Osteosarcoma (OS) was a malignant tumor occurring with unknown etiology that made prevention and early diagnosis difficult. Mesenchymal stem cells (MSCs), which were found in bone marrow, were claimed to be a possible origin of OS but with little direct evidence. We aimed to characterize OS cells transformed from human MSCs (hMSCs) and identify their association with human primary OS cells and patient survival. Genetic modification with p53 or retinoblastoma (Rb) knockdown and c‐Myc or Ras overexpression was applied for hMSC transformation. Transformed cells were assayed for proliferation, differentiation, tumorigenecity, and gene expression profile. Only the combination of Rb knockdown and c‐Myc overexpression successfully transformed hMSCs derived from four individual donors, with increasing cell proliferation, decreasing cell senescence rate, and increasing ability to form colonies and spheres in serum‐free medium. These transformed cells lost the expression of certain surface markers, increased in osteogenic potential, and decreased in adipogenic potential. After injection in immunodeficient mice, these cells formed OS‐like tumors, as evidenced by radiographic analyses and immunohistochemistry of various OS markers. Microarray with cluster analysis revealed that these transformed cells have gene profiles more similar to patient‐derived primary OS cells than their normal MSC counterparts. Most importantly, comparison of OS patient tumor samples revealed that a combination of Rb loss and c‐Myc overexpression correlated with a decrease in patient survival. This study successfully transformed human MSCs to OS‐like cells by Rb knockdown and c‐Myc overexpression that may be a useful platform for further investigation of preventive and target therapy for human OS. Stem Cells Translational Medicine 2017;6:512–526

Cell therapies using human mesenchymal stem cells (MSCs) have received much attention in the past decade. In pursuit of the therapeutic potential of MSCs, cell expansion is required to generate a great number of cells with desired phenotype and functionality. Long‐term expansion in vitro, however, can lead to altered functions. To explore the changes in DNA damage responses (DDR) in MSCs expanded, DDR pathways following irradiation were characterized in early‐ and late‐passage bone marrow MSCs. Seventy‐two hours after irradiation, the percentage of sub‐G1 cells in early‐passage MSCs did not change significantly. Reduced TUNEL staining was observed in early‐passage MSCs compared to late‐passage MSCs 4 h after irradiation. Comet assay also revealed that early‐passage MSCs were more resistant to irradiation or DNA damages induced by genotoxic agents than late‐passage MSCs. ATM phosphorylation and γ‐H2AX and phospho‐p53 increased in early‐passage MSCs while decreased in late‐passage MSCs. Through inhibition by KU55933, DDR pathway in early‐passage MSCs was shown to be ATM‐dependent. Higher levels of poly (ADP‐ribose) polymerase‐1 (PARP‐1) and PAR synthesis were observed in early‐passage MSCs than in late‐passage MSCs. Knockdown of PARP‐1 in early‐passage MSCs resulted in sensitization to irradiation‐induced apoptosis. Overexpression of PARP‐1 in late passage MSCs could render irradiation resistance. Lower activity of DDR in late‐passage MSCs was associated with rapid proteasomal degradation of PARP‐1. In conclusion, early‐passage MSCs are more irradiation‐resistant and have increased DDR activity involving PARP‐1, ATM and their downstream signals. Stem Cells Translational Medicine 2017;6:1504–1514

Abstract. Polo-like kinase 1 (PLK1), a serine/threonine kinase and an oncogene, is crucial in regulating cell cycle progression. PLK1 also has been demonstrated as a potential target of osteosarcoma (OS) by using short hairpin RNA libraries in lentiviral vectors for screening of protein kinase. In preclinical studies, GSK461364, a potent and selective ATP-competitive PLK1 inhibitor, showed antiproliferative activity against multiple tumor cell lines. In the present study, we evaluated the expression level of PLK1 in OS and explored the cytotoxic mechanism of GSK461364 against OS. PLK1 was significantly overexpressed in OS compared with normal osteoblasts and other types of sarcoma. GSK461364 inhibited PLK1 and caused mitotic arrest by inducing G2/M arrest in OS cells. Moreover, GSK461364 exerted a cytotoxic effect by inducing apoptosis in OS, and induced cellular senescence in OS cell lines, as indicated by an increased senescenceassociated β-galactosidase activity and enhanced DcR2 and interleukin-1α expression. In addition, we demonstrated a synergistic cytotoxic effect of GSK461364 and paclitaxel, possibly resulting from combined mitotic arrest. In conclusion, the present study revealed that PLK1 was overexpressed in OS and that GSK461364 exerted its cytotoxic effect on OS by inducing mitotic arrest and subsequent apoptosis and induced cellular senescence; therefore, senescence-associated markers can be used as treatment biomarkers, and a combination of GSK461364 and paclitaxel can potentially treat OS.

Based on the in vivo and in vitro demonstration of herb-drug interference in breast cancer cells, we conclude that physicians should pay more attention to such interference when treating patients with receptor-positive (estrogen receptor-positive, progesterone receptor-positive, or HER2) breast cancers.

Level IV, therapeutic study.

Osteosarcoma (OS) is the most common bone tumor in children and teenagers. The multidrug resistant property of OS produces a major obstacle to chemotherapy, since the effective drug dose cannot be achieved via conventional drug delivery routes without serious systemic cytotoxicity. Microbubbles in conjunction with ultrasound (US) has recently been shown to spatially and temporally permeabilize the cellular membrane, promoting drug penetration into tumors. Here, we investigated whether drug (doxorubicin, DOX)-loaded bubbles (DOX-bubbles) can serve as drug-loaded carriers in combination with US in order to facilitate tumor drug delivery. The proposed bubbles have a high payload capacity (efficiency of 69.4 ± 9.1%, payload of 1.4 mg/mL) for DOX. In vitro data revealed that when used in combination with US (1-MHz), these DOX-bubbles facilitate DOX entering into tumor cells. In tumor-bearing animals, DOX-bubbles + US could provide 3.7-fold suppression of tumor growth compared with the group without insonation (1.8 ± 0.9 cm3 vs. 8.5 ± 2.2 cm3) because of the acceleration of DOX-induced tumor necrosis. In the meantime, the tumor perfusion and volume can be monitored by DOX-bubbles with contrast-enhanced ultrasound imaging. Our data provide useful information in support of translating the use of theranostic US-responsive bubbles for regulated tumor drug delivery into clinical use.

IntroductionBreast cancer is the most frequently diagnosed cancer among women internationally and is the second highest cause of cancer-related death. The incidence of breast cancer varies hugely around the world, both in developed and developing countries (1). In Thailand, breast cancer is currently the most common cancer and involves 43% of all cancers diagnosed in women (2).Estrogen receptors, when binding with estrogen, have important physiological roles in cardiovascular protection, the humoral immune response, neuroprotection, and bone remodeling. In breast cancer cells, estrogen and/or progesterone receptor status is highly linked to the patients' prognosis after surgery (3). Although many selective estrogen receptor modulators have been studied (4, 5), at present, hormonal therapy with tamoxifen is considered a gold standard when preventing tumor recurrence in women with hormone-responsive breast cancer. Tamoxifen, a non-steroidal selective estrogen receptor modulator, is commonly used for the hormone therapy of receptors-positive breast cancers in pre- Chang-Gung Memorial Hospital, Taoyuan, 333, Taiwan, ROC Received February 2, 2014; Accepted May 8, 2014 Abstract. This study investigated the herb-drug interaction of xanthorrhizol and tamoxifen in human breast cancer cells. Using MCF-7 cell line as an in vitro model, the herb-drug interaction between xanthorrhizol and tamoxifen was measured by MTT assay, luciferase reporter assay, and cell cycle analysis. The effects of xanthorrhizol on growth/autophagy related signaling were determined by immunostaining, western blotting, and real time RT-PCR. Additionally, the in vivo effect of xanthorrhizol and tamoxifen on athymic nude mice implanted with MCF-7 cells was evaluated. When MCF-7 cells were co-treated with tamoxifen and xanthorrhizol, there were no significant changes in terms of cell number, luciferase activity, percentage S-phase cells and LC3-II expression. However, using the MCF-7 implanted nude mice model, it was possible to detect significantly increased tumor volumes, a larger tumor size, and increased protein expression of P38 and P27(Kip1) in the xanthorrhizol + tamoxifen group compared to the tamoxifen-alone group. It can be concluded that while there is no significant herb-drug interaction between xanthorrhizol and tamoxifen in vitro, there is such an interaction in tumor-bearing mice, which provides important information that affects breast cancer treatment translational research. In Vitro and In Vivo Effects of Xanthorrhizol on Human Breast Cancer MCF-7 Cells Treated With Tamoxifen

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