Cellular response to the combined effect of trifluoperazine and ionizing radiation: A molecular study.

Abstract

e13533 Background: It is known that some phenothiazines group of drugs have the potential to modulate ionizing radiation (IR) induced cellular damage. In our previous study, we have shown that Trifluoperazine (TFP), a phenothiazine derivative antipsychotic drug, modulate the effect of IR by inhibiting the IR induced DNA double strand break (DSB) repair, and raising the possibility of using TFP as an adjuvant to radiotherapy. In the present study we tried to explore the mechanism of IR induced DNA damage repair inhibition by TFP. Methods: Effect of TFP and IR on cell viability was determined by MTT assay. Study of possible signaling pathway leading to IR induced cell killing was checked using indirect immunolabeling and transfection. Results: It was observed that the use of TFP (2.5 µg/mL) reduces the LD50 from 13 Gy to 7.5 Gy. After transfecting the cells with GFP-tagged Ku80 plasmid, we have seen that in the gamma-irradiated cells, Ku80 was completely localized in the nucleus, indicating close association of Ku with the broken DNA ends. However, in the TFP (2.5 μg/mL) treated cells, Ku80 was retained in the cytoplasm after irradiation. Similarly, with indirect immunofluorescence experiments using antibody against DNA-PKcs, the catalytic component of DNA-PK, large bright dots of DNA-PKcs was present in the nucleus when cells were treated with IR alone. But, when the cells were irradiated in the presence of TFP, DNA-PKcs migrated to the nucleus partially, even after 4 h of irradiation. Conclusions: Thus, TFP prevents the trafficking of DNA-PK complex from cytoplasm to nucleus and thereby inhibits the DNA-PK mediated NHEJ. Taken together, our results suggest that TFP sequestered the DNA-PK complex, which is a major player in non homologous end joining (NHEJ) and enhance the effect of IR.