Acetyl Shikonin To Prostate Cancer DNA Damage Related To Oxidative Stress Signal Pathway

ObjectivePC3 and DU145,two androgen independent prostate cancer(AIPC)cell lines,were used to clarify the role of ATM/ATR signal network in oxidative stress and DNA damage induced by acetyl shikonin elucidate,validate acetyl shikonin as a novel leading compound used in AIPC therapy.Methods1.PC3 and DU145 cells were seeded in 96-well plates,and treated with acetyl shikonin alone for 24 h,48 h or 72 h or with NAC.Cell viability was monitored with MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)assay.Cells were incubated with 10μL/well MTT for 4 h after treatment,and the absorbance was detected by measuring at 570 nm on a plate reader.2.DU145 and PC3 cells were treated with acetyl shikonin(0,2.5,5,10μM or 8μM acetyl shikonin+5 mM NAC)for different time(6 h,12 h,24 h).The cells were collected and incubated with Mitosox or C11-BODIPY,at 37℃,for 15 min in dark room.The fluorescence intensity,which means ROS lelves,was monitored by flow cytometry.Moreover,DU145 after acetyl shikonin treatment was incubated with DCFH-DA and then monitored by flow cytometry.3.To detect the influence of acetyl shikonin on DNA damage repair.Nuclear protein was separated after acetyl shikonin treatment for 24 h.DNA end-joining was investigated.4.After treatment with acetyl shikonin alone or acetyl shikonin+chemicals,cells lysed with RIPA buffer containing fresh protease inhibitors.Proteins were quantified by the BCA protein method.Equal amounts of proteins were separated by SDS-PAGE and electro-transferred onto nitrocellulose membrane.After being blocked with 5%non-fat milk in TBST buffer(20 mM Tris–HCl,137 mM NaCl,and 0.1%Tween 20,pH 8.0)for 1h at room temperature,the membrane was incubated with specific primary antibodies overnight at 4 ~oC,respectively,followed by HRP-conjugated secondary antibodies.The immuno complexes were visualized by enhanced chemiluminescence detection system and followed by exposure to X-ray films.5.After acetyl shikonin(0,2.5,5,8,10,NAC+8μM)treatment for indicated time,the cells were incubated with annexin-V/PI at dark room for 15 min.using flow cytometry to detect and analysis the apoptosis rate of cells.6.Different concentrations of acetyl shikonin(0,5,10μM)were added to DU145cells for 24 hours,nuclear proteins were extracted with nuclear protein extraction kit,the activity of caspase3 was detected with Caspase-3 enzyme activity kit.Result1.After acetyl shikonin were added with cells for 24 h,the IC50 of PC3 cells was8.59±0.42μM;the IC50 of DU145 cells was 7.5±0.38μM.After the addition of 10 mM of the ROS scavenger NAC,The viability of PC3 cells which treated with 10μM ROS scavenger NAC were obviously higher than the cells which were treated only with acetyl shikonin(8μM).2.After acetyl shikonin were added with cells for 6 h,12 h,24 h,dyed by BODIPY,detected that intracellular ROS level had no obvious change.After acetyl shikonin were acted on cells for 6 h,12 h,24 h,dyed by DFCH-DA,detected that intracellular ROS level increased significantly in a time and dose dependent manner.After acetyl shikonin were acted on cells for 6 h,12 h,24 h,dyed by Mitosox,detected that intracellular ROS level increased significantly in a time and dose dependent manner.But intracellular ROS level of NAC treated group was significantly decreased.3.Different concentrations of acetyl shikonin had effect on inhibiting DNA damage repair of DU145 cells in a time-dose dependent manner.4.After different concentrations of acetyl shikonin were added with DU145/PC3 cells for 24 h,the protein expression level ofγ-H2AX,PARP-1,p-Chk2 were clearly on the rise on the contrary,the protein levels of RAD51,KU80/KU80 were down regulated.The addition of NAC could make the proteins expression level down regulated significantly.After the addition of ATM inhibitor,the expression ofγ-H2AX was down regulated.5.After acetyl shikonin were added on PC3/DU145 cells for 6 h,12 h and 24 h,cells apoptosis ratio were increased significantly.The apoptotic ratio decreased significantly when 5 mM NAC was added into DU145 and 10 mM NAC was added into PC3.6.The activity of caspase3 presents a notable positive correlation with the action time of acetyl shikonin.ConclusionThe proliferation of PC3 and DU145 cells were inhibited by Acetyl Shikonin with a dose and time-dependent manner.Acetyl Shikonin promotes prostate cancer cells to produce ROS,in which superoxide from mitochondria was the major form.The Acetyl Shikonin-induced apoptosis of prostate cancer cells was dependent on ROS-mediated ATM/Chk2 activation and DNA damage.