BackgroundOsteosarcoma 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.MethodsMTT 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.ResultsThe 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).ConclusionsShikonin 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.

Osteosarcoma (OS) is the most common primary malignant bone tumor, notorious for its metastasis. We have recently shown that shikonin, an effective constituent extracted from Chinese medicinal herb, induces necroptosis in OS cells. Nevertheless, the effects of low-dose shikonin on the invasiveness of OS cells are unknown. Here, we showed that shikonin dose-dependently decreased OS cell invasiveness in both scratch wound healing assay and transwell cell migration assay. Moreover, the direct target of shikonin on cell invasiveness was found to be matrix metalloproteinase (MMP)-13. Further, the inhibitory effects of shikonin on cell invasiveness were completely abolished in MMP13-overexpressing OS cells. Together, these data suggest that shikonin may suppress OS invasiveness through MMP13 suppression. Thus, our data highlight a previous unappreciated role for shikonin in suppressing OS cell metastasis.

Background/Aims: Osteosarcoma (OS) is a primary malignant bone tumor in humans, and is notorious mainly for its distal metastases. We have recently shown that Shikonin, an effective constituent extracted from Chinese medicinal herb, inhibits OS cell invasion through suppression of matrix metalloproteinase 13 (MMP13). However, the underlying mechanisms remain unknown. Methods: Here, we studied the levels of tumor necrosis factor (TNF)-alpha-induced protein 8-like 2 (TIPE2) in OS cells upon Shikonin treatment. TIPE2 levels were adapted in OS cell lines through transfection with plasmids carrying transgene or short-hairpin interference RNA (shRNA), and the effects of TIPE2 adaptation on MMP13 and cell invasiveness were evaluated by RT-qPCR, Western blot, ELISA and transwell cell migration assay, respectively. TIPE2 levels in OS specimens from patients were examined and correlated with cancer metastases and patient survival. Results: We found that Shikonin dose-dependently decreased MMP13 levels, and increased TIPE2 levels in two OS cell lines, U2OS and SaOS-2. Overexpression of TIPE2 in U2OS significantly suppressed MMP13 levels and cell invasiveness. Depletion of TIPE2 in SaOS-2 cells significantly increased MMP13 levels and cell invasiveness. Moreover, TIPE2 levels in OS specimens were significantly decreased, compared to adjacent non-cancer bone tissue. Lower TIPE2 levels correlated with higher incidence of metastases and worse 5-year survival. Conclusion: TIPE2 mediates the suppressive effects of Shikonin on MMP13 in osteosarcoma cells, and TIPE2 may be a novel therapeutic target for OS.

Patient-specific instrumentation (PSI) was designed to improve the accuracy of preoperative planning and postoperative prosthesis positioning in total knee arthroplasty (TKA). However, better understanding needs to be achieved due to the subtle nature of the PSI systems. In this study, 3D printing technique based on the image data of computed tomography (CT) has been utilized for optimal controlling of the surgical parameters. Two groups of TKA cases have been randomly selected as PSI group and control group with no significant difference of age and sex ( > 0.05). The PSI group is treated with 3D printed cutting guides whereas the control group is treated with conventional instrumentation (CI). By evaluating the proximal osteotomy amount, distal osteotomy amount, valgus angle, external rotation angle, and tibial posterior slope angle of patients, it can be found that the preoperative quantitative assessment and intraoperative changes can be controlled with PSI whereas CI is relied on experience. In terms of postoperative parameters, such as hip-knee-ankle (HKA), frontal femoral component (FFC), frontal tibial component (FTC), and lateral tibial component (LTC) angles, there is a significant improvement in achieving the desired implant position ( < 0.05). Assigned from the morphology of patients' knees, the PSI represents the convergence of congruent designs with current personalized treatment tools. The PSI can achieve less extremity alignment and greater accuracy of prosthesis implantation compared against control method, which indicates potential for optimal HKA, FFC, and FTC angles.