| To study the inhibitory effect and IC50 of isoflavonoid Genistein on androgen-independent DU145 prostate cancer cells in vitro. The radiosensitizing effect and the underlying mechanisms were also investigated through combined use of Genistein with irradiation.MethodsProstate cancer cell line DU145 was used in this experiment. IC50 and the synergistic action of Genistein with irradiation on DU145 were studied through clonogenic assay, which was applied to compare the survival fractions of DU145 cells after treatments with Genistein alone and/or graded IR. TUNEL(Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling), DAPI (4',6-Diamidino-2-phenylindole) stain and DNA ladder were applied to detect cell apoptosis both morphologically and semi-quantatively. Flow cytometry was used to observe cell cycle alterations and apoptosis/hyperdiploid cell populations. Immunoblotting was used to detect the cell-cycle related protein expression including cyclin Bl,cdc-2 and p21cip1.ResultsThe IC50 of Genistein on DU145 cells was about 30 umol/L. Clonogenic assaydemonstrated that Genistein, even at low to medium concentrations, enhanced the radiosensitivity of DU145 cells. After treatments with IR and/or Genistein for 24 h, apoptosis was mainly seen with Genistein at high concentration and was minimally dependent on IR. Apoptosis also occurred after treatments for 72 h with lower concentrations of Genistein, especially when combined with IR. While IR or Genistein led to a G2/M cell cycle arrest, combination of them further increased DU145 cells at G2/M phase. This G2/M arrest was largely maintained at 72 h, and accompanied by increasing apoptosis and hyperdiploid cell populations. Cell-cycle related protein analysis disclosed biphasic changes in cyclin Bl, less markedly increased cdc-2 and stably elevated p21cip1 levels with increasing Genistein concentrations.DiscussionOur results showed that combined treatment of IR with Genistein even at low to medium concentrations synergistically increased the radiosensitivity of DU145 prostate cancer cells. While early-stage apoptosis was mainly induced by Genistein at high concentration, prolonged observation revealed that significant apoptosis also occurred with Genistein at lower concentrations, especially when combined with IR. Genistein plus IR led to more cells arrested at G2/M phase and a time-dependent increase in hyperdiploid cells, which might have also contributed to the enhancement of radiosensitivity by Genistein on DU145 cells.Our results showed that early-stage apoptosis of DU145 was mainly affected by the concentration of Genistein rather than IR, and DU145 cells seemed resistant to IR induced apoptosis. Mechanisms regarding the radiore si stance of tumors have not been fully elucidated, and might involve the increase in survival gene expressions. NF-kappaB is a well known survival factor that can be activated by many stimuli including IR, and in vitroexperiments exhibited its anti-apoptotic action. There were reports that Genistein inhibited NF-kappaB activation in prostate cancer cells, which might partly explain the dose/time-dependent induction of apoptosis in DU145 cells by Genistein. Flow cytometry results also provided evidence that parts of the above apoptotic cells might have derived from the sustained G2/M arrest or hyperdiploid cells.G2/M checkpoint arrest has been proposed to be a major determinant of cellular radio-and chemosensitivity. We found that the combination of Genistein (at 5-30 umol/L) with IR (6 Gy) further increased DU145 cells at G2/M phase. Genistein at 100 umol/L plus IR did not further increase, but rather decreased cells at G2/M phase as compared to using Genistein alone. This might be explained by the fact that a part of the G2/M arrested cells have gone through the process of apoptosis, as evidenced by our results of TUNEL/DNA ladder analyses. It was also likely that an increase of cells at S phase with 100 umol/L of Genistein plus IR treatment might have partly reflected the phenomenon of pre-...
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