The Mechanism Of Gambogic Acid Increasing Chemosensitivity And Inducing Autophagy In Pancreatic Cancer Cells

Objective:1.To investigate whether gambogic acid inhibits the growth of pancreatic cancer cells;2.To investigate whether gambogic acid can promote the chemosensitivity of pancreatic cancer cells,and to study its mechanism;3.To investigate whether gambogic acid induces autophagy and to clarify its character in in pancreatic cancer.Methods: 1.MTT,cell cycle and apoptosis assays were performed to demonstrate the effect of gambogic acid against pancreatic cancer cells;2.Pancreatic cancer cells were treated with combination of gambogic acid and gemcitabine,the effects of gambogic acid on the chemosensitivity of them to gemcitabine were analyzed by MTT and cell apoptosis assays.The effects of gambogic acid on the expression of resistance genes RRM1 and RRM2 were analyzed by q-PCR and Western-Blot.xenograft tumor model constructed with nude mice was used to detect the efficacy of the treatment of gambogic acid and gemcitabine;3.Autophagy of pancreatic cancer cells was detected by Western-Blot,acridine orange staining and transmission electron microscopy.Autophagic flux was observed through transfecting GFP/RFP LC3-Ⅱ lentiviral vector to pancreatic cancer cells.Inhibition of autophagy by using chloroquine,3-MA or downregulating Atg-7 with to observe the effects of autophagy on gambogic acid against pancreatic cancer cells.Subcutaneous xenograft tumor model was constructed to verify the in vitro studies.Results: 1.The results showed that gambogic acid effectively inhibited the proliferation and cell cycle of pancreatic cancer cell and induced cell apoptosis;2.The results showed that combination of gambogic acid and gemcitabine could synergistically inhibit the growth of pancreatic cancer cells.Gambogic acid inhibited the phosphorylation of ERK and AKT protein and simultaneously supressed the expression of RRM2,but not RRM1.In addition,E2F1 was inhibited by gambogic acid,and down-regulation of E2F1 reduced the expression of RRM2.In vivo study verified that combination of gambogic acid and gemcitabine synergistically repressed the growth of subcutaneous tumors;3.The results showed that gambogic acid induced the expression of autophagy-related protein LC3-Ⅱ,and inhibited the expression of p62,and promoted the formation of autophagosomes and acidic vesicular organelles and autophagic flux in pancreatic cancer cells.Inhibition of autophagy increased the anti-tumor effect of gambogic acid in pancreatic cancer cells.Combination of gambogic acid and chloroquine promoted the cell apoptosis through increasing the ROS accumulation in pancreatic cancer cells.The in vivo study verified the significant anti-tumor effect of the combination treatment of gambogic acid and chloroquine in pancreatic cancer.Conclusions: 1.Gambogic acid can effectively inhibit the growth of pancreatic cancer cells;2.It promotes the sensitivity of pancreatic cancer cells to gemcitabine by inhibiting the expression of RRM2;3.Gambogic acid can induce protective autophagy in pancreatic cancer cells.Combination of chloroquine and gambogic acid can effectively treat pancreatic cancer through increasing the accumulation of ROS in cells.