Dexamethasone Enhances Trichosanthin-Induced Apoptosis in the HepG2 Hepatoma Cell Line by Inhibition of NF-κB Activation
|Course||Human Anatomy and Embryology|
|Keywords||Trichosanthin NF-κB Apoptosis HepG2 cells Dexamethasone Hepatoma cells|
Role in HepG2 cells of the first part NF-κB/IκB signaling pathway in the TCS anti-Background: trichosanthin (trichosanthin, TCS), the separation of effective components extracted from the tuberous root of herbaceous TCS, belonging to type Ⅰ ribosome inactivating protein with induction of labor, immunomodulatory, anti-tumor and anti-viral variety of biological effects. The anti-tumor activity in recent years, TCS concern multiple apoptotic mechanisms involved in TCS-induced apoptosis of tumor cells. Compared with cervical cancer, choriocarcinoma TCS highly sensitive tumor cell lines HepG2 cells sensitive to the low degree of TCS, the mechanism has not been elucidated. Nuclear transcription factor kappa B (nuclear factor kappa B, NF-κB) is widely present in eukaryotes, involved in cell growth, differentiation, gene expression and regulation of inflammation and immune responses, and apoptosis. Currently, NF-κB activation is involved in the process of TCS antitumor, and mechanism of action in the process, has not been reported. Research Objective: In our study, the low degree of TCS sensitive HepG2 cells as a cell model to study the impact of the TCS NF-κB activation in HepG2 cells, NF-κB activation in TCS induced apoptosis in HepG2 cells, revealing HepG2 cells TCS low sensitive mechanism for the TCS the antitumor experimental study, providing a theoretical basis. Research Methods: MTT assay in HepG2 and the MIHA cells treated with various concentrations the TCS treatment cell viability after 48 h. Extract the cytoplasm and nucleus of the cell protein processing by TCS Application Western blot assay cytoplasmic IκB-a protein, NF-κB p65 subunit, Cox-2 protein levels, as well as the nuclei of NF-κB p65 subunit level . Using the dual luciferase reporter gene assay system detects the transcriptional activity of NF-κB. The application by Hoechst33258 TCS-induced apoptosis, detected and statistical rate of apoptosis. IKB-a protein by inhibition of NF-κB activation inhibitor (IκB-DM) biological and chemical inhibitors (PDTC), observed changes in the p65 subunit of NF-κB, and cell viability and apoptosis rate changes. Findings: (1) after 48 h of treatment by 200μg/ml TCS, HepG2 and MIHA cell growth inhibition rate was approximately 48% and 17%, indicating that TCS has a selective inhibitory effect on HepG2. (2) TCS rapid induction of hepatoma HepG2 IKB-a protein levels decreased significantly, accompanied by reduced levels of cytoplasmic NF-κB protein and nuclear protein levels of NF-κB, nuclear translocation of NF-κB. The TCS can significantly promote the transcriptional activity of NF-κB, a time-dependent manner. And TCS can induce NF-κB downstream Cox-2 gene expression. (3) by stable transfection of IκB-DM and blank control (pEGFP-N1) HepG2 cell lines stably expressing. Blank plasmid control group cytoplasmic IκB-a and NF-κB p65 protein trend similar to HepG2 cells not transfected plasmid; IKB-a protein basal level of IκB-DM group increased significantly, and TCS treatment lasted for 3h nor See degradation. IκB-DM group showed changes in the level of nuclear proteins NF-kappa B p65 protein in the cytoplasm. By the MTT assay, 50μg/ml TCS after 48 h of treatment, cell viability IκB-DM group (43%) lower than the blank plasmid group (63%) 20% (P <0.05). Application PDTC pretreatment of HepG2 cells to inhibit NF-κB activation, Hoechst 33258 staining observed rate of apoptosis (PDTC TCS) group group increased by 16% compared with TCS. Conclusions: (1) TCS HepG2 cells with selective cytotoxicity, TCS has a better application prospects of the anti-tumor. (2) the TCS can cause IKB-a protein levels in the cytoplasm of HepG2 cells, leading to NF-κB is released, the occurrence of nuclear transfer, and to regulate downstream gene expression. (3) NF-κB activation can inhibit the TCS-induced apoptosis in HepG2 cells and HepG2 sensitive to the low degree of TCS; effective inhibition of NF-κB signaling pathway HepG2 cells can enhance the sensitivity of TCS, promoting TCS anti-tumor effect. The second part of dexamethasone promote trichosanthin induced human hepatoma HepG2 cells apoptosis research background: dexamethasone (dexamethasone) is a glucocorticoid hormones (glucocorticoids), has pharmacological effects such as anti-inflammatory and immunomodulatory. Dexamethasone through the inhibition of NF-κB activation induced by a variety of lymphoma cell apoptosis. In recent years, scholars have found that dexamethasone can be effective in preventing chemotherapy drugs cause nausea, vomiting, and promote the anti-tumor effect of the chemotherapy drug 5 - fluorouracil, cisplatin. However, dexamethasone has not been reported on TCS-induced apoptosis of human hepatoma cell regulation. Objective: This study dexamethasone can promote TCS induced HepG2 cells apoptosis, and further confirm the role of NF-κB activation in TCS-induced apoptosis in HepG2 cells for TCS alone or drug joint clinical trials of the anti-tumor research provide the basis. Research Methods: HepG2 cells by dexamethasone (1μM, 24 h) after pretreatment with different concentrations of TCS MS for 48 h, the MTT assay cell survival; applications detected by Western blot cytoplasmic IκB-a protein levels ; Hoechst 33258 staining to detect apoptosis, and statistical rate of apoptosis. Results: (1) TCS alone, IC50 more than 200μg/ml TCS with dexamethasone, IC50 approximately 50μg/ml, showed that dexamethasone promote TCS induced apoptosis in HepG2 cells. (2) after dexamethasone pretreatment, IKB-a protein is significantly increased, and associated with TCS, IKB-a protein levels did not change significantly. Conclusion: (1) TCS with dexamethasone, together with the increased sensitivity of HepG2 cells TCS, promote TCS induced apoptosis in HepG2 cells. (2) dexamethasone can significantly improve the basal level of IKB-a protein, the dexamethasone by promoting the transcription levels of the IκB-a Step IKB-a protein increases, thus inhibiting the dissociation of NFκB in the cytoplasm, and ultimately inhibit of NFκB activation, and promote the anti-tumor effect of the TCS.