Autophagy of SO-Rb50 cells induced by arsenic trioxide

Abstract

Background Cellular autophagy is a non-apoptosis death form of tumor tissue. Research determined that arsenic trioxide （As2O3） leads to apoptosis of tumor cells. But whether As2O3 induce autophagy of SO-Rb50 cells or not is unclear. Objective This study was to assess the effects of As2O3 on autophagy of SO-Rh50 cells. Methods As2O3 with the concentration of 0,0. 5,1.0,2.0,4.0 p.mol/L was used to treat the SO-Rb50 cell line for 48 hours,and the growth and proliferation of SO-Rb50 ceils were detected using MTT assay （A570 ） . pGFP- LC3 ,a marker of autophagy,was constructed to transfer SO-Rb50 cells, and the cells were then divided into RPMI- 1640 culture group （ untreated group） , As2O3 ＋ RPMI-1640 culture group （ As2O3 treated group） and rapamycin culture group （positive control group）. Autophagy of SO-Rh50 cells was examined by laser confocal microscope and monodansylcadaverine （MDC） influorescence staining, respectively,48 hours following cell culture, ghrastructural features of autophagy were examined with transmission electron microscope （TEM）. The percentage of autophagy positive cells in different concentrations of As203 treated groups was calculated with flow eytometer. Results The A570 values of SO-Rb50 cells were 2. 194-＋0. 066,1. 841-＋0. 213,1. 035-＋0. 046,0. 374-＋0. 042 and 0. 167-＋0. 019 in 0,0.5,1.0,2.0,4.0 p.mol/L As2 03 treated groups, with a significant difference among these 5 groups （ F = 547. 636,P〈0. 05 ）, and those of 0. 5,1.0,2. 0,4.0 μmol/L As203 treated groups were significantly reduced in comparison with untreated group （P = 0. 000）. The positive granular spots for GFP-LC3 chimeric protein were seen to aggregate in autophagic vacuoles in the As2O3 treated group and positive control group,but diffuse cytoplasmic signal for GFP-LC3 was found in the untreated group. Normal ultrastructure of SO-Rb50 cells was exhibited in the untreated group, and many double-membrane-like bound vesicles and autlysosomes were documented in the As203 treated group and positive control group under the TEM. A lots of MDC fluorescence granule were found in the As2O3 treated group and positive control group rather than the untreated group. Flow cytometry showed that the percentages of SO-Rb50 cells were 0,15.6% ,42.7% ,57.9% ,79.5% and 89.0% in the 0,0.5,1.0,2.0,4.0 μmol/L As2O3 groups and positive control group,respectively,showing a As203 concentration-dependent increase. Conclusions As2O3 can induce the autophagy of SO-Rb50 cells and inhibit the proliferation of SO-Rb50 cells. Autophagic response of SO-Rb50 cells appears prior to the nuclear change after exposed toAs2O3. The degree of autophagy of SO-Rb50 cells is associated with As2O3 dose. Key words: Retinal blastoma; Autophagy; Chemotherapy; Microtubules related protein light chain 3