Supplementary Material for: Artemisinin-Daumone Hybrid Inhibits Cancer Cell-Mediated Osteolysis by Targeting Cancer Cells and Osteoclasts

<b><i>Background/Aims:</i></b> Bone metastasis of cancer cells decreases patient survival and quality of life. Hybridization via the covalent coupling of two bioactive natural products is a useful strategy for developing more potent anticancer agents by enhancing their bioavailability and avoiding drug resistance<b><i>.</i></b> <b><i>Methods:</i></b> The <i>in vivo</i> activities of artemisinin-daumone hybrid 15 (ARTD) were estimated in cancer cell-inoculated mice and ovariectomized mice. The viability, migration, and invasion of cancer cells were measured via MTT, wound-healing, and transwell invasion assays. ARTD-regulated transcription factors were detected with an RT<sup>2</sup> profiler PCR array kit and Western blotting. Osteoclastogenesis and osteoclast activity were detected with tartrate-resistant acid phosphatase staining, a pit formation assay, gelatin zymography, and a cathepsin K ELISA assay. <b><i>Results:</i></b> ARTD blocked cancer-associated osteolysis more potently than artemisinin in mice with intratibially inoculated breast cancer or lung cancer cells. ARTD inhibited the viability, migration, and invasion of breast and lung cancer cells in the absence or presence of transforming growth factor-β1. ARTD treatment induced the expression of tumor suppressive activating transcription factor 3 and inhibited oncogenic E2F transcription factor 1 expression at the mRNA and protein levels. ARTD inhibited receptor activator of nuclear factor kappa-B ligand-induced osteoclast formation and bone resorbing activity by reducing the secreted levels of matrix metalloproteinase-9 and cathepsin K. Furthermore, ARTD prevented estrogen deficiency-induced bone loss in ovariectomized mice. <b><i>Conclusion:</i></b> ARTD may be a promising candidate for inhibiting cancer-induced bone destruction. The application of ARTD may be extended to patients with chemotherapy-induced ovarian failure or postmenopausal osteoporosis.