| Liver cancer is one of the most common non-communicable diseases world-wide,and its incidence is on rising.Current treatments for liver cancer such as chemotherapy often do not produce desirable outcomes,thus,there is a need to seek alternative and complementary therapies.Natural compounds are the main source of anticancer drug development,the importance of which has long been recognized.Annona muricata is known as “ Cancer killer ” and can induce apoptosis of various cancer cells.However,the underlying molecular mechanisms remain largely unknown.The present study investigated whether and how ethanol extract of Annona muricata leaves could cause apoptosis using liver cancer HepG2 cells as an experimental model.The main results are listed below:1.The extract was able to inhibit the viability of the liver cancer HepG2 and colon cancer HCT116 cells in a dose-and time-dependent manner in MTT assay.The results of flow cytometry showed that the herbal extract increased the amount of sub-G1 cells,which evidences apoptosis.TUNEL assay confirmed that the extract caused apoptosis of the cancer cells.2.Proteomic analysis showed that the extract caused changes in 14 peptide spots on the two dimensional gels by comparing the samples from the treatment group and those from the control group.Peptide mass finger printing technology was used to identify the differently expressed proteins.Then,we used the 14 identified proteins for bioinformatic annotation for molecular pathways.We found endoplasmic reticulum(ER)stress pathway was axtivated,which involved 3 proteins which were GRP94,HSP70 and PDI-related protein 5.3.Further investigations using Western blot analysis showed that that the herbal extract did increase the protein levels of Bip and CHOP as well as enhanced the phosphorylation of PERK and eIF2α in a time-dependentmanner.Knockdown of protein level of CHOP using siRNA technology inhibited the herbal extract-elicited apoptosis.4.In order to further study the molecular mechanism of extract induced liver cancer cell apoptosis,we try to test whether the extract has caused the increase of reactive oxygen species(ROS).The level of intracellular ROS level was quantified using a fluorescent probe DCFH-DA,and antioxidant NAC was used to further verify the results.The results showed that after treatment for 2 h,the ROS level was significantly increased in the cancer cell treated with the extract,and NAC was able to inhibit the extract-induced ROS and cell apoptosis.In conclusion,our results indicate that:1.The ethanol extract of Annona muricata leaves can inhibit the viability of the liver cancer HepG2 in a dose-and time-dependent manner,due to the extract-induced apoptosis,which suggests that the extract can potentially be developed into an alternative and complementary treatment for cancers such as liver cancer.2.The ethanol extract of Annona muricata leaves induced liver cancer cell apoptosis through ER stress signaling pathway.3.The ethanol extract of Annona muricata leaves could also trigger ROS production for apoptosis. |
