Study On The Radiosensitizing Effects Of Genistein And Corresponding Mechanisms

Aims：The aim of this work was to investigate the radiosensitization effects ofgenistein and corresponding mechanisms in vitro and in vivo. There are two parts inthe thesis. First, the radiosensitizing effect of genistein on breast cancer cells withdifferent estrogen receptor (ER) and p53statuses and corresponding mechanismswere examined; second, the radiosensitizing effect of genistein on mice sarcoma andthe underlying mechanisms was studied.Materials and Methods：Cell cycle distribution, cell apoptosis were detected by flow cytometry; Theinhibition effect of genistein on cell proliferation was determined with the MTT andcell counting assay; Cell survival fraction was detected with the standardcolony-formation assay; The distribution of γ-H2AX、Rad51、Ku70/80、DNA-PKcsin cells were measured using immunofluorescence staining; The expression of G2/Mphase checkpoint and cell apoptosis associated proteins were detected with westernblot technique; In addition, the cell apoptosis in paraffin embedding tissue sectionwas detected with the tunel method. Results：Part1:1. The cell proliferation determined with the MTT assay showed that genisteinhad relatively weak toxicity to both breast cancer cell lines at concentrations in therange of5-20μM. Both cell lines were pretreated with0,5,10and20μM genisteinfor24h followed by4Gy X-ray irradiation. Cell survival fraction decreased with theincrease of genistein concentration. When cells were pretreated with10μM genistein,the radiation enhancement ratios of the cells exposed to X-rays at a10%cell survival(IC10) were1.43for MCF-7and1.36for MDA-MB-231cells, respectively. Theγ-H2AX foci increased significantly in a genistein concentration-dependent manner,suggesting that the DNA damage was seriously exacerbated by genistein. The resultsmentioned above indicated that genistein had a good radiosensitizing effect on bothbreast cancer cell lines.2. When both cell lines were pretreated with10μM genistein then exposed toX-ray irradiation, the cell cycles were significantly arrested at12h post-irradiation.However, the fraction of cells in G2/M phase decreased sharply at24hpost-irradiation. The co-localization results of γ-H2AX and Rad51foci indicated thatgenistein could block the recruitment of Rad51to DNA injured sites and theapoptotic rates increased remarkably. These data illustrated that genisteinpretreatment exacerbated the G2/M phase arrest at12h post-irradiation and amajority of damaged cells in G2/M phase could not be repaired and then underwentdeath in an apoptosis-related manner.3. Genistein combined with X-rays induced G2/M phase arrest via the activationof ATM/Chk2/Cdc25C/Cdc2checkpoint pathway; With the increase of genisteinconcentration, the Bcl-2/Bax rate decreased in both cell lines, the expression ofwild-type p53was almost no changes (MCF-7cells) while the mutant p53expression was down-regulated remarkably (MDA-MB-231cells). However, theexpression of p73increased obviously in both cell lines. Therefore, our studyindicated that the cell apoptosis induced by the combination of genistein and X-rays in the breast cancer cells occurred through the mitochondrial apoptosis pathway,probably the p73mediated pathway.Part2：1. The results of cell counting showed that10μM genistein had no obviousinhibitory effect on the growth of S180cells. Using the dosage of10μM genistein,the radoation enhancement ratio of cells after exposure to X-rays at a10%cellsurvival (IC10) was1.38. Cell apoptotic rate increased significantly in the group ofgenistein combined with X-ray treatment.2. Balb/c mice inoculated with S180sarcoma were used in the following study.Mice were sacrifced by cervical dislocation at24h after irradiation and then thetumor tissues were immediately removed. The tumor tissues of the control group (C)had plenty of blood vessels. But in the genistein and X-ray cotreatment group(D+IR), the vessels reduced obviously. The tumor size and volume in the D+IRgroup decreased significantly than those in the other groups. The H&E stainingresults showed that the cell density in the D+IR group was low. The tunel detectionresults illustrated that there were a large number of cells undergoing death via anapoptosis pathway.3. Bax expression was up-regulated significantly and Bcl-2expression wasdown-regulated in the mitochondria, and lots of cytochrome c was transferred to thecytoplasm in the cotreatment group (D+IR). With the lapse of time after irradiation,the expression of Rad51in the D+IR group decreased significantly while γ-H2AXwas expressed stably, suggesting that the HR repair was blocked but the DNAdamage persisted. The results of Ku70/80, DNA-PKcs and Rad51colocalizationshowed that genistein combined with X-rays inhibited the activity of DNA-PKcs sothat the injuried-DNA sites were dominated by Ku70/80, leading to theincompleteness of the NHEJ and HR repairs and the occurrence of cell apoptosiseventually. Conclusions：1. Genistein has a radiosensitizing effect on MCF-7（ER+, wild-p53）andMDA-MB-231（ER-, mut-p53）breast cancer cell lines regardless of its estrogenreceptor or p53statuses. The potential mechanisms are the activation ofATM/Chk2/Cdc25c/Cdc2checkpoint pathway and the induction of G2/M phasearrest. The damaged cells might not be repaired but undergo apoptosis probably viathe p73induced mitochondria-mediated pathway.2. The experiments in vitro and in vivo indicate that genistein show aradiosensitizing effect on mice S180sarcoma. The underlying mechanism is theinhibition the activity of DNA-PKcs so that the NHEJ repair is limited. Meanwhile,the damaged-DNA sites are dominated by Ku70/80, resulting in the incompletenessof the HR repair. Therefore, the damaged cells could not be repaired so as to sufferapoptosis eventually.