Flavonoids Inhibits Hypoxia-inducible Factor-1? Expression And Enhances Radiosensitivity Of Hypoxic SKOV3 Human Ovarian Cancer Cells

Ovarian cancer is one of the three malignant tumors of female genital system,its mortality rate ranks the first place in gynecological tumors,and has become a serious threat to women's life and health.Radiotherapy is the third road to ovarian cancer treatment in addition to surgery and chemotherapy,and often usd as auxiliary or palliative therapy after surgery.However,the sensitivity to radiation differs in the different organizational sources of ovarian cancer cells,in addition,the existence of hypoxic cells in the tumor cells limits the curative effect.Hypoxia appears in the process of rapid growth of tumor because of oxygen diffusion block and poor blood flow.Hypoxic cells are resistant to radiation,seriously affect the quality of radiotherapy,and is strongly associated with poor prognosis of tumor.Thus,how to improve the radiosensitivity of hypoxic tumor cells has become a hot project of cancer radiotherapy for a long time.HIF-1 is the first hypoxia response factor found in mammalian cells,and is closely related to tumor radiosensitivity.When tumor cells exposured to irradiation,more than 100 genes regulated by HIF-1?are activated.These genes involved in energy metabolism,angiogenesis and cell proliferation,metabolism,movement,autophagy,apoptosis,and other links,make tumor cells quickly adapt to hypoxic microenvironment,and improve the resistance to radiation.So we can improve the radiosensitivity of hypoxic cells by intervening the activity of HIF-1 alpha.The study cultured SKOV3 cells in the hypoxic condition and took it as research objects,investigated the radiosensitizing effects of four kinds of flavonoids such as genistein,chrysin,quercetin and breviscapine to hypoxic SKOV3 cells,and took irisquinone,a commercially available and natural radiosensitizer to hypoxic cells as positive control,chose an agent who had strongest radiosensitizing effect to explore the effect of flavonoids on HIF-1alpha and radiosensitizing mechanism.The study aims at providing new ideas and methods to seek radiosensitizer to hypoxic cells and tumor radiotherapy.The details are as follows:1.Establishing and measuring the hypoxic SKOV3 cellular model in vitroThe hypoxic SKOV3 cellular model in vitro was established and tested by three methods such as clonogenic assay,MTT assay and hypoxia marker.The results showed that though the three methods had advantages and disadvantages,they obtained a consistent result that the hypoxic SKOV3cellular model in vitro was successfully established.2.The growth inhibition effects of flavonoids on normoxic and hypoxic SKOV3 cellsMTT assay was used to research the growth inhibition effects of irisquinone and flavonoids such as genistein,chrysin,quercetin and breviscapine on normoxic and hypoxic SKOV3 cells.The results showed that in the normoxic and hypoxic condition,a certain concentration range of genistein,chrysin,quercetin,breviscapine and irisquinone all inhibited SKOV3 cell survival rate with a concentration-dependent manner?P<0.05?.In the normoxic condition,IC₅₀s of the five agents functioned on SKOV3 cells for 72h were 47.45?g/ml,29.15?g/ml,75.98?g/ml,148.78?g/ml,38.77?g/ml,and in the hypoxic condition,they were 45.70?g/ml,24.72?g/ml,75.68?g/ml,151.46?g/ml,33.26?g/ml.3.The radiosensitizing effects of flavonoids on normoxic and hypoxic SKOV3 cellsWe chose high,medium and low concentration of flavonoids in the range of non-toxic concentration,and use MTT assay to investigate the radiosensitizing effects of flavonoids on normoxic and hypoxic SKOV3 cells,we ranked the five agents based on SER.The results showed that the radiosensitizing effects in a order from strong to weak were irisquinone,genistein,chrysin,quercetin and breviscapine,SER of breviscapine was less than 1.6.So we selected genistein to investigate the radiosensitization mechanism on hypoxic SKOV3 cells.4.The influence of genistein on the expression of HIF-1?in hypoxic SKOV3cells and the related signaling pathwayCells were treated with genistein or media for 24h and hypoxia for 12h,then irradiated.Western blot and immunofluorescence staining was performed to investigate the effect of genistein on HIF-1?activity and the related signaling pathway.The results showed that the HIF-1?protein weakly expressed in normoxic SKOV3 cells,and significantly upregulated under hypoxia.When the hypoxic SKOV3 cells were pretreated with genistein,hypoxia-induced accumulation of HIF-1?was attenuated,and significantly attenuated in the IR plus genistein group compared with the control group under hypoxia.Thus,preincubation with genistein inhibited accumulation of hypoxia-induced HIF-1?protein,and the radiosensitizing effects of genistein may be related to it.The inhibition of HIF-1?by genistein may be concerned with inhibiting Ras/Raf/MEK-1/ERK1-2 signal pathway and enhancing p53level but may be not correlated with PI3K/Akt/mTOR pathway.5.The radiosensitization mechanism of genistein on hypoxic SKOV3 cellsCells were treated with genistein or media for 24h and hypoxia for 12h,then irradiated.Hoechst 33342 staining and the fluorescence microscope were used to investigate the cell apoptosis.PI staining and the flow cytometry were used to investigate the cell cycle.DSB was analyzed by measuring the percentage of?-H2AX foci in SKOV3 cells.The results indicated that genistein used alone or in combination with radiation increased the apoptosis rate of hypoxic SKOV3 cells,and the effect was concentration dependent?P<0.05?.Western blot and immunofluorescence staining was performed on?-H2AX protein,suggesting that genistein may increase DSB and inhibit DNA damage repair in the irradiated cells.The above two kinds of effects were both related to the upregulation of p53 protein by genistein.In addition,genistein induced a G₂/M cell cycle arrest in the irradiated cells.