Abstract
BackgroundOsteosarcoma is the most common bone tumor and is associated with a poor prognosis. Conventional therapies, surgery and chemotherapy, are still the standard but soon reach their limits. New therapeutic approaches are therefore needed. Conventional aqueous mistletoe extracts from the European mistletoe (Viscum album L.) are used in complementary cancer treatment. These commercial extracts are water-based and do not include water-insoluble compounds such as triterpenic acids. However, both hydrophilic and hydrophobic triterpenic acids possess anti-cancer properties. In this study, a whole mistletoe extract viscumTT re-created by combining an aqueous extract (viscum) and a triterpene extract (TT) was tested for its anti-cancer potential in osteosarcoma.MethodsTwo osteosarcoma cell lines were treated with three different mistletoe extracts viscum, TT and viscumTT to compare their apoptotic potential. For this purpose, annexin/PI staining and caspase-3, −8 and −9 activity were investigated by flow cytometry. To determine the mechanism of action, alterations in expression of inhibitors of apoptosis (IAPs) were detected by western blot. Apoptosis induction by co-treatment of viscum, TT and viscumTT with doxorubicin, etoposide and ifosfamide was examined by flow cytometry.ResultsIn vitro as well as ex vivo, the whole mistletoe extract viscumTT led to strong inhibition of proliferation and synergistic apoptosis induction in osteosarcoma cells. In the investigations of mechanism of action, inhibitors of apoptosis such as XIAP, BIRC5 and CLSPN showed a clear down-regulation after viscumTT treatment. In addition, co-treatment with doxorubicin, etoposide and ifosfamide further enhanced apoptosis induction, also synergistically.ConclusionViscumTT treatment results in synergistic apoptosis induction in osteosarcoma cells in vitro and ex vivo. Additionally, conventional standard chemotherapeutic drugs such as doxorubicin, etoposide and ifosfamide were able to dramatically enhance apoptosis induction. These results promise a high potential of viscumTT as an additional adjuvant therapy approach for osteosarcoma.
Highlights
Osteosarcoma is the most common bone tumor and is associated with a poor prognosis
Both the aqueous mistletoe extract viscum and the triterpene extract TT were solubilized in phosphate buffered saline Phosphate-buffered saline (PBS) resulting in a final concentration of 2 μg/mL intact mistletoe lectin (ML)-I, < 1 μg/mL viscotoxins, 4000 μg/mL OA and 0.35 μg/mL betulinic acid (BA) (TT)
TT and viscumTT did not induce early cytotoxicity but inhibit cell proliferation in vitro Early cytotoxicity was analyzed by Cytotoxicity Detection Kit after 2 h whereby Lactate dehydrogenase (LDH) release was investigated
Summary
Osteosarcoma is the most common bone tumor and is associated with a poor prognosis. Conventional therapies, surgery and chemotherapy, are still the standard but soon reach their limits. Conventional aqueous mistletoe extracts from the European mistletoe (Viscum album L.) are used in complementary cancer treatment. These commercial extracts are water-based and do not include water-insoluble compounds such as triterpenic acids. A whole mistletoe extract viscumTT re-created by combining an aqueous extract (viscum) and a triterpene extract (TT) was tested for its anti-cancer potential in osteosarcoma. Conventional treatment of osteosarcomas is still surgery with subsequent combined chemotherapy (multidisciplinary therapy). The basic mechanism of action of Viscum album L. is not fully understood Both the European mistletoe as well as the Korean mistletoe mediated apoptosis induction by activation of the PI3K/ AKT pathway [15], JNK/p38/MAPK [16] signalling and caspase cascades [13, 17]. Activation of the mitochondrial apoptotic pathway was demonstrated inter alia in AML [19], ALL [18] and other cancer cell lines [20]
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