Two Anthraquinone Derivatives Induce HCT116 Cells Death Through ROS-JNK Signaling Pathway

Anthraquinone compounds which mainly exist in the giant knotweed,rhubarb,aloe and other Chinese herbal medicine,have antibacterial,hypotensive,antioxidation,antitumor and other biological activities.And some drugs have been applied in clinical.There are some common disadvantages that natural anthraquinone compounds are lower contents and cardiotoxicity,but also extract and separate quite difficult.Therefore,combined with the mechanism of antitumor drugs and the structure-effect relationship of anthraquinones derivatives,synthetic derivatives which are high efficiency and low toxicity is an important approach to the development of new drugs.Seven novel anthraquinone-amide acid derivatives were designed and synthesized in our lab previously.MTT assay detected the inhibition of the seven derivatives on eight cancer cells growth.The results showed that five compounds whose replace groups were glycine,leucine,proline,valine,phenylalanine can dramatically inhibit cell proliferation of esophageal cancer EC9706 cells,bile duct cancer QBC-939 cells,liver cancer HepG2 cells,stomach cancer SGC-7901 cells,colon cancer HCT116 cells,which replace of leucine and valine for anthraquinone derivatives(marked as:C2 and C3)displayed the most significantly proliferation inhibition effect on HCT116 cells.To investigate the effect and mechanisms of C2 and C3 induced HCT116 cells death,our studies as follows:1.The interactions with BSA and DNA of C2 or C3 were investigated via UV-Vis spectroscopy,fluorescence spectroscopy and circular dichroism(CD)methods.The results showed that two derivatives combined with BSA made its endogenous fluorescent static quenching,and destroyed the alpha helical structure of BSA.The binding mode of the derivatives with calf thymus DNA belongs to intercalation action with weakening the energy of base pair.Then two derivatives weakened the interaction between their bases by intercalation with DNA interactions.C2 and C3 interaction with BSA and DNA was quite significant,which laid the theoretical foundation for further testing C2 and C3 anticancer activity.2.The result indicated that C2 and C3 could inhibit the proliferation of HCT116 cells in a dose-dependent manner by MTT assay.The morphological changes of HCT116 cells were observed using an inverted microscope,after cells were treated with different concentration of two derivatives for twenty-four hours.Found that with increasing concentration of derivatives,cell shrinkage occurred significantly,rounded and cell spacing increases.Furthermore,4',6'-diamidino-2-phenylindole staining(DAPI staining)indicated nuclear condensation in nuclear morphology,and nuclei exhibited different sizes and shapes.Apoptosis and cell cycle in HCT116 cells were analyzed by flow cytometry.It was well appreciated that cell cycle was arrested and the phenomenon of apoptosis was occurred.Owing to the phenomenon of apoptosis is not notable,we supposed derivatives induced autophagy in HCT116 cells.The results suggested that two derivatives significantly induced autophagy in HCT116 cells through the MDC dyeing and GFP-LC3 detection.Because of similar structures of the two compounds,the same mechanism,in case of C3 on cells,then western blotting was used to detect the expression of the changes of apoptosis-related protein including PARP?Cleaved caspase 9?Cleaved caspase 3?Bcl-2?p-Bcl-2?Bcl-xL?p-Bcl-xL?Bax,and the changes of autophagy-related protein such as P62?LC3??LC3??and Beclin-1.After a certain concentration of C3 treated on cells over time,we validated that apoptosis-related proteins including Cleaved PARP?Cleaved caspase 9?Cleaved caspase 3?p-Bcl-2?p-Bcl-xL and Bax increased significantly,vice versa,autophagy-related protein,Bcl-2?Bcl-xL,decreased remarkably,suggesting that C3 significantly induced HCT116 cells apoptosis;furthermore,the expression of autophagy related protein P62 decreased significantly and the expression of LC3?,Beclin 1 increased significantly learned that C3 significantly induced autophagy HCT116 cells occur.3.To further test the mechanism of C3-induced autophagy and apoptosis in HCT116 cells,using flow cytometry detects the change of ROS levels after different drugs treated HCT116 cells.It turned out that after different concentrations of C3 treated HCT116 cells,ROS levels of cells increased significantly.However,ROS was decreased by NAC and C3 treated,compared with the role of C3 to HCT116 cells.In the meantime,the expression of p-JNK1and p-JNK2 increased notably in HCT116 cells in a time-dependent manner by western blotting.When NAC inhibiting ROS,protein expressions of p-JNK1 and b-J-NK2 were significantly reduced.Accordingly,the derivatives-provoked autophagy and apoptosis of HCT116 cells may be via the activation of ROS-JNK signaling pathway.After the specific inhibitor of JNK phosphorylation SP6500125 and C3 treated HCT116 cells,the levels of Cleaved caspase 9.Cleaved caspase 3?LC3? and LC3??Beclin-1 were down-regulated.The cell vitality significantly rises by MTT assay after being treated with SP6500125 and C3.We got the similar results after treating with JNK1/2siRNA.Obviously,C3 is likely to induce HCT116 cells autophagy and apoptosis through the ROS-JNK signaling pathways.The results of the study will lay the foundation for anthraquinone derivatives anticancer activity researches,and provide a scientific basis for the development of new drugs.