The Study On Antitumor Activity Of Platycodin D And Metal (Ruthenium,Iridium) Complexes

Objectives:In the treatment of cancer,chemotherapy,surgery and radiation therapy are still usually used treatments.In order to achieve a better therapeutic effect,in recent years,some active ingredients in Traditional Chinese Medicine in the anti-tumor effect have also been paid extensively attention.Platycodin D that is the main active ingredient of platycodon grandiflorum saponins has anti-inflammatory,antitussive and expectorant antioxidant activity,in addition,it has antitumor activity on tumor cells.However,the anti-tumor research is still not comprehensive,and the mechanism of action is not clear.Therefore,there are still many issues to be resolved.In addition,in terms of chemical drugs,it has been believed that ruthenium and iridium complexes have good antitumor potential.However,the antitumor activity of the complex and its signaling pathway are not definitely concluded.The antitumor mechanism of ruthenium and iridium complexes still needs further investigation.Methods:The platycodin D and ruthenium,iridium complexes designed and synthesized with antitumor activity were studied.MTT method was used to screen out the platycodin D and ruthenium and iridium complexes in vitro antitumor activity;endocytosis,AO/EB staining and flow cytometry were used to analyze whether the drug can induce apoptosis;DCFH-DA and JC-1 staining were used to detect the changes of intracellular reactive oxygen species and mitochondrial membrane potential by fluorescence microscopy and flow cytometry;single cell gel electrophoresis was carried out to detect whether the drug can damage cell DNA;AO staining was used to investigate whether the platycodin D can cause cell autophagy;flow cytometry was used to study whether the cell cycle was blocked after drug treatment;Transwell assay was performed to aasay whether drug influenced cell invasion;The changes of the expression of apoptosis related proteins in the cells were detected by western blot at the molecular level.Results:(1)MTT experiments show that platycodin D and ruthenium,iridium complexes synthesized have different antitumor activities,with strong anti-tumor activity of certain tumor cells.(2)AO/EB staining observed the morphological characteristic of apoptosis of tumor cells after treatment with drugs;cell apoptosis was detected by flow cytometry,and the apoptosis induced by drugs was further confirmed;the cell cycle was blocked in G0/G1 or G2/M.(3)The change of intracellular ROS level and mitochondrial membrane potential are detected by staining with DCFH-DA and JC-1 respectively.That the cells emitted bright green fluorescence was observed under the fluorescence microscope,indicating intracellular ROS level increased,mitochondrial membrane potential decreased;at the same time,the fluorescence intensity was detected by flow cytometry,and the results were in accordance with the results observed under microscope.(4)Single cell gel electrophoresis shows the platycodin D damaged DNA of PC-12 cells, leading to a fragment of DNA.AO staining showed that autophagy in PC-12 cells was induced by platycodin D.(5)Transwell assay shows that platycodin D and ruthenium,iridium complexes could effectively inhibit cell invasion ability.(6)Western blot illustrated that the expression of Caspase 3,Bid and Bak proteins was upregulated,while Procaspase 7,Bcl-2 and Bcl-x proteins were down regulated after the action of drugs,revealing that the platycodin D and ruthenium,iridium metal complexes may induce apoptosis through mitochondrial pathway.Come to a comprehensive analysis,we can infer that the platycodin D and the newly synthesized ruthenium and iridium metal complexes may cause the decline of mitochondrial membrane potential,the downregulation of expression of Bcl-x and Bcl-2protein,further activation of Caspase 3 to induce cell death and achieve anti-tumor effect.