ABSTRACT
OBJECTIVE@#To characterize the molecular mechanism underlying the antineoplastic activity of Celastrus orbiculatus Thunb. extracts (COE).@*METHODS@#The human hepatocellular carcinoma HepG2 cells with mammalian target of rapamycin (mTOR) knockdown expressed (HepG2/mTOR) were constructed using molecular biological technology. In vitro, the HepG2/mTOR cells were treated with COE at various concentrations (10, 20, 40, 80, 160 and 320 µ g/mL). Cell viability was determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. According to the half-maximal inhibitory concentration (IC) value (140 mg/L), the concentrations of COE in the subsequent experiment was set to alleviate cytotoxicity. The HepG2/mTOR cells were divided into 5 groups: negative control (untreated), COE treatment groups (40, 80, 120 mg/L COE) and positive control group (cisplatin, DDP, 2 mg/L), respectively. Wild-type HepG2 cells were used as a blank control. The effects of COE on the cell apoptosis were analyzed by flow cytometry and transmission electronic microscopy (TEM), respectively. The protein expression levels of mTOR signal pathways were determined by Western blotting. In vivo, HepG2/mTOR cells (2 × 10 cell/mice) were subcutaneously injected into the right flank of nude mice. Thirty-six female nude mice were randomly assigned to 6 groups according to body weight (6 mice per group) as follows: solvent vehicle control, Banmao Capsule treated group (BM, 195 mg/kg), Tegafur, Gimeracil and Oteracil Potassium Capsules (10 mg/kg) treated group, and different dosages of COE (10, 20, 40 mg/kg) groups. Tumor growth was monitored and immunohistochemical staining was used to examine the expression of apoptosis-related proteins in tumor tissues.@*RESULTS@#COE inhibited the proliferation significantly in a concentration-dependent manner in HepG2/mTOR cells (P<0.01). COE significantly induced the apoptosis of HepG2/mTOR cells (P<0.01), and the apoptotic bodies can be observed under TEM. COE significantly inhibits the proteins expression of mTOR-related signal pathways. In vivo, COE significantly inhibited tumor growth in nude mice (P<0.01). Moreover, the results showed that COE down-regulated the expression of Bcl-2 and Bcl-xL, and up-regulated the levels of Bax and caspase-3 protein (P<0.01).@*CONCLUSION@#COE was a potential chemotherapeutic drug in HCC treatments via targeting mTOR signal pathway.
ABSTRACT
OBJECTIVE@#To evaluate the effects of Celastrus Orbiculatus extracts (COE) on metastasis in hypoxia-induced hepatocellular carcinoma cells (HepG2) and to explore the underlying molecular mechanisms.@*METHODS@#The effect of COE (160, 200 and 240 µ g/mL) on cell viability, scratch-wound, invasion and migration were studied by 3-4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-H-tetrazolium bromide (MTT), scratch-wound and transwell assays, respectively. CoCl was used to establish a hypoxia model in vitro. Effects of COE on the expressions of E-cadherin, vimentin and N-cadherin were investigated with Western blot and immunofluorescence analysis, respectively.@*RESULTS@#COE inhibited proliferation and metastasis of hypoxia-induced hepatocellular carcinoma cells in a dose-dependent manner (P<0.01). Furthermore, the expression of epithelial-mesenchymal transition (EMT) related markers were also remarkably suppressed in a dose-dependent manner (P<0.01). In addition, the upstream signaling pathways, including the hypoxia-inducible factor 1 α (Hif-1 α) and Twist1 were suppressed by COE. Additionally, the Hif-1 α inhibitor 3-5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), potently suppressed cell invasion and migration as well as expression of EMT in hypoxia-induced HepG2 cells. Similarly, the combined treatment with COE and YC-1 showed a synergistic effect (P<0.01) compared with the treatment with COE or YC-1 alone in hypoxia-induced HepG2 cells.@*CONCLUSIONS@#COE significantly inhibited the tumor metastasis and EMT by suppressing Hif-1 α/Twist1 signaling pathway in hypoxia-induced HepG2 cell. Thus, COE might have potential effect to inhibit the progression of HepG2 in the context of tumor hypoxia.
Subject(s)
Humans , Biomarkers, Tumor , Metabolism , Carcinoma, Hepatocellular , Drug Therapy , Pathology , Celastrus , Chemistry , Cell Hypoxia , Cell Proliferation , Cell Shape , Cobalt , Down-Regulation , Epithelial-Mesenchymal Transition , Hep G2 Cells , Liver Neoplasms , Drug Therapy , Pathology , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplasm Proteins , Metabolism , Plant Extracts , Pharmacology , Therapeutic Uses , Signal TransductionABSTRACT
Objective To investigate the molecular mechanisms of Celastrus orbiculatus extracts (COE) of the invasion and metastasis inhibition in human hepatocellular carcinoma HepG2 cells by targeting mTOR. Methods The HepG2/mTOR- cells with mTOR knockout expression were constructed by using siRNA technology. The effect of COE on the proliferation of the HepG2/mTOR- cells was also studied. The HepG2/mTOR- cells were treated with COE in different concentrations (20, 40, 80, 160, and 320 mg/L) for 24 h. The cell reproductive capability of HepG2/mTOR- cells was detected by MTT. The effect of COE on the metastatic ability of HepG2/mTOR- cells in vitro was investigated by scratch assay and Transwell migration assay. The expression levels of molecular mechanisms related proteins MMP-2 and MMP-9 were assessed by Western blotting. Results The HepG2/mTOR- cells with mTOR knockout expression were successfully constructed. COE significantly inhibited the proliferation of HepG2/mTOR- cells in a concentration-dependent manner (P < 0.05). COE decreased the invasion and migration of HepG2/mTOR- cells. The results of Transwell experiment indicated that COE (80 mg/L) significantly reduced the number of transmembrane cells (P < 0.05). And the expression levels of MMP2 and MMP9 protein were significantly reduced in the HepG2/mTOR- cells after the treatment of COE. Conclusion COE can significantly inhibit the proliferation, invasion, and migration in the HepG2/mTOR- cells. Our data reveal that COE is a potential chemotherapeutic drug in human hepatocellular carcinoma treatments via targeting mTOR signal pathway.
ABSTRACT
<p><b>OBJECTIVE</b>Celastrus orbiculatus Thunb. has been used for thousands of years in China as a remedy against cancer and inflammatory diseases. This study aims to investigate whether C. orbiculatus extract (COE) could inhibit angiogenesis, which is the pivotal step in tumor growth, invasiveness, and metastasis.</p><p><b>METHODS</b>In this study, the extract from the stem of C. orbiculatus was used. Mouse hepatic carcinoma cells (Hepa1-6) were treated with COE in different nontoxic concentrations (10, 20, 40, 80, and 160 μg/mL). The mRNA and protein expression levels of vascular endothelial growth factor (VEGF) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively; the active fractions were further tested on C57BL/6 mice and human umbilical vein endothelial cells (HUVEC) for any antiangiogenic effects.</p><p><b>RESULTS</b>COE significantly inhibited proliferation and induced apoptosis in Hepa1-6 cells and inhibited VEGF expression at both mRNA and protein levels. Furthermore, this agent inhibited the formation of the capillary-like structure in primary cultured HUVEC in a dose-dependent manner. In vivo, COE significantly reduced the volume and weight of solid tumors with low adverse effects and decreased tumor angiogenesis.</p><p><b>CONCLUSIONS</b>In summary, COE could be used to treat hepatic carcinoma. The mechanisms of the antitumor activity of COE may be due to its effects against tumor angiogenesis by targeting the VEGF protein.</p>