Abstract A23: Pterostilbene (3',5'-dimethoxyresveratrol) inhibits cell growth and augments the efficacy of doxorubicin through stem cell phenotype suppression in osteosarcoma cells in vitro

Objective: Osteosarcoma (OS) is the most common primary malignant neoplasm of bone and mainly affects adolescents and young adults. Intensive multiagent chemotherapy and definitive surgery have dramatically improved its prognosis as well as functional outcome of affected limbs since the 1980s. However, survival rate of patients has remained around 70% for more than 30 years, and the prognosis remains especially poor in patients with metastatic disease or unresectable lesions. Thus, additional or alternative treatment strategy has long been desired for patients with such poor prognosis. Pterostilbene (PTE), a methyl ether of resveratrol derived from plants like grapes and blueberries, has recently attracted attention as a growing number of reports describe promising pharmacologic properties of antioxidative and antiaging effects and have shown its inhibitory effects against cancers including colon, oral cancer, and malignant lymphoma. However, there are very scarce reports about the effects of PTE against OS. Here, we show a very characteristic efficacy of PTE on human OS cells and propose its potential as a novel therapeutic intervention against OS. Materials and Methods: Effects of PTE on cellular growth were evaluated in human OS cell lines SaOS2, U2OS, and MG63 treated at concentrations ranging from 2 μM to10 μM (2, 5, 10 μM) for 48 hrs compared to nontreated cells by MTS assay. Additionally, we evaluated the effects of cotreatment of PTE with low-dose (0.2 μg/ml) of doxorubicin (DXR) against SaOS2 and U2OS. To investigate the efficacy of PTE on stemness, ability of spheroid formation in anchorage-independent 3D culture condition was evaluated in SaOS2 and U2OS cells treated by 10 μM PTE for 48 hrs and mRNA expression of stemness markers of Oct3 and CD44 was evaluated by RT-PCR at the same condition. Results: Treatment of PTE showed dose-dependent growth inhibitory effects on all cell lines and was especially significant on MG63 cells at the highest inhibition rate of 65% at the concentration of 10 μM for 48 hrs treatment. In contrast, PTE did not show growth inhibition on normal mesenchymal stromal cells, one of the candidates for OS cell of origin. Cotreatment of PTE with DXR indicated that PTE augmented the growth suppressive effects of DXR lifting the growth inhibition rate up to 515% from DXR alone against SaOS2 and U2OS (62-67% and 25-40%, respectively). PTE treatments decreased sphere-forming ability in both SaOS2 and U2OS cells in terms of sphere numbers and sphere sizes, and also decreased mRNA expression of stemness markers of Oct3 and CD44 in both cell lines. Discussion: Current results suggest that PTE not only inhibits cellular growth of OS but also suppresses stem cell phenotype which was defined by sphere-forming ability and mRNA expression of stemness markers. Mechanisms of stem cell phenotype suppression by PTE are still unknown, but it might be caused by either decrease or differentiation of stem cell subpopulation. Suppression of stem cell phenotype could lead to enhancing the efficacy of cytotoxic chemotherapeutic agents such as DXR. Previous reports suggested that antitumor activity of PTE against OS cells targeted the JAK2/STAT3 signaling pathways. The efficacy of PTE is cell-type dependent, possibly due to the mechanisms of cell death or cell metabolism. The molecular pathways involved in growth inhibition and stem cell phenotype suppression caused by PTE are under investigation using pathway inhibitors and metabolism modulators. Conclusion: PTE inhibits cellular growth and augments the efficacy of DXR against OS cells possibly through the suppression of stem cell characteristics represented by sphere-forming ability and mRNA expression of stemness markers. The current study suggests that PTE could be a possible candidate for a novel therapeutic intervention against OS. Citation Format: Shingo Kishi, Kanya Honoki, Shinji Tsukamoto, Hiromasa Fujii, Yumiko Kondo, Akira Kido, Toshifumi Tsujiuchi, Hiroki Kuniyasu, Yasuhito Tanaka. Pterostilbene (39,59-dimethoxyresveratrol) inhibits cell growth and augments the efficacy of doxorubicin through stem cell phenotype suppression in osteosarcoma cells in vitro [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A23.