Inhibitory Effects Of Two Traditional Chinese Medicine Monomers On Osteosarcoma Cells

Research background:Osteosarcoma(OS)is a mesenchymal malignant tumor,which is characterized by the formation of immature osteoid by tumor cells.It has a bimodal distribution,peaking after the ages of 10-20 and 65.It is the third most common cancer type among children and adolescents aged 12 to 18,second only to leukemia and lymphoma.83% of osteosarcoma patients are highly malignant,characterized by invasive growth and rapid hematogenous dissemination.Early lung metastasis is closely related to the second growth peak of long shaft.Before the 1970 s,the standard treatment for osteosarcoma was amputation,but the postoperative 5-year survival rate was less than 20%.With the application of chemotherapeutic drugs in the treatment of osteosarcoma,the 5-year survival rate of OS patients was significantly improved compared with the past,but the5-year survival rate of patients with lung metastasis was still less than 30%.Over the past 40 years,despite the rapid development of clinical oncology and the emergence of new antitumor drugs,no new drug can shake the position of the four first-line therapeutic drugs for osteosarcoma.However,chemotherapy drug treatment has multidrug resistance(MDR),and there is a great lack of follow-up effective drugs for osteosarcoma patients who fail to be treated with first-line chemotherapy drugs.They can only try new treatment schemes through clinical trials.Therefore,it is urgent to constantly explore new promising drugs for osteosarcoma clinical treatment [6].We screened two traditional Chinese medicine monomers to study their inhibitory effects on osteosarcoma:Ailanthus altissima is a plant of Ailanthus in Simaroubaceae.As a traditional Chinese medicine,it has a long history in China;For example,its bark,root bark and fruit have been used to treat Ascaris lumbricoides,diarrhea,spermatorrhea,bleeding and gastrointestinal diseases.Ailanthone(AIL)is a pentacyclic diterpene lactone extracted from Toona sinensis.It has significant clinical efficacy in the treatment of inflammation,malaria,allergy,tuberculosis,ulcer,and Amoeba related diseases,and has antitumor effect.A large number of in vitro studies have shown that AIL has inhibitory effects on various tumor cells,such as acute myeloid leukemia [14],bladder cancer,lung cancer,gastric cancer,liver cancer and breast cancer,vestibular schwannoma and prostate cancer.Curcumin is a hydrophobic polyphenol with small relative molecular weight extracted from the rhizomes of Zingiberaceae and Araceae traditional Chinese medicine.With the deepening of curcumin research,curcumin’s anti-inflammatory,antioxidant,control of diabetes,regulation of apoptosis and proliferation have been discovered.Due to its low toxicity and small adverse reactions,curcumin has shown potential clinical application in the treatment of liver fibrosis,atherosclerosis and cancer.Previous studies have shown that curcumin can inhibit the proliferation of tumor cells such as breast cancer,colon cancer,gastric cancer,prostate cancer and melanoma by inhibiting a variety of signal transduction pathways,regulating autophagy,inducing apoptosis,inhibiting the epithelial mesenchymal transition of tumor cells and thus reducing the migration ability of tumor cells.Research objectives:1.To explore the effects of different concentrations of Ailanthone on proliferation,apoptosis,migration and invasiveness of human osteosarcoma cells.2.Through the combined analysis of transcriptome,proteomics and metabolomics,to explore the effect of AIL on the metabolism of osteosarcoma cells.3.To explore the effects of different concentrations of curcumin on proliferation,apoptosis,migration and invasiveness of human osteosarcoma cells.Research methods:Part Ⅰ:1.The effects of different concentrations of Ailanthone on the proliferation,migration,invasion and apoptosis of osteosarcoma cells were studied by CCK-8,Wound Healing Assay,Transwell and Flow cytometry.2.Flow cytometry combined with PI staining was used to evaluate the effect of different concentrations of AILe on the cell cycle distribution of osteosarcoma.3.Metabolomics and proteomics were used to screen the metabolic and protein differences of AILtreated at IC50 concentration for 24 hours.4.q RT-PCR and Western blot were used to detect the gene and protein differences caused by different concentrations of AIL treatment for 24 hours.5.The inhibitory effect of AIL on osteosarcoma in vivo was detected by mouse xenotransplantation experiment.Part Ⅱ:1.Curcumin inhibited proliferation,clonal formation,migration and invasion of osteosarcoma cells and induced apoptosis in a concentration-dependent manner.2.Western blot showed that Cleaved-Caspase-3,BAX,and BCL-2 expression were significantly increased and Cleaved-Caspase-3 and BAX expression were significantly decreased after 24 h of curcumin treatment.Research results:Part Ⅰ:1.The proliferation,migration and invasion of OS cells were inhibited in a concentration dependent manner after 24 h treatment with different concentrations of Ailanthone.2.To on one inhibits the growth of subcutaneous transplanted tumor of OS.3.Treatment with different concentrations of AIL for 24 hours induced OS cell apoptosis and cell cycle arrest in a concentration dependent manner.4.Proteomic analysis showed that 68.6% of the differential proteins were related to metabolic processes,among which the change of serine biosynthesis and metabolism pathway was the most significant.5.The results of q RT-PCR and Western blot were consistent with the gene expression of a-seq based on rnail,which could reduce the expression of serine biosynthetic pathway(SSP).PHGDH,PSAT1 and PSPH m RNA of the two cells were significantly down regulated in a concentration dependent manner.6.Treatment with ail resulted in the reduction of intermediate metabolites of SSP and its downstream pathway.7.Overexpression of PHGDH reversed the effect of ail on OS cells.Part Ⅱ:Curcumin inhibited the proliferation,clonogenesis,migration,invasion and apoptosis of osteosarcoma cells in a concentration dependent manner.Figure 12 Table 2 Reference 141...