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Research on the efficacy of Celastrus Orbiculatus in suppressing TGF-β1-induced epithelial-mesenchymal transition by inhibiting HSP27 and TNF-α-induced NF-κ B/Snail signaling pathway in human gastric adenocarcinoma.

Zhu Y, Liu Y, Qian Y, Dai X, Yang L, Chen J, Guo S, Hisamitsu T - BMC Complement Altern Med (2014)

Bottom Line: The non-cytostatic concentrations of COE effectively inhibited TGF-β1 induced EMT process in SGC-7901 cells, which is characterized by prevented morphological changes, increased E-cadherin expression and decreased Vimentin, N-cadherin expression.Interestingly, overexpression of HSP27 significantly decreases the inhibitory effect of COE on EMT and the NF-κB/Snail pathway.Taken together, these results suggest that COE inhibits the EMT by suppressing the expression of HSP27, correlating with inhibition of NF-κB/Snail signal pathways in SGC-7901 cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Combining Chinese Traditional and Western Medicine, Medical College, Yangzhou University, Yangzhou, Jiangsu 225001, China. Liuyanqing2014@163.com.

ABSTRACT

Background: Celastrus 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.

Methods: The 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 β1 (TGF-β1) 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.

Results: The non-cytostatic concentrations of COE effectively inhibited TGF-β1 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-β1. 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 κB (NF-κB)/Snail signaling pathway induced by tumor necrosis factor-α (TNF-α) was also attenuated under the pretreatment of COE. Interestingly, overexpression of HSP27 significantly decreases the inhibitory effect of COE on EMT and the NF-κB/Snail pathway. Furthermore, COE significantly reduced the number of peritoneal metastatic nodules in the peritoneal gastric cancer xenograft model.

Conclusions: Taken together, these results suggest that COE inhibits the EMT by suppressing the expression of HSP27, correlating with inhibition of NF-κB/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 role of HSP27 in COE-mediates inhibition of EMT and NF-κB/Snail signal pathway. (A) Overexpression of HSP27 was performed by transfected with the recombinant retroviral expression plasmid and the cells were selected by flow cytometry for GFP + cells. Nearly 100% transduction efficiency was achieved as shown by fluorescent microscope. (B) Expression of HSP27, E-cadherin, N-cadherin, Vimentin were analyzed by western blot in SGC-7901 cells that were transfected with control (empty) or HSP27 vectors or treated with TGF-β1 (10 ng/mL). β-actin was served as an internal control of protein level. The relative density was normalized to β-actin, which was determined by densitometric analysis. Values are expressed as means±SD of three independent experiments. **P <0.01, compared with control. ##P <0.01, compared to MIGR1 group. (C) The overexpression HSP27 cell line and control cell line were treated with or without COE (20 μg/mL) for 1 h followed by TGF-β1 (10 ng/mL) for 24 h, and the protein expression of HSP27, E-cadherin, N-cadherin, Vimentin were detected by western blot analyses. β-actin was served as an internal control of protein level. (D) Cell invasion and migration were analyzed by transwell assay. The quantitative data was presented as means±SD of three independent experiments. (E) The overexpression HSP27 cell line and control cell line were treated with or without COE (20 μg/mL) for 1 h followed by TNF-α (10 ng/mL) for 24 h, and the protein expression of IκBα, p-IκBα, NF-κB p65 and Snail were detected by western blot analyses. β-actin and H3 were served as an internal control of protein level. **P <0.01 compared with the respective untreated control (MIGR1 or MIGR1-HSP27). ##P <0.01 compared COE-treated MIGR1 group compared with COE-treated MIGR1-HSP27 group.
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Fig4: The role of HSP27 in COE-mediates inhibition of EMT and NF-κB/Snail signal pathway. (A) Overexpression of HSP27 was performed by transfected with the recombinant retroviral expression plasmid and the cells were selected by flow cytometry for GFP + cells. Nearly 100% transduction efficiency was achieved as shown by fluorescent microscope. (B) Expression of HSP27, E-cadherin, N-cadherin, Vimentin were analyzed by western blot in SGC-7901 cells that were transfected with control (empty) or HSP27 vectors or treated with TGF-β1 (10 ng/mL). β-actin was served as an internal control of protein level. The relative density was normalized to β-actin, which was determined by densitometric analysis. Values are expressed as means±SD of three independent experiments. **P <0.01, compared with control. ##P <0.01, compared to MIGR1 group. (C) The overexpression HSP27 cell line and control cell line were treated with or without COE (20 μg/mL) for 1 h followed by TGF-β1 (10 ng/mL) for 24 h, and the protein expression of HSP27, E-cadherin, N-cadherin, Vimentin were detected by western blot analyses. β-actin was served as an internal control of protein level. (D) Cell invasion and migration were analyzed by transwell assay. The quantitative data was presented as means±SD of three independent experiments. (E) The overexpression HSP27 cell line and control cell line were treated with or without COE (20 μg/mL) for 1 h followed by TNF-α (10 ng/mL) for 24 h, and the protein expression of IκBα, p-IκBα, NF-κB p65 and Snail were detected by western blot analyses. β-actin and H3 were served as an internal control of protein level. **P <0.01 compared with the respective untreated control (MIGR1 or MIGR1-HSP27). ##P <0.01 compared COE-treated MIGR1 group compared with COE-treated MIGR1-HSP27 group.

Mentions: 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 (Figure 4A). 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-β1 (Figure 4B). 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. As shown in Figure 4C, 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 (Figure 4D). More importantly, we sought to determine whether HSP27 is involoved in the COE-mediated inhibition of NF-κB/Snail signal pathway. As shown in Figure 4E, 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-κB/Snail signal pathway.Figure 4


Research on the efficacy of Celastrus Orbiculatus in suppressing TGF-β1-induced epithelial-mesenchymal transition by inhibiting HSP27 and TNF-α-induced NF-κ B/Snail signaling pathway in human gastric adenocarcinoma.

Zhu Y, Liu Y, Qian Y, Dai X, Yang L, Chen J, Guo S, Hisamitsu T - BMC Complement Altern Med (2014)

The role of HSP27 in COE-mediates inhibition of EMT and NF-κB/Snail signal pathway. (A) Overexpression of HSP27 was performed by transfected with the recombinant retroviral expression plasmid and the cells were selected by flow cytometry for GFP + cells. Nearly 100% transduction efficiency was achieved as shown by fluorescent microscope. (B) Expression of HSP27, E-cadherin, N-cadherin, Vimentin were analyzed by western blot in SGC-7901 cells that were transfected with control (empty) or HSP27 vectors or treated with TGF-β1 (10 ng/mL). β-actin was served as an internal control of protein level. The relative density was normalized to β-actin, which was determined by densitometric analysis. Values are expressed as means±SD of three independent experiments. **P <0.01, compared with control. ##P <0.01, compared to MIGR1 group. (C) The overexpression HSP27 cell line and control cell line were treated with or without COE (20 μg/mL) for 1 h followed by TGF-β1 (10 ng/mL) for 24 h, and the protein expression of HSP27, E-cadherin, N-cadherin, Vimentin were detected by western blot analyses. β-actin was served as an internal control of protein level. (D) Cell invasion and migration were analyzed by transwell assay. The quantitative data was presented as means±SD of three independent experiments. (E) The overexpression HSP27 cell line and control cell line were treated with or without COE (20 μg/mL) for 1 h followed by TNF-α (10 ng/mL) for 24 h, and the protein expression of IκBα, p-IκBα, NF-κB p65 and Snail were detected by western blot analyses. β-actin and H3 were served as an internal control of protein level. **P <0.01 compared with the respective untreated control (MIGR1 or MIGR1-HSP27). ##P <0.01 compared COE-treated MIGR1 group compared with COE-treated MIGR1-HSP27 group.
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Fig4: The role of HSP27 in COE-mediates inhibition of EMT and NF-κB/Snail signal pathway. (A) Overexpression of HSP27 was performed by transfected with the recombinant retroviral expression plasmid and the cells were selected by flow cytometry for GFP + cells. Nearly 100% transduction efficiency was achieved as shown by fluorescent microscope. (B) Expression of HSP27, E-cadherin, N-cadherin, Vimentin were analyzed by western blot in SGC-7901 cells that were transfected with control (empty) or HSP27 vectors or treated with TGF-β1 (10 ng/mL). β-actin was served as an internal control of protein level. The relative density was normalized to β-actin, which was determined by densitometric analysis. Values are expressed as means±SD of three independent experiments. **P <0.01, compared with control. ##P <0.01, compared to MIGR1 group. (C) The overexpression HSP27 cell line and control cell line were treated with or without COE (20 μg/mL) for 1 h followed by TGF-β1 (10 ng/mL) for 24 h, and the protein expression of HSP27, E-cadherin, N-cadherin, Vimentin were detected by western blot analyses. β-actin was served as an internal control of protein level. (D) Cell invasion and migration were analyzed by transwell assay. The quantitative data was presented as means±SD of three independent experiments. (E) The overexpression HSP27 cell line and control cell line were treated with or without COE (20 μg/mL) for 1 h followed by TNF-α (10 ng/mL) for 24 h, and the protein expression of IκBα, p-IκBα, NF-κB p65 and Snail were detected by western blot analyses. β-actin and H3 were served as an internal control of protein level. **P <0.01 compared with the respective untreated control (MIGR1 or MIGR1-HSP27). ##P <0.01 compared COE-treated MIGR1 group compared with COE-treated MIGR1-HSP27 group.
Mentions: 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 (Figure 4A). 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-β1 (Figure 4B). 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. As shown in Figure 4C, 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 (Figure 4D). More importantly, we sought to determine whether HSP27 is involoved in the COE-mediated inhibition of NF-κB/Snail signal pathway. As shown in Figure 4E, 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-κB/Snail signal pathway.Figure 4

Bottom Line: The non-cytostatic concentrations of COE effectively inhibited TGF-β1 induced EMT process in SGC-7901 cells, which is characterized by prevented morphological changes, increased E-cadherin expression and decreased Vimentin, N-cadherin expression.Interestingly, overexpression of HSP27 significantly decreases the inhibitory effect of COE on EMT and the NF-κB/Snail pathway.Taken together, these results suggest that COE inhibits the EMT by suppressing the expression of HSP27, correlating with inhibition of NF-κB/Snail signal pathways in SGC-7901 cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Combining Chinese Traditional and Western Medicine, Medical College, Yangzhou University, Yangzhou, Jiangsu 225001, China. Liuyanqing2014@163.com.

ABSTRACT

Background: Celastrus 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.

Methods: The 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 β1 (TGF-β1) 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.

Results: The non-cytostatic concentrations of COE effectively inhibited TGF-β1 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-β1. 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 κB (NF-κB)/Snail signaling pathway induced by tumor necrosis factor-α (TNF-α) was also attenuated under the pretreatment of COE. Interestingly, overexpression of HSP27 significantly decreases the inhibitory effect of COE on EMT and the NF-κB/Snail pathway. Furthermore, COE significantly reduced the number of peritoneal metastatic nodules in the peritoneal gastric cancer xenograft model.

Conclusions: Taken together, these results suggest that COE inhibits the EMT by suppressing the expression of HSP27, correlating with inhibition of NF-κB/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.

Show MeSH
Related in: MedlinePlus