OBJECTIVE: To explore the molecular mechanism of sorafenib resistance in hepatoma cells and identify for new targets to reverse drug resistance. METHODS: THP-1 cells were induced into M2 tumor-associated macrophages (M2-TAMs) in vitro and identified by immunofluorescence. SMMC-7721 cells were co-cultured with M2-TAMs with or without sorafenib treatment. CCK-8 assay was used to observe the inhibitory effect of sorafenib on the cell proliferation. Annexin V/PI double staining and protein immunoblotting were used to assess the effect of sorafenib on the proliferation, apoptosis and the expressions of apoptosis-related proteins and autophagy-related protein in SMMC-7721 cells co-cultured with M2-TAMs in the presence or absence of the autophagy inhibitor chloroquine (CQ). RESULTS: The IC of sorafenib at 48 h was 2.25 μmol/L in SMMC-7721 cells cultured alone, and increased to 4.72 μmol/L in the cells co-cultured with M2-TAMs. Compared with the cells cultured alone, the co-cultured SMMC-7721 cells showed significantly reduced apoptosis rate in response to sorafenib ( < 0.01) and significantly increased expression of Bcl-2 and Bcl-2/Bax ratio ( < 0.05) with also increased LC3-II/LC3-I ratio ( < 0.001) and lowered expression of p62 ( < 0.05), suggesting a significantly enhanced level of autophagy. CQ treatment significantly inhibited the proliferation of the co-cultured SMMC-7721 cells ( < 0.05), increased the cell apoptosis ( < 0.05) and reduced the Bcl-2/Bax ratio ( < 0.01). CONCLUSIONS M2-TAMs can attenuate the inhibitory effect of sorafenib on the proliferation of hepatoma cells by increasing the level of autophagy, suggesting a new strategy for reversing sorafenib resistance induced by the tumor microenvironment by inhibiting autophagy.
Apoptosis ; Autophagy ; Carcinoma, Hepatocellular ; Cell Line, Tumor ; Humans ; Liver Neoplasms ; Macrophages ; Sorafenib

OBJECTIVE: To evaluate the regulatory effect of berberine on autophagy and apoptosis balance of fibroblast-like synoviocytes (FLSs) from patients with in rheumatoid arthritis (RA) and explore the mechanism. METHODS: The inhibitory effect of 10, 20, 30, 40, 50, 60, 70, and 80 μmol/L berberine on RA-FLS proliferation was assessed using CCK-8 method. Annexin V/PI and JC-1 immunofluorescence staining was used to analyze the effect of berberine (30 μmol/L) on apoptosis of 25 ng/mL TNF-α- induced RA-FLSs, and Western blotting was performed to detect the changes in the expression levels of autophagy- and apoptosis-related proteins. The cells were further treated with the autophagy inducer RAPA and the autophagy inhibitor chloroquine to observe the changes in autophagic flow by laser confocal detection of mCherry-EGFP-LC3B. RA-FLSs were treated with the reactive oxygen species (ROS) mimic H₂O₂ or the ROS inhibitor NAC, and the effects of berberine on ROS, mTOR and p-mTOR levels were observed. RESULTS: The results of CCK-8 assay showed that berberine significantly inhibited the proliferation of RA-FLSs in a time- and concentration-dependent manner. Flow cytometry and JC-1 staining showed that berberine (30 μmol/L) significantly increased apoptosis rate (P < 0.01) and reduced the mitochondrial membrane potential of RA-FLSs (P < 0.05). Berberine treatment obviously decreased the ratios of Bcl-2/Bax (P < 0.05) and LC3B-II/I (P < 0.01) and increased the expression of p62 protein in the cells (P < 0.05). Detection of mCherry-EGFP-LC3B autophagy flow revealed obvious autophagy flow block in berberine-treated RA-FLSs. Berberine significantly reduced the level of ROS in TNF-α-induced RA-FLSs and upregulated the expression level of autophagy-related protein p-mTOR (P < 0.01); this effect was regulated by ROS level, and the combined use of RAPA significantly reduced the pro-apoptotic effect of berberine in RA-FLSs (P < 0.01). CONCLUSION Berberine can inhibit autophagy and promote apoptosis of RA-FLSs by regulating the ROS-mTOR pathway.
Humans ; Synoviocytes ; Berberine/metabolism* ; Reactive Oxygen Species/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Hydrogen Peroxide/metabolism* ; Sincalide/metabolism* ; Cell Proliferation ; Arthritis, Rheumatoid/metabolism* ; Signal Transduction ; TOR Serine-Threonine Kinases/metabolism* ; Apoptosis ; Fibroblasts ; Autophagy ; Cells, Cultured