The anti-tumor effect of shikonin on osteosarcoma by inducing RIP1 and RIP3 dependent necroptosis

Abstract

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.

Alternol, a natural compound, exerts an anti‐tumour effect on osteosarcoma by modulating of STAT3 and ROS/MAPK signalling pathways

“…Cell cycle and apoptosis proceeded as we previously described 21. Apoptosis was detected by FCM and analysed using BD Cellquest software (BD Biosciences, San Jose, CA, USA).…”

TIPE2 Mediates the Suppressive Effects of Shikonin on MMP13 in Osteosarcoma Cells

“…Shikonin has demonstrated anti-tumor potent, in which it induces cancer cell apoptosis and necroptosis [23][24][25]. Recently, we reported that high-dose of Shikonin had prompt but profound anti-tumor effect on both primary and metastatic OS, through inducing RIP1 and RIP3-dependent cancer cell necroptosis [26]. Moreover, Shikonin inhibits OS cell invasion through suppression of MMP13 [27].…”

Lycorine induces programmed necrosis in the multiple myeloma cell line ARH-77

“…However, due to the overexpression of anti-apoptotic proteins, many cancers have been found to develop resistance mechanisms against apoptosis-inducing chemotherapeutic agents [12][13][14]. In this regard, programmed necrosis, or type III programmed cell death, is expected to be a promising approach, and several agents have recently been found to induce necrosis as an anticancer mechanism [15][16][17][18][19]. Early-phase clinical trials for therapeutics that target regulators of necrotic cell death are under way [20].…”