HepG2, HLF, and SMMC-7721 cells were more sensitive to GA than LM3, Hep3B, and HLE cells

HepG2, HLF, and SMMC-7721 cells were more sensitive to GA than LM3, Hep3B, and HLE cells. IRE1 may be of benefit to enhance the antitumor activity of GA. Introduction Hepatocellular carcinoma (HCC) is the sixth most common malignant tumor, and the third leading cause of cancer mortality worldwide. Despite the introduction of new chemotherapeutic drugs, surgery remains the most effective way to treat HCC. However, surgery is limited by a high incidence of recurrence and intrahepatic/extrahepatic metastasis1C3. Glycyrrhizin and glycyrrhetinic acid (GA) are the most important chemical components of the traditional Chinese medicine, and and anti-proliferative activity of GA using the Cell Counting Kit-8 (CCK-8) assay on a panel of human HCC cell lines including HepG2, SMMC-7721, HLF, HLE, LM3 and Hep3B. Incubation of cells with increasing concentrations of GA for 24 or 48?h led to reduction in viability (Fig.?1a). The average inhibition by 150?M GA treatment for 24?h was 46.2, 45.1, 32.1, 27.1, Rabbit polyclonal to UBE2V2 5.8 and 21.3% in HepG2, SMMC-7721, HLF, HLE, LM3 and Hep3B cells, respectively. IC50 values for these HCC cells for 48?h were 124.0??5.0, 88.3??2.5, 131.5??4.5, 137.3??6.5, 218.0??38.0, and 162.0??3.5?M, respectively (Supplementary Fig.?1a). GA treatment for 24 and 48?h had a slight effect on 7701 normal liver cells (Supplementary Fig.?1b). HepG2, HLF, and SMMC-7721 cells were more sensitive to GA than LM3, Hep3B, and HLE cells. These three HCC cell lines were selected for subsequent studies. To test the effect of GA on HCC cell tumorigenicity, colony formation assays were conducted. GA reduced the numbers of colonies of all HCC cell lines tested in a dose-dependent manner (Fig.?1b and Supplementary Fig.?1c). These data demonstrated that GA reduced HCC cell proliferation in a dose-dependent manner and (Supplementary Fig.?2b). Taken together, these data demonstrated that GA induced autophagy and in HCC cells. Autophagy inhibition augments apoptotic cell death induced by GA We determined whether GA-induced autophagy was cytoprotective or cytotoxic. Autophagy was inhibited pharmacologically using CQ or genetically by siRNA against autophagy protein ATG5 and ATG7. As expected, CQ increased accumulation of LC3BII (Fig.?4e). Treatment with CQ significantly increased GA-induced apoptosis in HepG2 and HLF cells (Fig.?4aCd). This was associated with increased cleaved poly (ADP-ribose) polymerase (PARP) in GA/CQ-treated cells compared with GA-treated cells when measured by western blotting (Fig.?4e). To confirm these results, we used siRNA to silence ATG5 and ATG7 expression, which blocked autophagy initiation. Western Gatifloxacin hydrochloride blotting showed that that ATG7 and ATG5 were efficiently decreased (Supplementary Fig.?2d). The expression of the autophagy marker LC3BII was reduced and cleaved PARP accumulated in ATG-silenced HepG2 and HLF cells with GA treatment (Fig.?4f,g). These changes were associated with a significant increase in apoptosis (Fig.?4aCd). To investigate further whether autophagy mediated P62 degradation, we used the autophagy inhibitor, CQ or ATG7 siRNA, to block autophagy. P62 expression was increased further by autophagy inhibition (Supplementary Fig.?3a). This suggested that autophagy was responsible for degradation of some P62. To prove that autophagy was antagonistic for apoptosis, we used a Gatifloxacin hydrochloride classic autophagy inducer, rapamycin, which promoted autophagy by inhibiting the activity of the mammalian target of the rapamycin complex 123. HepG2 cells were treated with GA in the presence or absence of rapamycin for 24? h and then subjected to the CCK8 assay and western blotting. We observed that rapamycin partially reduced GA-induced cell death and expression of cleaved PARP (Supplementary Fig.?3b,c). Collectively, these data indicated that the GA-induced autophagic response played a cytoprotective role in counteracting apoptosis. Open in a separate window Figure 4 Autophagy inhibition augments apoptotic cell death induced by GA. (aCd) HepG2 and HLF cells were transfected with ATG5/7 siRNA and negative control for 48?h, or pretreated with or without CQ for 1?h and then with GA (200?M) for 24?h. Apoptosis was examined by FACS. (e) Cells were pretreated with or without CQ for 1?h prior to treatment with GA for 24?h. PARP was analyzed by western blotting. (f,g) Cells were transfected with ATG5/7 siRNA and the negative control Gatifloxacin hydrochloride for 48?h prior to GA treatment for 24?h. PARP was analyzed by western blotting. Data presented as mean??SEM from three independent experiments. *(Supplementary Fig.?2b). Previous studies have reported that reactive oxygen species (ROS) generation could trigger ER stress25,26. Pretreatment with ROS inhibitor N-acetylcysteinepartially blocked expression of ATF4, CHOP, and IRE1 in GA-treated HepG2 cells (Supplementary Fig.?2e). These data indicated that GA induced ER stress via ROS production. Open in a separate window.