Identification And Characterization Of Anti-osteosarcoma Small Molecule Compounds

Osteosarcoma is the most common form of primary malignant bone cancer inchildren and adolescents and the rate of5-year survival in osteosarcoma patients is61.6%. Although in the last decades, multiagent chemotherapy in combination withsurgery resulted in an improvement in patient survival rates, which increased to70%,osteosarcoma is still characterized by frequent relapse and metastatic disease.Exploring novel therapeutic agents and their molecular mechanisms are, therefore,necessary for improving the outcome of osteosarcoma treatment.In this study, a total of400small molecule compounds including305fromnatural source, were tested for their anti-osteosarcoma activity. We found17bioactivecompounds with promising anti-osteosarcoma activity out of which isoalantolactoneand VK4were selectedfor further mechanistic studies.1、Isoalantolactone, isolated from Inula spp., has been reported to inhibit growthof several cancer cells. However, nopriorresearchhas been done to demonstrate theanti-proliferative potential of isoalantolactone on osteosarcoma. This study is the firstto investigate the effects of isoalantolactone on cell viability in human osteosarcomaU2OS, MG-63and Saos-2cells, and its mechanism of actionin U2OS cells. Ourresults demonstrated that isoalantolactone triggered S and mainly G2/M phase arrest,accompanying the down-regulation of expression level of cyclin B1at protein andmRNA level. Moreover, isoalantolactone induced apoptosis that was associated withreactive oxygen species (ROS) generation, the dissipation of mitochondrial membranepotential (MMP). Furthermore, our results indicated that this compound up-regulatesDR5, FADD, and cleaved caspase-8, increases the combination between DR5andFADD, and inhibits the expression of nuclear NF-κBp65.We also foundthatisoalantolactone-induced apoptosis was associated with the down-regulation of Bcl-2 and up-regulation of Bax, which finally led to the activation of caspase-3and itsdownstream substrate, PARP, in osteosarcoma U2OS cells. Isoalantolactone-inducedapoptosis is markedly abrogated when the cells were pretreated with N-acetylcysteine(NAC), a specific ROS inhibitor, suggesting that the apoptosis-inducing effect ofisoalantolactone in osteosarcoma cells is mediated by reactive oxygen species.Takentogether, our data demonstrate that isoalantolactone induces ROS-dependent apoptosisin U2OS cells via a novel mechanism involving inhibition of NF-κBp65and providethe rationale for further in vivo and preclinical investigation of isoalantolactoneagainst osteosarcoma.2、Vitamin K4(VK4) is a water-soluble menadione which is synthetic derivativesof VK2, it was primarily used as a clinical coagulation. The goal of this study is toinvestigate the inhibitory activity of VK4on osteosarcoma cell line U2OSproliferation and the mechanisms involved. Inhibitory effect of VK4on humanosteosarcoma cell line U2OS cells was measured using MTT method. Cell apoptosis,cell cycle distribution, intracellular ROS, mitochondrial membrane potential (MMP)were evaluated by flow cytometry method. Hoechst33342staining was used toevaluate nuclear DNA fragmentation. Western blot analysis was used to investigateeffect of VK4on major mitochondrial apoptosis regulatory proteins. MTT resultsshowed that VK4dose-dependently decreased the viability of osteosarcoma U2OScells. IC50value of VK4was around25μM. Hoechst33342results showed that VK4caused significant nuclear condensation in osteosarcoma U2OS cells. AnnexinV-FITC/PI double staining showed that VK4induced apoptosis in U2OS cells. Cellcycle analysis showed that VK4arrested the cell cycle at S phase in a dose-dependentmanner. Further mechanistic studies revealed that VK4-mediated induction ofapoptosis in U2OS cells is associated with disruption of mitochondrial membranepotential, down-regulation of Bcl-2, and up-regulation of Bax and activation ofcaspase-3. Moreover, we found that VK4inhibited osteosarcoma U2OS cellsmigration in vitro, however; further study is needed to explore the anti-invasive andanti-metastatic activity of VK4in detail. In conclusion, we identified a panel of anti-osteosarcoma small moleculecompounds and explored the action mechanisms of isoalantolactone and VK4,providing new lead compounds for the treatment of osteosarcoma.