Influence Of Blocking Irradiation-induced Autophagy By Chloroquine On Radiosensitivity Of Esophageal Cancer Cells

Objective: Our study is to examine whether blockingirradiation-induced autophagy by chloroquine (CQ) can sensitizeesophageal cancer cells TE-1to ionizing radiation (IR) and its furthermechanism. Materials/Methods: Effect of CQ on cell viability of TE-1cells was determined by MTT methodology. IC10and IC50of CQ werecalculated by software and the concentration of IC10was chosen for thefollowing experiments. We designed two groups of different dose of IRalone, different dosing time of CQ combined with IR. After treated withIR, expression of autophagy ralated protein LC3and Beclin-1wasdetected by Western-blot in different time. The formation of acidicvesicular organelles (AVOs) was determined by fluorescence stainingwith double staining of Lyso-Tracker Red DND-99and Hochest33258.Early stage apoptosis of6h,24h after different treatment was measuredby Annexin-V/PI staining through flow cytometry. Clonogenic survival ofTE-1cells on the treatment with addition of CQ before or after exposureto IR was examined by clonogenic forming assay, and the radiobiologyparameters were estimated from the curve fitted by the single-hit multitarget model [SF=1-(-e-D/D0)N] and linear-quadratic model [SF=e^(-αD-βD2)]. Results: With the treatment of different concentration ofCQ on TE-1cells for48hours, cell viability reduced in a dose-dependentmanner and its values of IC50and IC10were72.332±5.277μmol/L and15.416±3.327μmol/L, respectively. The concentration of15μmol/L waschosen for the following experiments. Western-blot showed theexpression of auophagy related protein Beclin-1and LC3, and conversion of LC3-I to LC3-II, reached a peak at time point of6h, markedlyincreased in irradiated TE-1cells. Fluorescence staining showed theextent of AVOs accumulation, fluorescence intensity in the cytoplasm ofTE-1cells markedly increased at6h and12h after IR. Compared with IRalone and group of treat with CQ before IR (Pre-CQ), group of treat withCQ after IR (Post-CQ) decreased IR-induced expression of autophagyrelated protein Beclin-1and conversion of LC3-I to LC3-II, obviously at6h and12h after IR, differences were statistically significant (P<0.05).Likewise, the extent of AVOs accumulation decreased and fluorescenceintensity weakened in the cytoplasm, differences were statisticallysignificant (P<0.0001). FCM showed that early stage apoptosis increasedwith the treatment of different IR dose on TE-1cells after24h, anddifferences were statistically significant (P<0.0001). With the addition ofCQ before or after IR for24h later, early stage apoptosis increased ingroup of treat with CQ after IR (2Gy+CQ), and differences werestatistically significant (P<0.0001). However, there is no statisticallysignificant difference for6h (P=0.8489). The values of D0, Dq, N, SF2, α,β and α/β of TE-1cells received IR alone were1.9964Gy,0.5533Gy,1.8930,0.5795,0.1537,0.0491and3.1284, respectively. The values ofD0, Dq, N, SF2, SERD0, α, β and α/β of TE-1cells received CQ before IR(Pre-CQ) were1.9260Gy,0.5285Gy,1.8810,0.5604,1.0366,0.1605,0.0532and3.0688, respectively. Those of TE-1cells received CQ afterIR (Post-CQ) were1.3872Gy,0.2040Gy,1.4030,0.3152,1.4392,0.4061,0.0638,6.3652, respectively. Conclusions: Our findings revealedthat IR can induce autophagy and increase cell apoptosis in esophagealcancer cells TE-1. CQ would sensitize esophageal cancer cells TE-1to IRthrough blocking IR-induced autophagy and increasing cell apoptosis.Application of CQ could sensitize esophageal cancer cells TE-1to ionizing radiation.