| Objective: Laryngeal carcinoma is one of the most common type of head and neck malignant cancer with the 5-year relative survival rates presented a downtrend to 63%.Radiation therapy plays a crucial role in laryngeal cancer treatment,however,the intrinsic or acquired irradiation resistance contributes to treatment failure of patients with laryngeal carcinoma.Genistein,a kind of isoflavonoid derived from soy products,presents pleiotropic anti-cancer effect through inhibiting cell growth,apoptosis,invasiveness,inducing G2/M phase arrest.Epidemiological survey showed that,compared with western diet,soy rich diet in Asian population had a lower incidence of prostate cancer and breast cancer.Our previous reserch on genistein showed that it presented anticancer effect in HEp-2 cells through inducing apoptosis,involvement of PI3K/Akt pathway.Besides,genistein is a potential radiosensitizer in several types of cancer,such as prostate cancer,breast cancer and cervical cancer.However,no reserch on whether genistein potentiate HEp-2 cells irradiation was found so far.In this study,we test whether genistein could potentiate irradiation effect in laryngeal carcinoma and illustrate the mechanisms underlying the radiosensitivity effect.Methods:1 CCK-8 assay was used to detect anti-proliferation effect of varies doses of genistein in 24,48 and 72 h after treatment.2 Clonegenic assay was used to detect the long-term effect of HEp-2 cells proliferation.Clonogenic assay to perform the radiosensitivity effect of genistein on HEp-2 cells and the data was fitted into the classic mathematical model.3 Ed U assay to assess the synergistic effect of genistein and irradiation on the DNA synthesis of HEp-2 cells.4 DSBs were quantified by ?H2AX foci immunofluorescence to detect the DSB induced by genistein and irradiation.5 Flow cytometry were used to analyse cell cycle distribution of genistein and irradiation treatment on HEp-2 cells.6 Western blot was used to semiquantified the expression of cell cycle related proteins and PI3K/Akt pathway proteins.Results:1 Genistein inhibited HEp-2 cells growth.Genistein inhibited cell growth in a dose-dependent manner after 24 h,48h and 72 h treatment.2 The treatment of HEp-2 cells with 10~100?M genistein resulted in a dose-dependent inhibition of colony formation.A significant inhibition of~80% was observed at 50 and 100 ?M genistein treatment.Whereas at the lower concentrations,25?M genistein promoted approximately 50% reduction and no significant effect was obtained by 10?M genistein.3 Genistein potentiated irradiation-induced inhibition of clonogenic survival in HEp-2 cells.Survival curves indicated that pretreatment with 10 and 25 ?M genistein suppress clonogenic survival of HEp-2 cells.As the parameter showed,genistein greatly enhanced the radiosensitivity of HEp-2cell line.4 Genistein had a synergistic effect with irradiation inhibiting DNA synthesis.The anti-proliferation effect of genistein on HEp-2 cells was in a dose-dependent manner.It is worth mentioning that the percentages of proliferating phase cells were not significantly decreased althoug the dose of irradiation added.The combination of irradiation and genistein had a synthegistic effect of anti-proliferation on HEp-2 cells.5 Genistein augmented irradiation-induced DNA damage in HEp-2 cells.we observed increased ?-H2 AX foci after irradiation.In contrast to irradiation treatment alone,cells pretreated with genistein for 24 h showed a signifcant increase.Moreover,the foci numbers remaining high at 24 h post irirradiation.6 Genistein induced G2/M arrest of HEp-2 cells in a dose and time dependent manner.We found that the significant increase in G2/M phase in adose and time dependent manner within varying concentrations of genistein.Moreover,genistein induced the sub-G1 phase apoptotic peak greater in 72 h and 96 h after treatment.7 Cell cycle distribution after irradiation combined with genistein and the checkpoint regulator proteins activated in HEp-2 cells.Remarkable increase in G2/M phase was found in the combination treatment with genistein and IR compared with IR alone in HEp-2 cells.Cell cycle chechpoint regulator proteins p-Chk2 and p-Chk1 expressing levals dramatically increased compared with IR alone.8 Genistein modulated the expression levels of PI3 K signaling proteins in HEp-2 cells.Compared with irradiation alone,pAkt levels decresed while the levels of p-PTEN increased when pretreated with genistein.Conclusions:These findings revealed that genistein could be a potential radiosensitizer of HEp-2 cells and possibly with the mechanisms of augmenting DSBs after irradiation and induces cell cycle arrest to apoptosis as well as inhibiting PI3K/Akt pathway. |
PDF Full Text Request