Dexamethasone Enhances Trichosanthin-Induced Apoptosis In The HepG2 Hepatoma Cell Line By Inhibition Of NF-κB Activation

Part I The role of NF-κB/IκB signaling pathway in trichosanthin-induced apoptosis in the HepG2 hepatoma cell lineBackground:Trichosanthin (TCS) is a type I ribosome-inactivating protein, extracted from the Chinese medicinal herb trichosanthes kirilowii, and has various pharmacological activities, including abortifacient, immunoregulatory, anti-tumor, and anti-viral activity. In recent years, TCS has been the subject of much research because of its potential antitumor activities. Many reports have revealed that various mechanisms participated in TCS-induced apoptosis. TCS-induced cytotoxicity of HepG2 cells was not as significant as that of JAR and HeLa cells, the underlying mechanisms remain to be elucidated. Nuclear factor-kappa B (NF-κB) is an important transcription factor that regulates the expression of various genes involved in inflammation, cell proliferation and apoptosis. So far, it is need to elucidate that whether NF-κB activation participated in TCS-induced apoptosis and the potential role of NF-κB in the low sensitivity of hepatoma cells to TCS.Objective:TCS low-sensitive hepatoma cell line HepG2 was employed for investigation. The present study investigated whether NF-κB activation was induced in TCS-induced apoptosis of HepG2 cells and the potential role of NF-κB in the low sensitivity of hepatoma cells to TCS-induced apoptosis.Methods:After treatment with various concentrations of TCS for 48 h, cell viability assays were performed using the MTT method. HepG2 cells were treated with 50μg/ml TCS at different time intervals, and cytoplasmic and nuclear extracts were prepared. The levels of IκB-αand Cox-2 were determined in cytoplasmic extracts, and the level of NF-κB p65 was detected in both cytoplasmic extracts and nuclear extracts by Western blot assay. Luciferase reporter assay detected the NF-κB transcriptional activity induced by TCS. And apoptosis was evaluated with Hoechst 33258 staining. Additionally, we took advantage of dominant-negative IκB (IκB-DM) over-expression and chemical inhibitor PDTC to inhibit NF-κB activation, and analyzed the changes of cell viability and apoptosis induced by TCS.Results:(1) After treatment with 200μg/ml TCS for 48 h, the cell growth inhibition of HepG2 and MIHA cells were about 48% and 17%, respectively. TCS had relatively low toxicity to HepG2 cells, but could still discriminate HepG2 cells and MIHA cells. (2) TCS could rapidly decrease the level of IμB-αprotein, and also decrease the level of cytoplasmic NF-κB p65 subunit and lead to an elevation of the p65 subunit level in the nuclei, indicating nuclear translocation of NF-κB. and decrease of COX-2 expression in HepG2 cells. TCS could induce NF-κB transcriptional activity in a time-dependent manner. In addition, TCS could gradually increase the level of downstream factor, Cox-2 protein. (3) It is established stably-transfected HepG2 cell lines expressing either IκB-DM or a mock control transfected with plasmid pEGFP-N1. After treatment with TCS, the changes of the level of IκB-αprotein and NF-κB p65 subunit in the mock control cells were similar to that observed in untransfected HepG2 cells. In contrast, the basal level of IκB-αprotein increased significantly in IκB-DM-transfected cells. In addition, there was no obvious cytoplasmic down-regulation of IκB-αprotein and p65 subunit in cells induced by TCS-induced for 3 h, and there was also no obvious elevation of the p65 subunit in the nuclei. MTT assays demonstrated that the viability of the IKB-DM-transfected cell group treated with 50μg/ml TCS for 48 h was 43%, compared with 63% in the mock-transfected cell group (P＜0.05). In addition, HepG2 cells were pretreated with inhibitor of NF-κB, PDTC, and stained with Hoechst 33258. There were 16% more apoptotic cells in the PDTC pretreatment group compared with the TCS alone group (P<0.05).Conclusion:(1) TCS can discriminate tumor cells (HepG2 cells) and normal cells (MIHA cells), which make TCS possessed a good application prospect of anti-tumor. (2) TCS can cause rapid down-regulation of IKB-a protein in the cytoplasm of HepG2 cells, liberation of NF-κB dimmers, entrance to the nucleus, and activation of NF-κB. (3) NF-κB activation played an anti-apoptotic role in TCS-treated cells. At least in part, by inhibiting the NF-κB signaling pathway can enhance trichosanthin-induced apoptosis in the HepG2, and thus strengthening the antitumor effects of TCS.PartⅡDexamethasone enhances trichosanthin-induced apoptosis in the HepG2 hepatoma cell lineBackground:Dexamethasone, a synthetic glucocorticoid, is widely used in clinical settings for its anti-inflammatory and immunomodulatory effects. Dexamethasone can treat cancers, such as lymphocytic leukemia, Hodgkin's disease and non-Hodgkin's myeloma, through effective inhibition of NF-κB activity and induction of apoptosis. In recent years, researchers have tested the effect of dexamethasone co-treated with antitumor drugs, such as 5-fluorouracil, cisplatin and so on, on solid cancers. The modulatory effects of dexamethasone on TCS mediated apoptosis in human hepatoma cells have not yet been reported.Objective:The present study investigated the modulatory effects of dexamethasone on TCS induced apoptotic death in the human HepG2 hepatoma cell line, which highlights the possibility of combined drug application of TCS and dexamethasone in the clinical treatment of hepatoma.Methods:After pretreated with or without 1μM dexamethasone for 24 h, and then treated with different concentrations of TCS, cell viability assays were performed using the MTT method. The levels of IκB-a in cytoplasmic extracts was detected by Western blot assay. And apoptosis was evaluated with Hoechst 33258 staining.Results:(1) Our results demonstrated that dexamethasone could enhance TCS induced apoptosis in the hepatoma cell line HepG2, decreasing IC50 values from in excess of 200μg/ml to 50 ug/ml. (2) Dexamethasone increased the level of IκB-αprotein and effectively inhibited TCS-induced down-regulation of IκB-α.Conclusion:(1) Dexamethasone was able to enhance the sensitivity of hepatoma HepG2 cells to TCS, and promote trichosanthin-induced apoptosis in the HepG2. (2) Dexamethasone might promote the transcription of the IκB-αgene to increase the expression of IκB-αprotein, and efficiently inhibited TCS induced down-regulation of IκB-αprotein, and enhance the antitumor effect of TCS itself.