Astragaloside Ⅱ Increases Cisplatin-induced Apoptosis By Regulating HMGB1-PI3K/Akt/mTOR-lysosomal Functions In Human Hepatoma Cancer Cells

Primary hepatocellular carcinoma is one of the most common malignant tumor diseases in china. Its mortality rank the second place of all malignant tumors in china. The conventional methods of clinical treatment include surgery and chemotherapy for liver cancer. Because of secret in liver cancer, most of the patients have been diagnosed in Advanced hepatocellular carcinoma. Chemotherapy has been considered a main means for Advanced hepatocellular carcinoma. As increase of chemotherapy drugs using time and frequency, the sensitivity of tumor cells to drugs significantly decreased. Study finds traditional Chinese medicine extracts to improve the life quality,extend survival time and increase the drug efficacy in patients. However, Chinese medicine extracts have the limited ability in cancer treatment. It is significant means that exploring Chinese medicine monomer combined with chemotherapy in treatment of liver cancer and clearing its mechanism.Autophagy is a protective phenomenon in cells, which is widely cognitive by one of lysosome way in degradation of proteins and organelles. Tumor cells can produce a large number of autophagic vacuole in the treatment of chemotherapy drugs in this stage of experiment research. Autophagosome make damage organelles and protein recurrently used by parcel, devouring, decomposition,which can Avoid tumor cell death. Autophagy have gotten wide attention by the researchers as a new drug therapy of cancer target. In the recent years, the endogenous HMGB1 may an important target protein in regulation of autophagy in cancer cells. Tumor cells can increase sensitivity to chemotherapy drugs by increasing the expression of cytoplasm HMGB1. The regulation mechanism of autophagy in cytoplasm HMGB1 may be associated with regulating the autophagy pathway PI3K/AKT/mTOR-lysosome.Our previous studies have confirmed using the chloroquine autophagy-lysosome inhibitors can inhibit protective autophagy in breast cancer cell line MCF-7 and significantly increase the MCF-7 sensitivity of chemotherapy drugs cisplatin.Astragaloside Ⅱ is the biological active monomers of traditional Chinese medicine astragalus extract. Our previous studies have found that Astragaloside Ⅱ can increase the sensitivity of 5-fluorouracil in Human hepatocarcinoma cell lines Bel-7402/FU. Whether the effect of increasing tumor cells sensitive to chemotherapy drugs by Astragaloside Ⅱ are universal? What is the specific mechanism? We design experiment to explore the effect and mechanism of Astragaloside Ⅱ for the autophagy and apoptosis by cisplatin in Human hepatocarcinoma cell lines HepG2 and SMMC-7721.Objectives:To investigate the Astragaloside Ⅱ increasing inhibition and apoptosis rate of human hepatic cancer cells by cisplatin and its possible mechanism of suppression in protective autophagy. By comparing the autophagy inhibitor chloroquine,we explore the autophagy inhibition mechanism of Astragaloside II is related to HMGB1-PI3K. /AKT/mTOR-lysosome.Methods:1. The HepG2 and SMMC-7721 inhibiting rate by cisplatin after alone or jointly Astragaloside II acting on cells was detected by MTT method.2. The expression of autophagosome was detected by Acridine orange staining and content of autophagy protein in twe live cancer cells was detected by Western blotting after different concentration of cisplatin.3. The expression of PI3K, AKT, p-AKT,mTOR, p-mTOR protein were determined by Western blot in HepG2 and SMMC-7721 cells treated with increasing time of cisplatin4. The change of apoptosis rate in human hepatic cancer cells by cisplatin after Chloroquine acting was detected by Hoechst33342,Flow cytometry and Western blotting.5. The change of apoptosis rate in human hepatic cancer cells by cisplatin after Astragaloside Ⅱ acting was detected by Hoechst33342,Flow cytometry and Western blotting.6. P62,LC3Ⅰ/Ⅱ expression in HepG2 and SMMC-7721 cells treated Chloroquine or Astragaloside Ⅱ with the indicated time of cisplatin were analysised by Western blotting7. Western blot was performed to test the expression of HMGB1,PI3K,AKT,p-AKT, mTOR,p-mTOR protein in HepG2 and SMMC-7721 cells treated with Astragaloside Ⅱ and cisplatin.8. Lysosomes fuction was tested by LysoTracker and Acridine orange. CSTB/CSTL protein was detected by Western blotting.Results:1. Astragaloside Ⅱ increases inhibition rate of HepG2 and SMMC-721 cells by cisplatin.2. Cisplatin induces autophagy in HepG2 and SMMC-7721 cancer cells.3. Cisplatin induces autophagy by PI3K/AKT/mTOR pathway in HepG2 and SMMC-7721 cancer cells.4. Chloroquine enhances inhibition and apoptosis of HepG2 and SMMC-7721 cells to cisplatin by inhibiting autophagy.5. Astragaloside Ⅱ sensitizes HepG2 and SMMC-7721 cancer cells to cisplatin.6. Astragaloside Ⅱ inhibit cisplatin-induced autophagy,which is similar to Chloroquine.7. Astragaloside Ⅱ block cisplatin-induced HMGB1 transfering from nucleus to cytoplasm.8. Astragaloside Ⅱ inhibit cisplatin-induced autophagy by up-regulating PI3K-AKT-mTOR pathway.9. Astragaloside Ⅱ inhibit autophagy by affecting lysosomal function.1. Astragaloside Ⅱ could increase inhibition and apoptosis rate of human hepatic cancer cells by cisplatin.2. Astragaloside Ⅱ sensitizes HepG2 and SMMC-7721 cancer cells to cisplatin by inhibiting cisplatin-induced protective autophagy.3. Astragaloside Ⅱ was capable of inhibiting autophagy with HMGB1-PI3K-AKT-mTOR-lysosomal functions in HepG2 and SMMC-7721 cells.