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.

Aim: To explore the mechanism by which long non-coding RNA (lncRNA) TTN-AS1 regulates osteosarcoma cell apoptosis and drug resistance via the microRNA miR-134-5p/malignant brain tumour domain containing 1 (MBTD1) axis.Results: The lncRNA TTN-AS1 was highly expressed in osteosarcoma and was associated with poor prognosis. The lncRNA TTN-AS1 promoted cell viability and inhibited apoptosis. MiR-134-5p targeted MBTD1, which was regulated by lncRNA TTN-AS1. MBTD1 was highly expressed in osteosarcoma and was associated with poor prognosis. MBTD1 promoted cell viability and inhibited apoptosis, and knockdown of MBTD1 reversed the cancer-promoting effects of lncRNA TTN-AS1. Downregulation of lncRNA TTN-AS1 reduced drug resistance.Conclusion: In osteosarcoma, lncRNA TTN-AS1 promoted the expression of MBTD1 by targeting miR-134-5p and regulated cell growth, apoptosis and drug resistance.Methods: The expression characteristics of genes in osteosarcoma patients were analysed using bioinformatics. Plasmid transfection technology was applied to silence or overexpress lncRNA TTN-AS1, miR-134-5p and MBTD1. Western blotting and quantitative polymerase chain reaction (qPCR) were used to detect protein and RNA, respectively. A cell counting kit 8 (CCK-8) and flow cytometry were used to detect cell viability and apoptosis. The effects of lncRNA TTN-AS1 and MBTD1 on osteosarcoma in vivo were studied by using a tumour burden assay.

Osteosarcoma (OS) has an unfavorable prognosis and tends to metastasize to lung tissue. Although the CXCL12-CXCR4 axis appears to affect progression and metastasis in numerous tumors, its mechanism and downstream pathways in OS remain unclear. We used western blotting and flow cytometry to detect CXCR4 and CXCR7 expression in two OS cell lines (LM8 and Dunn). An MTT assay was used to evaluate the effects of CXCL12 and AMD3100, a specific CXCR4 antagonist, on cell viability. Flow cytometry was utilized to analyze changes in apoptosis induced by serum deprivation following treatment with CXCL12 and AMD3100. A Transwell assay was used to assess cell migration in response to CXCL12 and AMD3100. Western blotting was performed to identify the phosphorylation of signaling molecules (JNK, c-Jun, Akt, p38 and Erk1/2) and expression of caspase-3 and -8, and PARP. Mouse models were employed to evaluate AMD3100 inhibition of primary OS growth and lung metastasis in vivo. CXCR4 expression was detected in LM8 but not Dunn cells, and neither cell line expressed CXCR7. The addition of CXCL12 induced the survival and migration of serum-starved CXCR4+ LM8 cells activating JNK and Akt pathways, which were abrogated by adding AMD3100. However, similar results were not observed in CXCR4− Dunn cells. CXCL12 protected LM8, but not Dunn cells, from apoptosis induced by serum deprivation by suppressing PARP cleavage, which was partly reversed by AMD3100. In a mouse model, AMD3100 reduced primary tumor growth and lung metastasis compared with the controls. Thus, the CXCL12-CXCR4 axis regulated OS survival and metastasis through the JNK and Akt pathways, and blocking them with AMD3100 was found to be a potential OS treatment.