoma (SW872, T778) cell lines as well as HT1080. Primary fibroblasts were not affected adversely by the two compounds in RTCA. Conclusion: Curcumin and VAE can inhibit the proliferation and viability of STS cells.

Anti‑proliferative activity of epigallocatechin‑3‑gallate and silibinin on soft tissue sarcoma cells.

Disseminated soft tissue sarcomas (STS) present a therapeutic dilemma. The first-line cytostatic doxorubicin demonstrates a response rate of 30% and is not suitable for elderly patients with underlying cardiac disease, due to its cardiotoxicity. Well-tolerated alternative treatment options, particularly in palliative situations, are rare. Therefore, the present study assessed the anti-proliferative effects of the natural compounds epigallocatechin-3-gallate (EGCG), silibinin and noscapine on STS cells. A total of eight different human STS cell lines were used in the study: Fibrosarcoma (HT1080), liposarcoma (SW872, T778 and MLS-402), synovial sarcoma (SW982, SYO1 and 1273) and pleomorphic sarcoma (U2197). Cell proliferation and viability were analysed by 5-bromo-2′-deoxyuridine and MTT assays and real-time cell analysis (RTCA). RTCA indicated that noscapine did not exhibit any inhibitory effects. By contrast, EGCG decreased proliferation and viability of all cell lines except for the 1273 synovial sarcoma cell line. Silibinin exhibited anti-proliferative effects on all synovial sarcoma, liposarcoma and fibrosarcoma cell lines. Liposarcoma cell lines responded particularly well to EGCG while synovial sarcoma cell lines were more sensitive to silibinin. In conclusion, the green tea polyphenol EGCG and the natural flavonoid silibinin from milk thistle suppressed the proliferation and viability of liposarcoma, synovial sarcoma and fibrosarcoma cells. These compounds are therefore potential candidates as mild therapeutic options for patients that are not suitable for doxorubicin-based chemotherapy and require palliative treatment. The findings from the present study provide evidence to support in vivo trials assessing the effect of these natural compounds on solid sarcomas.

Abstract 344: Antimitotic effect and complex of nonmitotic effect on tumor biology of eribulin mesilate in soft tissue sarcoma model

Abstract Background and objectives: Eribulin mesilate (Eribulin), a first-in-class halichondrin B-based microtubule dynamics inhibitor, has been shown to have novel mechanism of actions (reversion of epithelial-mesenchymal transition of tumor cell and promotion of vascular remodeling) beyond its established antimitotic activity in breast cancer models, leading to decrease of tumor metastatic potency. Recently, eribulin showed trends towards greater overall survival compared to Dacarbazine in subjects with leiomyosarcoma and liposarcoma patients in phase 3 trial. The objective of this study was to investigate the effect of eribulin on tumor cell proliferation of soft tissue sarcoma (STS). Furthermore, the effects of eribulin on morphology and gene expression of differentiation markers against STS and tumor blood perfusion in a leiomyosarcoma xenograft model were examined. Methods: Anti-proliferation activity of eribulin was examined in 6 human STS cell lines. Anti-tumor activity of eribulin was examined in xenografts of A-673 (Ewing's sarcoma), SK-LMS-1 (leiomyosarcoma), SW 872 (liposarcoma), and HT-1080 (fibrosarcoma) at several doses. To analyze the effect of eribulin on in vitro morphological change, 3 human STS cell lines were treated with eribulin at 1, 3, and 10 nmol/L for 7 days. Gene expression change was analyzed by microarray and differentiation marker genes in 2 cell lines were confirmed by qPCR analysis. For tumor blood perfusion analysis, eribulin was administered at doses of 0.38, 0.75, and 1.5 mg/kg one shot in SK-LMS-1 xenografts, and tumor blood perfusion was calculated as Hoechst-positive areas in tumor cryosections 5 days after the administration. Results: Eribulin showed anti-proliferation activity in vitro against all 6 cell lines in a dose dependent manner with 50% inhibitory concentration values of around 1 nmol/L. Eribulin showed significant antitumor activity against 4 xenografts in a dose dependent manner. Eribulin treatment for 7 days induced apparent morphological change in 3 cell lines. Of these, expression level of smooth muscle differentiation marker gene CNN1 was 7.1-fold upregulated in SK-UT-1 and adipocyte differentiation marker genes MYLK, C/EBPβ, and KIF23 were 1.9-, 1.4-, and 1.3-fold upregulated in SW 872, respectively, by 10 nmol/L treatment compared to vehicle control. In addition, eribulin significantly enhanced tumor blood perfusion compared to vehicle control 5 days after the administration in SK-LMS-1 xenografts at all doses.Conclusions: Eribulin showed anti-proliferation activity against all of STS cell lines we tested in vitro and in vivo. Furthermore, eribulin might cause differentiation of STS cells and remodeling of tumor vasculature to enhance tumor blood perfusion. These data supported that eribulin showed mitotic effect and non-mitotic effect, which may decrease tumor metastatic potency in STS, as well in breast cancer. Citation Format: Satoshi Kawano, Makoto Asano, Yusuke Adachi, Junji Matsui. Antimitotic effect and complex of nonmitotic effect on tumor biology of eribulin mesilate in soft tissue sarcoma model. [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 344.

Down-Regulation of AKT Signalling by Ursolic Acid Induces Intrinsic Apoptosis and Sensitization to Doxorubicin in Soft Tissue Sarcoma.

Several important biological activities have been attributed to the pentacyclic triterpene ursolic acid (UA), being its antitumoral effect extensively studied in human adenocarcinomas. In this work, we focused on the efficacy and molecular mechanisms involved in the antitumoral effects of UA, as single agent or combined with doxorubicin (DXR), in human soft tissue sarcoma cells. UA (5–50 μM) strongly inhibited (up to 80%) the viability of STS cells at 24 h and its proliferation in soft agar, with higher concentrations increasing apoptotic death up to 30%. UA treatment (6–9 h) strongly blocked the survival AKT/GSK3β/β-catenin signalling pathway, which led to a concomitant reduction of the anti-apoptotic proteins c-Myc and p21, altogether resulting in the activation of intrinsic apoptosis. Interestingly, UA at low concentrations (10–15 μM) enhanced the antitumoral effects of DXR by up to 2-fold, while in parallel inhibiting DXR-induced AKT activation and p21 expression, two proteins implicated in antitumoral drug resistance and cell survival. In conclusion, UA is able to induce intrinsic apoptosis in human STS cells and also to sensitize these cells to DXR by blocking the AKT signalling pathway. Therefore, UA may have beneficial effects, if used as nutraceutical adjuvant during standard chemotherapy treatment of STS.

NF-κB acts as a molecular link between tumor cells and Th1/Tc1 T cells in the tumor microenvironment to exert radiation-mediated tumor suppression.

Abstract Radiation is a local treatment for many types of solid cancers. About two thirds of cancer patients require radiation during the course of their disease treatment. Radiation modulates both tumor cells and immune cells in the tumor microenvironment to exert anti-tumor activity, but the molecular connection between tumor cells and immune cells that mediates radiation-exerted tumor suppression activity is largely unknown. We report here that radiation induces rapid activation of the p65/p50 and p50/p50 NF-κB complexes in human soft tissue sarcoma (STS) cells. Radiation-activated p65/p50 and p50/p50 bind to the TNFα promoter to activate its transcription in STS cells. Radiation-induced TNFα then induces tumor cell death in an autocrine manner. Smac mimetic BV6 induces cIAP1 and cIAP2 degradation to increase tumor cell sensitivity to radiation-induced cell death in vitro and to enhance radiation-mediated suppression of STS xenografts in vivo. Inhibition of caspases, RIP1, or RIP3 blocks radiation/TNFα-induced cell death, whereas inhibition of RIP1 blocks TNFα-induced caspase activation, suggesting that caspases and RIP1 act sequentially to mediate the non-compensatory cell death pathways. We determined in a syngeneic sarcoma mouse model that radiation up-regulates the NF-κB target genes IRF3, IFNβ, and the T cell chemokines CCL2 and CCL5 in the tumor microenvironment, which is associated with activation and increased infiltration of Th1/Tc1 T cells in the tumor microenvironment. Consequently, combined BV6 and radiation completely suppressed tumor growth in vivo. Radiation-induced NF-κB functions as a molecular link between tumor cells and immune cells in the tumor microenvironment for radiation mediated tumor suppression.

EP-2026: Effect of PARP-1 inhibition on human soft tissue sarcoma cells radiosensitivity

EP-2026: Effect of PARP-1 inhibition on human soft tissue sarcoma cells radiosensitivity

NF-κB functions as a molecular link between tumor cells and Th1/Tc1 T cells in the tumor microenvironment to exert radiation-mediated tumor suppression.

Radiation modulates both tumor cells and immune cells in the tumor microenvironment to exert its anti-tumor activity; however, the molecular connection between tumor cells and immune cells that mediates radiation-exerted tumor suppression activity in the tumor microenvironment is largely unknown. We report here that radiation induces rapid activation of the p65/p50 and p50/p50 NF-κB complexes in human soft tissue sarcoma (STS) cells. Radiation-activated p65/p50 and p50/p50 bind to the TNFα promoter to activate its transcription in STS cells. Radiation-induced TNFα induces tumor cell death in an autocrine manner. A sublethal dose of Smac mimetic BV6 induces cIAP1 and cIAP2 degradation to increase tumor cell sensitivity to radiation-induced cell death in vitro and to enhance radiation-mediated suppression of STS xenografts in vivo. Inhibition of caspases, RIP1, or RIP3 blocks radiation/TNFα-induced cell death, whereas inhibition of RIP1 blocks TNFα-induced caspase activation, suggesting that caspases and RIP1 act sequentially to mediate the non-compensatory cell death pathways. Furthermore, we determined in a syngeneic sarcoma mouse model that radiation up-regulates IRF3, IFNβ, and the T cell chemokines CCL2 and CCL5 in the tumor microenvironment, which are associated with activation and increased infiltration of Th1/Tc1 T cells in the tumor microenvironment. Moreover, tumor-infiltrating T cells are in their active form since both the perforin and FasL pathways are activated in irradiated tumor tissues. Consequently, combined BV6 and radiation completely suppressed tumor growth in vivo. Therefore, radiation-induced NF-κB functions as a molecular link between tumor cells and immune cells in the tumor microenvironment for radiation-mediated tumor suppression.

Effects of 25-O-acetyl-23,24-dihydro-cucurbitacin F on cell viability, cell cycle distribution and apoptosis induction in human soft tissue sarcoma cells

Effects of 25-O-acetyl-23,24-dihydro-cucurbitacin F on cell viability, cell cycle distribution and apoptosis induction in human soft tissue sarcoma cells

624 Inhibition of poly(ADP-ribose) polymerase-1 radio-sensitizes human soft tissue sarcoma cells

624 Inhibition of poly(ADP-ribose) polymerase-1 radio-sensitizes human soft tissue sarcoma cells

25-O-acetyl-23,24-dihydro-cucurbitacin F induces cell cycle G2/M arrest and apoptosis in human soft tissue sarcoma cells

Ethnopharmacological relevanceQuisqualis indica is used in traditional Chinese medicine to treat cancer and related syndromes and also known for its anthelminthic effects. Aim of the studySoft tissue sarcomas represent a rare group of malignant tumors that frequently exhibit chemotherapeutic resistance and increased metastatic potential. In this study, we evaluated the cytotoxic, apoptosis inducing and cell cycle arresting effects of 25-O-acetyl-23,24-dihydro-cucurbitacin F which has been isolated from leaves and twigs of Q. indica. Material and methodsThe present study investigates the effects of 25-O-acetyl-23,24-dihydro-cucurbitacin F (1) on cell viability, cell cycle distribution, and apoptotic induction of three human sarcoma cell lines of various origins by using the CellTiter 96® AQueous One Solution Cell Proliferation Assay, flow cytometrical experiments, real-time RT-PCR, Western blotting, and the Caspase-Glo® 3/7 Assay ResultsWe could show that 1 reduced cell viability in a dose-dependent manner and arrested the cells at the G2/M interface. The accumulation of cells at the G2/M phase resulted in a significant decrease of the cell cycle checkpoint regulators cyclin B1, cyclin A, CDK1, and CDK2. Interestingly, 1 inhibited survivin expression significantly, which functions as a key regulator of mitosis and programmed cell death, and is overexpressed in many tumor types including sarcomas. Moreover, 1 induced apoptosis in liposarcoma and rhabdomyosarcoma cells caspase-3 dependently. ConclusionOur data strongly support 1 as a very interesting target for further investigation and development of novel therapeutics in sarcoma research.

Abstract 725: Combination therapy of telomerase-specific oncolytic adenovirus and zoledronic acid in human osteosarcoma cells

Abstract Osteosarcoma is one of the primary malignant bone tumors that often occur around the ages of 15 to 20. Despite advances in combined chemotherapy and surgical resection, 30% of osteosarcoma patients still die from tumor or metastasis. Therefore, new treatment strategy is being required. We previously reported that telomerase-specific, replication-competent oncolytic adenovirus (Telomelysin, OBP-301), showed the in vitro and in vivo antitumor effects in human bone and soft tissue sarcoma cells; however OBP-301 could not efficiently prevent bone destruction in an orthotopic xenograft tumor model. Recently, third-generation bisphosphonates, zoledronic acid (ZOL), is widely used to inhibit bone destruction in metastatic bone tumors in the clinical settings. Moreover, ZOL has been shown to induce the antitumor effect synergistically in combination with chemotherapeutic agents in human sarcoma cells. In this study, we investigated the in vitro and in vivo antitumor effects of combination therapy with ZOL and OBP-301 in human osteosarcoma cells. Four human osteosarcoma cell lines, HOS, SaOS-2, U2OS and MNNG/HOS were used. We used XTT assay to examine the antitumor effect of ZOL and OBP-301 individually and combinatory on days 2,3,5. ZOL was treated at concentration of 0 to 10µM, and OBP-301 was infected at multiplicity of infections (MOI) of 0 to 100 plaque forming units (PFU)/cell. Combination index was calculated with the CalcuSyn software (BioSoft) and combined antitumor effect between ZOL and OBP-301 was analyzed. XTT assay revealed that combination treatment of ZOL and OBP-301 showed synergistic and additive effects. To analyze the molecular mechanism in the synergistic effect of ZOL and OBP-301, western blot analysis for apoptosis (PARP) and autophagy (LC3, p62) was performed. Treatment with ZOL induced apoptosis, which was confirmed by the cleavage of PARP, and OBP-301infection induced both apoptosis and autophagy, which is confirmed by conversion of LC3- I to LC3- II and p62 downregulation. Combination treatment showed increased apoptotic cell death in all human osteosarcoma cells. Moreover, in vivo antitumor effect of combination therapy was investigated in an orthotopic xenograft tumor model, in which MNNG/HOS cells (2×106 cells per site) were inoculated into the left tibias of female athymic nude mice. ZOL and OBP-301 were injected subcutaneously and intratumorally, respectively, for three times every week. Tumor volume was measured once a week and bone destruction status was analyzed using 3D-CT imaging system at final assessment. Combination treatment showed increased antitumor effect and efficiently prevented bone destruction compared to monotherapy in an orthotopic xenograft MNNG/HOS tumor model. These results suggest that combination treatment of ZOL and OBP-301 is a promising antitumor strategy for improvement of clinical outcome in patients with osteosarcomas. Citation Format: Yasuaki Yamakawa, Joe Hasei, Hiroshi Tazawa, Toshinori Omori, Shuhei Osaki, Tsuyoshi Sasaki, Aki Yoshida, Toshiyuki Kunisada, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Combination therapy of telomerase-specific oncolytic adenovirus and zoledronic acid in human osteosarcoma cells. [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 725. doi:10.1158/1538-7445.AM2014-725