The Study Of The Effect Of Autophagy On Stem Chemotheragy In CD133~+ Colorectal Carcinoma Cells

Objective:To observe the autophagy of CD133+ CSCs induced by 5-FU,and explore the effect of autophagy on the cytotoxic effect of 5-FU.Methods:(1) The cell proliferative inhibition rates and IC50 of 5-FU on human colon cancer cell lines SW480 were measured by MTT assay.(2) CD133+ CSCs were purified with MACS(magnetic activated cell-sorting system) CD 133 cell isolation kit, and were enriched by Serum-free medium under floating culture system.(3) The morphplpgical features of cells were observed using transmission election microscopy(TEM),and the autophagic vacuoles(AV) was observed using fluorescent microscope by monodansylcadaverin(MDC) staining.The intensity of autophagy was evaluated by flow cytometery.The expression level of LC3-Ⅱwas evaluated by Western blotting after 5-FU treatment.(4) The effect of autophagy on the chemotherapeutic was evaluated by cell cycle analysis,cell viability assay,sphere formation assay,Boyden chamber assay.Results:(1) The vibility of human colon cancer cell line SW480 was obviously inhibited with different contrations of 5-FU in time- and dose-dependent manners,half of inhibition concentration(IC50) value was (1.09±0.18)μg/mL.(2) Autophagsomes were observed under transmission election microscope,autophagy vesica was observed using fluorescent microscope and the expression levels of autopha related protein LC3-Ⅱwere significantly increased following 5-FU treatment in CD133+CSCs.(3) After the combination treatment of 5-FU and 3-MA,Cell cycle was evidently change.The stages'of G0/G1 and G2/M were shorten,and the stages'of S was evidently increased (P<0.05),compared with 5-FU alone. At the same time,the viability of CD133+ CSCs was significantly decreased from (81.8±4.6)% to (56.3±5.5)%(P<0.05), and the colony-forming efficiency from (81.1±3.4)% to (64.4±4.8)%(P<0.05). But the ability of invasion was not significantly changed. Conclusion:(1) 5-FU may induce autophagy in CD133+ CSCs.(2) In 5-FU-treated CD133+ CSCs, autophagy was closely correlated with cell proliferation,but not correlated with the ability of invasion. Autophagy may play a role as a self-defense mechanismand its inhibition could be employed to increase the cytotoxic effect of 5-FU to CD133+ CSCs.