The Role Of Formononetin On Turmor Growth In The Human Osteosarcoma And The Study Of Its Mechanism

BackgroundOsteosarcoma, the most common non-hematologic primary malignant neoplasm of the bone, is characterized by the development of bone or osteoid substance by the tumor cells. The disease is mainly found in young patients between 10 and 25 years old, and it is one of the most frequent causes of cancer-related deaths in childhood. The traditional treatment is amputation with low 5-year survival rate. Distant metastasis is the main lethal of osteosarcoma. Chemotherapy is the key treatment to control the metastasis. however, the side-effects of chemotherapy is the dominating obstruction for the clinic use.Plant-derived phytoestrogens display estrogenic properties due to the similar molecular structures between phytoestrogens and estrogens binding to the estrogen receptor (ER). There is also evidence to suggest that osteosarcoma is an ER-positive cancer and that phytoestrogens can mediate estrogen-like effects in human osteosarcoma cells.Endocrinotherapy as a new way to tumor therapy, which through inhibit the synthesis and decrease the activity of hormone to achieve the effect of antitumor, is now attract extensive study. The study was designed to explore the anti-tumor effects of Formononetin and its mechanism in xenograft transplanted by human osteosarcoma cell line in nude mice. Furthermore, the paper intends to evaluate the side effects of Formononetin and provide a new way for target therapy of osteosarcoma.Objectives1. To study the role of Formononetin in the proliferation and induce cell apoptosis of U2OS cells in vitro.2. To study the role of Formononetin in the inhibition on human osteosarcoma cell line U2OS in vivo and to obsever the side effect of Formononetin to the nude mice.3. To describe the mechanism of the inhibition of Formononetin on human osteosarcoma cell line U2OS.Methods1. U2OS cells were treated with different concentrations of formononetin(0,20,40 and 80μM) and the proliferation of the cells was measured using an MTT assay. Cell apoptosis was examined by flow cytometry. Analysis the fluorescence intensity by flow cytometry instrument (FACSCalibur), and quantitative detection the percentage of the cell cycle in G0-G1, S, and G2-M in each group. The levels of miR-375, Bax and Bcl-2 protein expression in treated cells were determined by Western blot and RT-PCR.2. Human endometrial carcinoma exnograft models were established in nude mice. The antitumor activity of formononetin was also evaluated in vivo in nude mice bearing orthotopic tumor implants. The levels of miR- 375, Bax and Bcl-2 protein expression in treated cells were determined by immunohistochemistry and RT-PCR.Results1. The MTT showed that High concentrations of formononetin significantly suppress the proliferation of cells. Formononetin inhibit the proliferation of U2OS cell increases with increased formononetin dose and time. The rate of inhibition was 71% after treated 72h after formononetin in group 80μM. The ratio of GO/Glwas significantly higher in the cell cycle, and increase with the increased formononetin dose.2. Flow cytometry showed that high concentrations of formononetin significantly induce cell apoptosis. After formononetin treatment of U2OS cells, the ratio of apoptotic cells with 40 and 80 μM formononetin was 23% and 34% respectively (p<0.05). The results showed that formononetin-induced cell apoptosis increases with increased formononetin dose.3. The Hoechst 33258 images showed that U2OS cell apoptosis increases with increased formononetin dose.4. Western blot and Real-time PCR showed that the expression of Bcl-2 and miR-375 decreases with formononetin in the U2OS cells, while Bax increases, compared with control group in vitro.5. Formononetin significantly inhibition on human osteosarcoma cell line U2OS in nude mice, The tumor volume was inhibited by 41.12% (80μM group), and the tumor weight was inhibited by 39.53%. Compared with the control group, that difference was statistically significant(P< 0.05). Moreover, nude mice in all group were in good state, in spirit and behavior. There was no significant difference in liver and kidney function between the groups.6. The expression of Bcl-2 weakly positive with formononetin in the U2OS cells, while Bax strongly positive, compared with control group by immunohistochemistry.7. The expression of miR-375 decreases with formononetin in the U2OS cells, compared with control group by RT-PCR in the vivo. The expression of Bcl-2 decreases while Bax increases, compared with control group by Western blot in the vivo.Conclusions1. The study demonstrated a dose-dependent and time-dependent inhibition proliferation of Formononetin in U2SO cells.2. The study demonstrated a dose-dependent and time-dependent induction apoptosis of Formononetin in U2SO cells.3. Formononetin significantly downregulation expression of Bcl-2, miR-375, and upregulation of Bax in vitro and vivo.4. The antitumor effect of Formononetin is significant in nude mice, however, no significant side effect to the nude mice was observed5. The miRNA-375 may involved in the mechanism of the inhibition of Formononetin on human osteosarcoma cell line U2OS.