A Group Of Rare Ginsenosides And Paclitaxel Induce Apoptosis Synergistically In A549 Cells

Lung cancer is one of the most common malignant tumors in the world with the highest morbidity and mortality.Globally,there are about 2 million new cases of lung cancer each year,of which non-small cell lung cancer（NSCLC）accounts for more than80 percent.Despite advances in lung cancer treatment,the five-year survival rate for patients with NSCLC is only 15.5%.Therefore,it is urgent to develop more sensitive and effective treatment strategies for lung cancer.Panax ginseng is a rare plant that can be used as medicine and food.It has the characteristics of good anti-tumor effect and low toxicity,and attracts extensive attention in the medical field.Ginsenoside is the prime active constituent of ginseng,which has anti-inflammation,cytotoxicity of cancer cells,immunity enhancement and other functions.The"Group of Rare Ginsenosides"（GRGs）used in this study was provided by Jilin Hyecoda Biotechnology Development Co.,Ltd.Its main components are Rh4,Rg3,Rg5,Rk1 and Rk3,accounting for 58%of the total saponins.Paclitaxel（PTX）,a diterpenoid compound extracted from Taxus chini,is a first-line drug widely used in the treatment of lung cancer.The cytotoxic effect of paclitaxel is due to its binding toβ-tubulin,which stabilizes the microtubule structure and blocks mitosis,leading to cell apoptosis.However,paclitaxel treatment has severe side effects such as leukopenia,neutropenia,and peripheral neurotoxicity,and has limitations such as poor specificity to tumor tissue and the tendency of tumor cells to develop drug resistance.By analyzing the mechanism of GRGs and paclitaxel on cancer cells,we speculated that the two drugs may synergistically inhibit the growth of NSCLC.In order to determine whether GRGs and paclitaxel have synergistic effects on the growth inhibition of A549 cells,we used MTT analysis to calculate the IC₅₀ of the two drugs,and designed a co-treatment experiment of the two drugs to calculate the dose-reduction index（DRI）and the combination index（CI）.The results showed that the IC₅₀ values of GRGs and paclitaxel against A549 cells were 71.78μg/m L and 10.15 n M,respectively,and the combination of GRGs and paclitaxel synergistically inhibited the growth of A549 cells.DAPI staining and cell morphological observation showed that typical apoptotic morphological changes,such as cell shrinkage,cell membrane blistering,nuclear condensation and rupture,and apoptotic corpuscle formation,occurred in the combination drug group at 12 hours.Flow cytometry analysis showed that,compared with the single drug group,significant apoptosis occurred at 12 hours in the combination group.At 18 hours,PARP began to break.Studies on the pathway of cell apoptosis induced by GRGs and paclitaxel synergically showed that both Caspase-3 and-9 were significantly activated in A549cells treated with GRGs and paclitaxel.Compared with the control group,the activation of Caspase-3 and-9 was 13.75-fold and 8.47-fold at 24 hours,respectively,and no significant activation of Caspase-8 was observed.Western blot analysis showed that the fracture zone of Caspase-9 was obvious,while the fracture zone of Caspase-8 was not obvious.Mitochondrial membrane potential measurement showed that the mitochond-rial membrane potential depolarization occurred at 12 hours in the combination drug group.Western blotting analysis showed that GRGs promoted the translocation of pacetaxel-induced Bax and Bak to mitochondrial outer membrane,mediated the release of Cytochrome c（Cyto.c）and Smac,and activated Caspase-9,which eventually activated the Caspase-3.Therefore,GRGs and paclitaxel co-initiate mitochondria-mediated endogenous apoptosis pathways.We also detected the anti-apoptotic proteins of Bcl-2 family and IAPs family proteins,and found that the protein levels of Bcl-2,Bcl-x L,c-IAP2 and X-IAP decreased gradually in the combination group.These results indicated that the down-regulation of anti-apoptotic proteins further promoted the process of apoptosis.Notably,GRGs reduced the killing effect of paclitaxel on human umbilical vein endothelial cells（HUVEC）,indicating that GRGs inhibited the injury of paclitaxel to vascular endothelial cells.Therefore,GRGs may effectively improve the therapeutic effect of paclitaxel on lung cancer.