| Gastric cancer is the most common gastrointestinal cancer originating from the epithelium and is a serious threat to human health. Most patients are diagnosed at an advanced stage, with metastasis and poor prognosis. Metastasis of gastric cancer is not only a sign of deterioration, but also the major cause of treatment failure and death. Therefore, effective therapeutic agent targeting the cancer metastasis is significant for gastric cancer treatment.Epithelial-mesenchymal transition (EMT), the process of cells losing their epithelial phenotype and acquiring a migratory mesenchymal phenotype, has been accepted to play an important role in cancer metastasis in several human malignancies including gastric cancer. Thus, EMT could be a very promising therapeutic target, and the inhibition of EMT may prevent or restrain the metastasis of gastric cancer.Celastrus orbiculatus (Celastraceae), has been used as a folk medicine in China for the treatment of many diseases, including arthritis and other inflammatory diseases. It was found that the ethyl acetate extract of Celastrus orbiculatus (COE) displays anticancer effects in vitro and in vivo through the inhibition of proliferation, angiogenesis, invasion and metastasis ability. Although COE has been reported in various bioactivity assays, the molecular mechanism by which it modulates the EMT-mediated alteration of human gastric cancer cells has not been elucidated so far.In this study, we investigated the effects of COE on transforming growth factor β1 (TGF-β1) induced EMT in vitro and tumor growth and metastasis in vivo and explored the underlying molecular mechanism. The results indicated that COE inhibited the EMT and tumor growth and metastasis by suppressing the expression of heat shock protein 27 (HSP27), correlating with inhibition of nuclear factor κB (NF-κB)/Snail signal pathway in SGC-7901 cells. Therefore, the present findings suggest that COE might have potential therapeutic effect against gastric cancer.The findings will be described in three parts as follows.Part I COE Inhibits TGF-β1-induced EMT in SGC-7901 cellsObjective:To investigate the effect of COE on EMT in human gastric cancer cell line SGC-7901 induced by TGF-β1 and further analyze the underlying mechanisms.Methods:We cultivate gastric cancer cell line SGC-7901 and using the different concentrations (2.5,5,10 ng/mL) of TGF-β1 effect for 24 h, and then identify the EMT through cell shape and protein expression’s change. MTT method was used to detect the anti-proliferative effect of COE on SGC-7901 cells. The morphological changes were determined by optical microscopy. The expression of E-cadherin, N-cadherin, and Vimentin were analyzed using western blotting. The impact of COE on SGC-7901 cell invasion and migration was evaluated by transwell assay. The molecular targets of COE in SGC-7901 cells were investigated by two-dimensional gel electrophoresis (2-DE) and MALDI-TOF-TOF mass spectrometer.Results:Treating with different concentration of TGF-β1 for 24 h, the gastric cancer cell line SGC-7901 changed to the fusiform shape and the disappeared cell link. The expression of E-cadherin was down-regulated (P<0.05), whereas N-cadherin and Vimentin were up-regulated (P<0.05). This study demonstrates that using SGC-7901 induced by 10 ng/mL TGF-β1 in 24 h, EMT model can be successfully established. COE inhibited the viability of SGC-7901 cells in a concentration-dependent manner. Therefore, the non-cytostatic concentrations (below 20 μg/mL) of COE was chosen for further experiments to guarantee that inhibition of COE for TGF-β1 induced EMT was not due to inhibition of proliferation. The non-cytostatic concentrations of COE effectively inhibited TGF-(31 induced EMT process in SGC-7901 cells, which is characterized by increased E-cadherin expression and decreased Vimentin, N-cadherin expression. Moreover, COE inhibited invasion and migration induced by TGF-β1 (P<0.05). Using a comparative proteomics approach, there were four protein spots which were promoted in i treated group compared with control group, but depressed in the COE treated group. These differentially expressed spots were successfully identified by MALDI-TOF/TOF and MS/MS analysis. Four of them were identified as HSP27, Prohibitin, Cofilin-1, and Annexin A5. Conclusion:COE maybe plays an anti-EMT effects through comprehensively regulating the functional proteins such as HSP27, Prohibitin, Cofilin-1, and Annexin A5, et al.Part II The role of HSP27 in COE-mediates inhibition of EMT and NF-KB/Snail signal pathwayObjective:To confirm the role of HSP27 in COE inhibits EMT in SGC-7901 cells and to determine whether HSP27 is involoved in the COE-mediated inhibition of NF-κB/Snail signal pathway.Methods:After SGC-7901 cells were treated with different dose of COE, the protein and mRNA expression of HSP27 was detected by western blot and real-time PCR, respectively. Furthermore, an ubiqutin-proteasome inhibitor MG132 was applied to figure out the potential ways in which COE could degrade HSP27 protein. The activity of NF-kB and the expression of NF-KB/Snail signaling pathway were evaluated by using luciferase reporter gene and western blotting assay. The recombinant retro virus plasmid MIGR1-HSP27 containing full length of human HSP27 gene was successful constructed. The constructed plasmid MIGR1-HSP27, empty plasmid MIGR1 were transfected into SGC-7901 cells and the cells were selected by flow cytometry for GFP+cells. The effect of HSP27 overexpression on tumor cell EMT was assessed by western blot assay. To confirm the role of HSP27 in COE-mediates inhibition of EMT and NF-KB/Snail signal pathway, both the overexpression HSP27 cell line and control cell line were treated with COE and TGF-(3 or TNF-a at optimal concentration. The invasion, migration, the activity of NF-κB, the expression of EMT markers and NF-κB/Snail signaling pathway were evaluated by using transwell assay, luciferase reporter gene assay, and western blotting, respectively.Results:Treatment with COE down-regulated HSP27 protein expression in a dose-dependent manner, while the expression levels of HSP27 mRNA was not changed remarkably. These results showed that COE could decrease the HSP27 protein level without modulating the expression of its mRNA transcripts levels. In order to measure effects of COE on degradation of HSP27 protein, cells were treated with the proteasome inhibitor MG132. And we found that MG132 upregulated the level of HSP27 induced by TGF-β1 when treated with COE. This suggests that an increased degradation of HSP27 occured in SGC-7901 cells treated with COE. To confirm the role of HSP27 in COE inhibits EMT in SGC-7901 cells, the recombinant retrovirus plasmid MIGR1-HSP27 containing full length of human HSP27 gene was successful constructed. The constructed plasmid MIGR1-HSP27, empty plasmid MIGR1 were transfected into SGC-7901 cells and the cells were selected by flow cytometry for GFP+cells, a transduction efficiency of close to 100% was achieved. Western blot analysis indicated that HSP27 overexpression by recombinant retrovirus plasmid transfections in SGC-7901 cells not only increased HSP27 content but also reproduced EMT features classically induced by TGF-(31. Subsequently, the overexpression HSP27 cell line and control cell line were pretreated with or without COE (20 μg/mL) for 1 h and then stimulated with 10 ng/mL TGF-β1 for 24 h. COE treatment caused suppression of HSP27 level in control cells as well as in HSP27 overexpressed cells. The effects of COE on the upregulation of E-cadherin and downregulation of N-cadherin and Vimentin were also blocked by HSP27 overexpression. In addition, HSP27 overexpression blocked the effects of COE on cell invasion and migration. Our results also demonstrated that TNF-α (classic NF-κB pathway activator) promoted the expression of NF-κB p65, phosphorylation of IκBα, and Snail protein, accompanied by a decreased expression of IκBα, and this effect was abrogated by COE. Besides, the research also showed that the enhancement of NF-κB transcriptional activity by TNF-α was also inhibited by COE using an NF-kB mediated luciferase reporter gene assay. This results support previous hypothesis that COE might inhibit EMT via the regulation of the NF-KB/Snail pathway. More importantly, we sought to determine whether HSP27 is involoved in the COE-mediated inhibition of NF-KB/Snail signal pathway. The overexpression of HSP27 decreased the inhibitory effect of COE on the nuclear translocation of NF-κB p65 and Snail, as well as the phosphorylation of IκBα. These results indicated that HSP27 may play a critical role in COE inhibits EMT by inhibiting NF-KB/Snail signal pathway.Conclusion:HSP27 played a critical role in the process of COE-mediates inhibition of EMT and NF-κB/Snail signal pathway and offer a new perspective on the role of COE in preventing the procession of cancer.Part Ⅲ COE inhibits growth and metastasis of SGC-7901 cells in vivo xenograft modelObjective:To study the effects of COE on inhibition the growth and metastasis of tumor in nude mice model of gastric cancer and to explore the relative molecular mechanism.Methods:To evaluate the tumor growth and metastasis, cultured human gastric cancer SGC-7901 cells (2×106) were inoculated into the right flank or the peritoneal cavity of nude mice. After the nude mice models were established, the mice were randomly divided into blank control group, negative control group (1%DMSO), positive control group (xeloda,267 mg/kg), and low-dose, medium-dose and high-dose COE groups (10,20 and 40 mg/kg, respectively). Each group contained seven mice. Then, mice were intragastrically given COE for 21 days and xeloda was given for 1-14 consecutive days with one week rest. For in vivo tumor xenograft growth assay, the tumor dimensions were measured every three days by a digital caliper and tumor volume was calculated using the formula:V= length×width2×0.5. The body weight of each mouse was measured every three days after treatment. After 21 days of treatment, the mice were killed by cervical dislocation, and their tumors were excised and weighed. Tumors were collected, fixed with 4% formaldehyde, embedded in paraffin and sectioned for haematoxylin and eosin (H&E) staining according to standard histological procedures. For experimrntal tumor metastasis model, all the mice were sacrificed by cervical dislocation and laparotomy was performed at the end of treatment. Peritoneal dissemination, liver metastasis, lung metastasis, and ascites formation were examined. The number of mesentery nodules larger than 1 mm in diameter was also determined. The peritoneal nodules, liver and lungs were paraffin-embedded, sectioned, and subjected to H&E staining for histological analysis. In order to better understand the molecular mechanism of COE on tumor growth and metastasis in vivo, we examined the expression levels of E-cadherin, N-cadherin, Vimentin, HSP27, NF-κB p65 and Snail in the tumor tissue in subcutaneous and peritoneal dissemination tumor models by immunohistochemisty analysis.Results:Compared with the control group, the COE treated group showed a significantly inhibition of tumor growth. It was found that COE significantly decreased the tumor weight compared with the control group. Furthermore, light microscopy revealed that tumor tissue in mice receiving COE (40 mg/kg) displayed more severe necrosis than that in the control group, which was similar to the effect of xeloda. The results also showed that COE had the capability of reducing the peritoneal dissemination, since the number of macroscopic nodules in COE groups was much lower than that in the control group. The H&E stain of peritoneal nodules showed that the degree of tumor necrosis in high-dose COE group was higher than that in the control group. In order to test the effect of COE on organ metastasis, histological evaluation of the liver and lung sections from each mouse were performed. The metastatic lesions were distinguishable on H&E stained sections from the liver and lung tissue. We observed that COE treatment markedly reduced liver and lung metastases and dissemination to other tissues including omentum, parietal peritoneum, diaphragm, compared with the control group. Together, these results support the role of COE in suppression of gastric cancer growth and metastasis. The results of immunohistochemisty analysis revealed that COE could increase the expression E-cadherin and reduce the expression of N-cadherin and Vimentin in subcutaneous tumor models. Furthermore, it was also observed that the targeted proteins of HSP27 and NF-KB/Snail signaling were also diminished in COE treated tumors compared with solvent-treated animals. In addition, a similar result was observed in peritoneal dissemination tumor model. A novel mechanism of COE that suppressed EMT and inhibited HSP27 and NF-κB/Snail signaling pathway during treatment of gastric cancer was revealed by these data.Conclusion:Our findings provided new evidence that COE is an effective inhibitor of growth and metastatic potential of SGC-7901 cells in vivo through suppression of EMT. Based on these findings, COE may be considered a novel anticancer agent for the treatment of metastasis in gastric cancer. |
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