The Study On The Mechanisms Of SG511-BECN, A Novel Oncolytic Adenovirus, In K562 Cell Line

Part1:The Construction and Cultivation of LC3-EGFP-K562 Cell LineObjective:We constructed LC3-K562 cell line,which could be researched to confirm SG511-BECN may induce autophagy in leukemia cell line and implore their mechanisms.Methods:We transfected plasmid p^EGFP-LC3 to K562 cell line by electroblot. Then we used limiting dilution assay to construct LC3-K562 cell line,which could express LC3 resistantly.The transfection efficiency was measured via flow cytometry analysis.Furthermore we used fluorescence microscop to observed obvious green puncturing fluorescence after the treatment by Rapamycin--mTOR inhibitor.Results:(1)The transfection efficiency of p^EGFP-LC3 was 73.6%.(2) we observed obvious green puncturing fluorescence in cytoplasm after treatment with rapamycin. (3)We constructed LC3-K562 cell line successfully.(4)Rapamycin could induce autophagy in K562 cell line. Part2:The mechanisms and effects of SG511-BECN on k562 cell lineObjective:We investigated the antitumor activity of SG511-BECN against k562 cell line to comfirm SG511-BECN could induce K562 cell autophagy and kill K562 cell ultimately.By this study,we hope to find a target therapy for leukemia with the strategy of gene-viral therapy.Method:The infection rate of SG511 was measured by flow cytometry analysis.We detected autophagy induced by SG511-BECN via LC3-GFP-K562 ceil fluorescence chage and observed the expression of LC3â…¡via western blot in SG511-BECN-infected K562 cell.Subcellular localization of beclin-1 in K562 cell treated with SG511-BECN 25MOI was observed by confocal microscope.We used UVRAG-siRNA to interfere UVRAG gene in K562 cell to detect the effect with autophagy via green puncturing fluorescence change and further detect the cell viability by MTT assay.We tested the generation of ROS in K562 cell treated with SG511-BECN 25MOI or combined with ROS inhibitor Trion1mM/NAC4mM via ROS-Kit,further determined K562 cell viability by MTT assay.We used MTT assay to detect cell toxcicity with SG511-BECN on normal human mononuclear cells.Results:(1)SG511 can effectively infect leukemia cell lines in vitro.(2)We first confirmed Oncolytic Adenovirus SG511-BECN can induce autophagy in LC3-K562 cell.And beclin-1 localized in endoplasmic reticulum.(3)When LC3-K562 cell infected by SG511-BECN was treated with UVRAG-siRNA,autophagy would be suppressed obviously,and further detected that LC3-K562 cell was protected via MTT assay.(4)we used ros-kit to confirm during the process of autophagy,ros was an indispensable agent. (5)The cytotoxicity on normal human mononuclear cells with SG511-BECN was much. lower than K562 cell. Conclusion:A novel oncolytic adenovirus SG511-BECN can efficiently infect leukemia cell lines and cause K562 cell lysis as well as induce autophagic cell death.By comparison, the cytotoxicity of SG511-BECN on normal human mononuclear cells was lower.Our study determined UVRAG can induce K562 cell autophagy with beclin-1 together. During this procedure,autophagy induced the generation of ROS and after adding Trion or NAC(ros inhibitors),ROS was blocked,furthermore autophagy was inhibited.