Background: Osteosarcoma is the most frequent primary malignant bone tumor, notorious for its lung metastasis. Shikonin, an effective constituent extracted from Chinese medicinal herb, was demonstrated to induce necroptosis in some cancers. Methods: MTT assay was performed to detect cell survival rate in vitro. Flow cytometry was used to analyze cell cycle and cell death. Western blot was performed to determine the expression levels of RIP1, RIP3, caspase-3, caspase-6 and PARP. The tibial primary and lung metastatic osteosarcoma models were used to evaluate the anti-tumor effect of shikonin in vivo. Results: The cell survival rate was decreased in a dose and time dependent manner when treated with shikonin. No major change in cell cycle was observed after shikonin treatment. The cell death induced by shikonin could be mostly rescued by specific necroptosis inhibitor necrostatin-1, but not by general caspase inhibitor Z-VAD-FMK. The number of necrotic cells caused by shikonin was decreased after being pretreated with Nec-1 detected by flow cytometry in K7 cells. After 8-hour treatment of shikonin, the expression levels of RIP1 and RIP3 were increased while caspase-3, caspase-6 and PARP were not activated in K7 and U2OS cells determined by Western blot. Size of primary tumor and lung metastasis in shikonin treated group were significantly reduced. The protein levels of RIP1 and RIP3 in primary tumor tissues were increased by shikonin. The overall survival of lung metastatic models was longer compared with control group (p < 0.001). Conclusions: Shikonin had prompt but profound anti-tumor effect on both primary and metastatic osteosarcoma, probably by inducing RIP1 and RIP3 dependent necroptosis. Shikonin would be a potential anti-tumor agent on the treatment of primary and metastatic osteosarcoma.

Osteoarthritis (OA) is a now regarded as a worldwide whole joint disease with synovial inflammation, cartilage degeneration, and subchondral sclerosis. Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used drugs for OA treatment which only relieve the symptoms and restrain the progression of OA. However, various severe adverse effects often occur in patients with long-term NSAIDs use, which heavily burdens the healthcare system and impacts the quality of life. Therefore, it is much imperative to identify alternative drugs with increased efficacy. Syringaresinol (Syr), a naturally occurring phytochemical which belonging to the lignan group of polyphenols, shows anti-tumor and anti-oxidant activities, which to benefit human health. Studies has shown Syr can regulate the inflammatory response by modulating the secretion and expression level of cytokines IL-6, IL-8, and tumor necrosis factor (TNF)-α. it also shows the inhibitory effect on NF-κB pathway in mouse cells. In the present study, we aimed to demonstrate the anti-inflammatory effects of Syr in OA. In vitro Syr treatment in IL-1β-activated mouse chondrocytes significantly restrained the expression of NO, PGE2, IL-6, TNF-α, INOS, COX-2 and MMP-13. Moreover, it considerably ameliorated the degradation of aggrecan and collagen II. Furthermore, the phosphorylation of the NF-kB signaling pathway was significantly suppressed by Syr. Moreover, in vivo, the cartilage degeneration was attenuated and the increased Osteoarthritis Research Society International (OARSI) scores were reversed in the DMM + Syr group, comprared to those in the DMM group. In sum, our study demonstrated that Syr can attenuate the inflammation in vitro and further verified its effect on OA in vivo. Thus, Syr might be a potent therapeautic alternative for OA treatment.