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The Ethyl Acetate Extract From Celastrus orbiculatus Promotes Apoptosis of Gastric Cancer Cells Through Mitochondria Regulation by PHB.

ABSTRACT: Objective: To investigate the effect of ethyl acetate extract from Celastrus orbiculatus (COE) on gastric cancer cell apoptosis and reveal its underlying molecular mechanism. In addition, it was aimed to stablish a theoretical basis for the clinical application of Celastrus orbiculatus in the gastric cancer treatment. Material and Methods: Western blot and RT-qPCR were used to detect mRNA and protein expression of PHB in gastric cancer and adjacent tissues. MTT method was used to detect the COE effect on the proliferation of AGS cells and to determine the 50% inhibitory concentration COE on these cells. COE effect on AGS apoptosis was evaluated by flow cytometry. Changes in apoptosis-related proteins expression in AGS cells were detected by western blot and changes in mitochondrial membrane potential were detected by JC-1 fluorescence staining. PHB expression was knocked down in AGS cells by lentiviral-mediated RNA interference. The COE antitumor effect was assessed in vivo using a subcutaneous transplantation tumor model in nude mice and in vivo fluorescence tracing technique in small animals. Results: The clinical samples analysis results showed that the PHB expression in gastric cancer samples was significantly higher than in corresponding adjacent tissues. MTT results showed that the AGS cell proliferation was significantly inhibited. RT-qPCR and western blot results showed that COE can significantly inhibit the PHB mRNA and protein expression, respectively. Flow cytometry analysis showed that COE was able to significantly promote AGS cell apoptosis. Western blot results also indicated that apoptosis-related protein expression changed significantly; BCL-2 expression significantly reduced while the Caspase-3 and Bax expression significantly increased after COE treatment. JC-1 fluorescence staining results showed that COE changed the mitochondrial membrane potential and activated the mitochondrial apoptosis pathway. Furthermore, in vivo experiments results demonstrated that the growth of subcutaneous transplanted tumor was significantly inhibited by the PHB knockdown and by the COE intragastric administration. Conclusion: COE can significantly promote apoptosis of human gastric cancer cells, which can be achieved by inhibiting PHB expression, thus altering the structure and function of mitochondria and activating the mitochondria apoptosis pathway. The antitumor effect of COE has also been proved in vivo.

SUBMITTER: Tao L

PROVIDER: S-EPMC8194300 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundCelastrus orbiculatus has been used as a folk medicine in China for the treatment of many diseases. In the laboratory, the ethyl acetate extract of Celastrus orbiculatus (COE) displays a wide range of anticancer functions. However, the inhibition of the metastasis mechanism of COE in gastric cancer cells has not been investigated so far.MethodsThe present study was undertaken to determine if the anti-metastasis effect of COE was involved in inhibiting of epithelial-mesenchymal transition (EMT) of human gastric adenocarcinoma SGC-7901 cells. In vitro, a well-established experimental EMT model involving transforming growth factor ?1 (TGF-?1) was applied. Viability, invasion and migration, protein and mRNA expression of tumor cells were analyzed by MTT assay, transwell assay, western blot and real-time PCR, respectively. The molecular targets of COE in SGC-7901 cells were investigated by two-dimensional gel electrophoresis (2-DE) and MALDI-TOF-TOF mass spectrometer. Overexpression of heat shock protein 27 (HSP27) was performed by transfected with the recombinant retroviral expression plasmid. In vivo, the anti-metastasis mechanisms of COE in the peritoneal gastric cancer xenograft model was explored and the effect was tested.ResultsThe non-cytostatic concentrations of COE effectively inhibited TGF-?1 induced EMT process in SGC-7901 cells, which is characterized by prevented morphological changes, increased E-cadherin expression and decreased Vimentin, N-cadherin expression. Moreover, COE inhibited invasion and migration induced by TGF-?1. Using a comparative proteomics approach, four proteins were identified as differently expressed, with HSP27 protein being one of the most significantly down-regulated proteins induced by COE. Moreover, the activation of nuclear factor ?B (NF-?B)/Snail signaling pathway induced by tumor necrosis factor-? (TNF-?) was also attenuated under the pretreatment of COE. Interestingly, overexpression of HSP27 significantly decreases the inhibitory effect of COE on EMT and the NF-?B/Snail pathway. Furthermore, COE significantly reduced the number of peritoneal metastatic nodules in the peritoneal gastric cancer xenograft model.ConclusionsTaken together, these results suggest that COE inhibits the EMT by suppressing the expression of HSP27, correlating with inhibition of NF-?B/Snail signal pathways in SGC-7901 cells. Based on these results, COE may be considered a novel anti-cancer agent for the treatment of metastasis in gastric cancer.

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The Ethyl Acetate Extract From Celastrus orbiculatus Promotes Apoptosis of Gastric Cancer Cells Through Mitochondria Regulation by PHB.

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