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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.
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Objective: To study the effect and the mechanism of Celastrus orbiculatus extract (COE) on inhibiting the invasion and metastasis of human gastric cancer SGC-7901 cells. Methods: The groups of different cells were set up, such as negative control group, COE with different concentration groups, and positive control group. The effects of COE on the cytotoxicity and proliferation of human gastric cancer SGC-7901 cells were determined by MTT assay. The effect of COE on the metastatic ability of human gastric cancer SGC-7901 cells in vitro was observed by invasion assay and migration assay. The effects of COE on the expression of matrix metalloproteinases (MMPs) and their inhibitors in human gastric cancer SGC-7901 cells were examined by Western blotting. After the treatment in different groups, mRNAs of MMP2, MMP9, tissue inhibitors of metalloproteinases (TIMPs), TIMP1, and TIMP3 in cells were detected by RT-PCR. Results: Compared with the negative control group, human gastric cancer SGC-7901 cells treated with COE at different concentration exhibited a dose-dependent growth inhibition. COE obviously reduced the migration and invasion potential of human gastric cancer SGC-7901 cells. In addition, the expression levels of MMP2 and MMP9 were dramatically suppressed by COE in a concentration-dependent manner, while the expression levels of TIMP1 and TIMP3 were raised. RT-PCR showed that mRNAs of TIMP1 and TIMP3 were raised with the increase of drug concentration. Conclusion: COE could significantly inhibit the invasion and metastasis of human gastric cancer SGC-7901 cells via an up-regulation of the expression levels of TIMP1 and TIMP3 and down-regulation of the expression levels of MMP-2 and MMP-9.
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This study aimed at investigating the effects of the extract of Chinese herb,Nansheteng (C.orbiculatus Thunb.),on human HepG2 cells through the overexpression of mTOR.The GV238-mTOR recombinant plasmids were transfected into HepG2 cells using molecular biological technology.The expression level of mTOR was evaluated by means of relative activity of luciferase and western blot.Human hepatic carcinoma HepG2/mTOR++ cells were treated with C.orbiculatus extract in different concentrations (20,40,80,160 and 320 tg·mL-1) for 24 h.The mTOR protein expression was detected by western blot.It was found that the protein expression of mTOR in transfected HepG2 cells was significantly enhanced.C.orbiculatus extract significantly inhibited the proliferation of HepG2/mTOR++ cells.Simultaneously,C.orbiculatus extract inhibited mTOR at its protein level in a dose-dependent manner.In conclusion,we successfully constructed recombinant mTOR cloning vectors,and established the stable HepG2 cell line with the overexpression of mTOR.Besides,C.orbiculatus extract significantly inhibited mTOR protein expression in human hepatic carcinoma HepG2 cells.
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Objective: To establish an HPLC method for the determination of kaempferitrin in Celastri Orbiculati Fructus and to compare the quality of samples from different habitats in northeast China. Methods: The extracts were obtained by methanol ultrasonic method and kaempferitrin was determinated by HPLC. Chromatographic separation was achieved on an Agilent Zorbax Eclipse Plus C18 column (250 mm × 4.6 mm, 5 μm) at 30℃ and the mobile phase was a mixture of methanol- 0.01 mol/L solution of potassium dihydrogenphosphate -glacial acetic acid (40:60:15). The flow rate was 1.0 mL/min and the detection wavelength was 254 nm. Results: The linearity range was 0.092 7-0.926 7 μg (r = 0.999 8), average recovery was 100.8%, and RSD = 1.1% (n = 6). Conclusion: This method is validated in terms of selectivity, linearity, precision, and accuracy. It can be used as the effective method for quality control. The determination results show that the longer the fruit sits followed by picking, the less contents it has.
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Objective To discuss the antitumor mechanism preliminarily by observing effects of Celastrus orbiculatus extracts (COE) on vascular endothelial growth factor (VEGF) mRNA and protein expression in hepatoma (Hepal-6) cells of mice.Methods Hepa1-6 cells were treated with COE at different nontoxic concentration (0, 10, 20, 40, 80, and 160 μg/mL) for 16 h. The mRNA and protein expressions of VEGF were detected by reverse transcription-PCR and Western Blotting,respectively. Results COE significantly inhibited VEGF expression at both mRNA and protein levels in a dose-dependent manner. Conclusion COE can inhibit VEGF expression in Hepa1-6 cells, therefore suggest that VEGF could be chosen as an therapeutic target for COE in the context of cancer chemoprevention and anticancer therapy.
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AIM:To study the chemical constitutes of Celastrus orbiculatus Thunb. METHODS:Root of celastmus orbiculatus was macerated with cool ethanol to obtain the extract under the vaccum drying processing,extract obtained was extracted respectively by petroleum ester and ethyl acetate. Final extracts were purified by Silicagel column and analyzed by UV,IR,Ms and NMR to identify the chemical structural formula. RESULTS:Seven compounds were isolated from Celastrus orbiculatus Thunb,from which five constituents came from petroleum ester extract,and identified as sugiol (1),pristimerin (2),?-sitosterol (3),celastrol (4),from which two constituents came from ethyl acetate extract,salapermic acid(5),benzoic acid (6) and daucosterol (7),respectively. CONCLUSION:Compounds 1,5 were obtained from the plant for the first time.