Effects Of The Modulator Of Autophagy On The Chemotherapy Sensibility Of Gastric Cancer MGC803Cells And Its Mechanism

Objective: To investigate the effects of the therapy of5-FU combined with theautophagy inhibitor or the autophagy activator on the proliferation, autophagy andapoptosis of gastric cancer MGC803cells, to discuss the sensibilization of thechemotherapy by the modulator of autophagy and its mechanism.Methods: Human gastric cancer cells from MGC803cell strain were intervented by5-FU treatment group, RAD001treatment group which would up-regulated autophagyexpression, Bafilomycin a1treatment group which would down-regulated autophagyexpression,5-FU+RAD001combined treatment group,5-FU+Bafilomycin a1combinedtreatment group and control group. After the MGC803cells were intervented for24hours,48hours and72hours, the MTT assay was used to determine the inhibition effectson cell proliferation. The expression of autophagy was monitored throughmonodansylcadaverin(MDC) staining and immunefluorescence of LC3which is thespecific protein of autouphagy. PI-Annexin V staining with flow cytometry was took out toquantitating the cell apoptosis. The autophagy-related protein Beclin-1, P62and CathepsinB protein expression were detected by Western blot. The autophagy-related genes Atg7,Atg12and Lamp-2mRNA expression were detected by RT-PCR.Results: After the treatment of gastric cancer MGC803cells, the5-FU treatmentgroup,5-FU+RAD001combined treatment group and5-FU+Bafilomycin a1combinedtreatment group had inhibited the proliferation of MGC803cells significantly.Respectively in the5-FU+Bafilomycin a1combined treatment group, the inhibition ratio of24h,48h and72h were32.06%±3.25%,70.15%±2.36%and89.75%±2.76%, comparedwith other groups the inhibition rate of5-FU+Bafilomycin a1combined treatment group isthe highest (P<0.05). The monodansylcadaverin (MDC) staining displayed that the autophagic vacuoles of24h,48h and72h increased gradually in RAD001treatment groupand decreased in Bafilomycin a1treatment group. The number of autophagic vacuoles inthe5-FU+RAD001combined treatment group was the highest when that in the5-FU+Bafilomycin a1combined treatment group was the least. The immunefluorescenceexpressions of LC3was significantly up-regulation in the5-FU+RAD001combinedtreatment group and down-regulation in the5-FU+Bafilomycin a1combined treatmentgroup under the fluorescence microscope, compared with other groups. PI-Annexin Vstaining with flow cytometry showed the result that the gastric cancer MGC803cells underthe combined treatment of5-FU+Bafilomycin a1had more increased apoptosis rate thanother groups. It indicated that the5-FU+Bafilomycin a1combined treatment group reducedthe expression of autophagy-related protein Beclin1, enhanced the expression of P62andCathepsin B protein detected through western blot. The RT-PCR detected that theexpression of Atg7, Atg12and Lamp-2mRNA were reduced in the5-FU+Bafilomycin a1combined treatment group.Conclusion: Bafilomycin a1+5-FU combined treatment group has the highest cellularproliferation inhibition rate by down-regulated autophagy. The sensitization ofBafilomycin a1to chemotherapy is implemented by inhibiting the protective autophagy.The mechanism may be related to the regulation of lysosomes and the enhancement of cellapoptosis.