Extracts of Celastrus Orbiculatus Inhibit Cancer Metastasis by Down-regulating Epithelial-Mesenchymal Transition in Hypoxia-Induced Human Hepatocellular Carcinoma Cells
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摘要
Objective: To evaluate the effects of Celastrus Orbiculatus extracts(COE) on metastasis in hypoxiainduced hepatocellular carcinoma cells(Hep G2) and to explore the underlying molecular mechanisms. Methods:The effect of COE(160, 200 and 240 μg/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. Co Cl2 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 immuno?uorescence 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 1α(Hif-1α) and Twist1 were suppressed by COE. Additionally, the Hif-1α 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 Hep G2 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 Hep G2 cells. Conclusions: COE signi?cantly inhibited the tumor metastasis and EMT by suppressing Hif-1α/Twist1 signaling pathway in hypoxia-induced Hep G2 cell. Thus, COE might have potential effect to inhibit the progression of Hep G2 in the context of tumor hypoxia.
Objective: To evaluate the effects of Celastrus Orbiculatus extracts(COE) on metastasis in hypoxiainduced hepatocellular carcinoma cells(Hep G2) and to explore the underlying molecular mechanisms. Methods:The effect of COE(160, 200 and 240 μg/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. Co Cl2 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 immuno?uorescence 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 1α(Hif-1α) and Twist1 were suppressed by COE. Additionally, the Hif-1α 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 Hep G2 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 Hep G2 cells. Conclusions: COE signi?cantly inhibited the tumor metastasis and EMT by suppressing Hif-1α/Twist1 signaling pathway in hypoxia-induced Hep G2 cell. Thus, COE might have potential effect to inhibit the progression of Hep G2 in the context of tumor hypoxia.
Objective: To evaluate the effects of Celastrus Orbiculatus extracts(COE) on metastasis in hypoxiainduced hepatocellular carcinoma cells(Hep G2) and to explore the underlying molecular mechanisms. Methods:The effect of COE(160, 200 and 240 μg/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. Co Cl2 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 immuno?uorescence 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 1α(Hif-1α) and Twist1 were suppressed by COE. Additionally, the Hif-1α 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 Hep G2 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 Hep G2 cells. Conclusions: COE signi?cantly inhibited the tumor metastasis and EMT by suppressing Hif-1α/Twist1 signaling pathway in hypoxia-induced Hep G2 cell. Thus, COE might have potential effect to inhibit the progression of Hep G2 in the context of tumor hypoxia.
引文
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6.Zhang L,Huang G,Li X,Zhang Y,Jiang Y,Shen J,et al.Hypoxia induces epithelial-mesenchymal transition via activation of SNAI1 by hypoxia-inducible factor-1αin hepatocellular carcinoma.BMC Cancer 2013;13:108.
7.Pallier K,Cessot A,C?téJF,Just PA,Cazes A,Fabre E,et al.TWIST1 a new determinant of epithelial to mesenchymal transition in EGFR mutated lung adenocarcinoma.PLoSOne 2012;1:e29954.
8.Gajula RP,Chettiar ST,Williams RD,Thiyagarajan S,Kato Y,Aziz K,et al.The Twist box domain is required for Twist1-induced prostate cancer metastasis.Mol Cancer Res 2013;11:1541-7786.
9.Li G,Liu D,Zhang Y,Qian Y,Zhang H,Guo S,et al.Celastrol inhibits lipopolysaccharide stimulated rheumatoid fibroblast-like synoviocyte invasion through suppression of TLR4/NF-κB-mediated matrix metalloproteinase-9expression.PLoS One 2013;7:e68905.
10.Qian YY,Zhang H,Hou Y,Yuan L,Li GQ,Guo SY,et al.Celastrus orbiculatus extract inhibits tumor angiogenesis by targeting vascular endothelial growth factor signaling pathway and shows potent antitumor activity in hepatocarcinomas in vitro and in vivo.Chin J Integr Med 2012;10:752-760.
11.Zhang H,Qian YY,Liu YQ,Li GQ,Cui PF,Zhu YD,et al.Celastrus orbiculatus extract induces mitochondrialmediated apoptosis in human hepatocellular carcinoma cells.J Tradit Chin Med 2012;32:621-626.
12.Wang MR,Zhang X,Liu YQ.Acetoacetate extract from Celastrus orbiculatus Thunb inhibits growth of RFP-xenografted human liver carcinoma.Chin J Hepatol(Chin)2012;20:377-380.
13.Zhu Y,Liu Y,Qian Y,Dai X,Yang L,Chen J,et al.Research on the efficacy of Celastrus Orbiculatus in suppressing TGF-β1-induced epithelial-mesenchymal transition by inhibiting HSP27 and TNF-α-induced NF-κB/Snail signaling pathway in human gastric adenocarcinoma.BMC Complement Altern Med 2014;14:433.
14.Zhou G,Dada LA,Wu M,Kelly A,Trejo H,Zhou Q,et al.Hypoxia-induced alveolar epithelial-mesenchymal transition requires mitochondrial ROS and hypoxia-inducible factor 1.Am J Physiol Lung Cell Mol Physiol 2009;6:L1120-L1130.
15.Tsai JH,Yang J.Epithelial-mesenchymal plasticity in carcinoma metastasis.Genes Dev 2013;20:2192-2206.
16.Yeo EJ,Chun YS,Park JW.New anticancer strategies targeting HIF-1.Biochem Pharmacol 2004;68:1061-1069.
17.Kakodkar R,Soin AS.Liver transplantation for HCC:a review.Indian J Surg 2012;1:100-117.
18.Vil anueva DR,Llovet JM.Targeted therapies for hepatocel ular carcinoma.Gastroenterology 2011;5:1410-1426.
19.Hurst DR,Welch DR.Metastasis suppressor genes:at the interface between the environment and tumor cell growth.Int Rev Cell Mol Biol 2011;286:107-180.
20.Wang YF,Zhou BH.Epithelial-mesenchymal transition in breast cancer progression and metastasis.Chin J Cancer2011;9:603-611.
21.Tsuji T,Ibaragi S,Hu GF.Epithelial-mesenchymal transition and cell cooperativity in metastasis.Cancer Res2009;18:7135-7139.
22.Zhao Z,Cheng X,Wang Y,Han R,Li L,Xiang T,et al.Metformin inhibits the IL-6-induced epithelial-mesenchymal transition and lung adenocarcinoma growth and metastasis.PLoS One 2014;4:e95884.
23.Ponnusamy MP,Seshacharyulu P,Lakshmanan I,Vaz AP,Chugh S,Batra SK.Emerging role of mucins in epithelial to mesenchymal transition.Curr Cancer Drug Targets2013;9:945-956.
24.Lamouille S,Subramanyam D,Blelloch R,Derynck R.Regulation of epithelial-mesenchymal and mesenchymalepithelial transitions by microRNAs.Curr Opin Cell Biol2013;2:200-207.
25.Gonzalez DM,Medici M.Signaling mechanisms of the epithelial-mesenchymal transition.Sci Signal 2014;344:re8.
26.Huang SG,Zhang LL,Niu Q,Xiang GM,Liu LL,Jiang DN,et al.Hypoxia promotes epithelial-mesenchymal transition of hepatocellular carcinoma cells via inducing GLIPR-2expression.PLoS One 2013;10:e77497.
27.Huang SG,Zhang LL,Niu Q,Xiang GM,Liu LL,Jiang DN.Hypoxia-inducible factor prolyl-hydroxylase-2 mediates transforming growth factor beta 1-induced epithelialmesenchymal transition in renal tubular cells.Biochim Biophys Acta 2013;6:1454-1462.
28.Wu CH,Tang SC,Wang PH,Lee H,KoJ L.Nickelinduced epithelial-mesenchymal transition by reactive oxygen species generation and E-cadherin promoter hypermethylation.J Biol Chem 2012;30:25292-25302.
29.Oyanagi J,Ogawa T,Sato H,Higashi S,Miyazaki K.Epithelial-mesenchymal transition stimulates human cancer cells to extend microtubule-based invasive protrusions and suppresses cell growth in collagen gel.PLoS One2012;12:e53209.
30.Kang Y,Luczaj L,Kang J,Zhang S.Wild food plants and wild edible fungi in two valleys of the Qinling Mountains(Shaanxi,central China).J Ethnobiol Ethnomed 2013;9:26.
31.Li G,Liu D,Guo S,Sunagawa M,Hisamitsu T,Liu Y.Antiinvasive effects of Celastrus Orbiculatus extract on interleukin-1beta and tumour necrosis factor-alpha combination-stimulated fibroblast-like synoviocytes.Lement Altern Med 2014;14:62.
32.Zhu YD,Liu YQ,Qian YY,Zhang H,Li GQ,Yang L.Extracts of Celastrus Orbiculatus exhibit anti-proliferative and anti-invasive effects on human gastric adenocarcinoma cells.Chin J Integr Med 2014;11:1-8.
2.Colecchia A,Schiumerini R,Cucchetti A,Cescon M,Taddia M,MarascoG,et al.Prognostic factors for hepatocellular carcinoma recurrence.World J Gastroenterol2014;20:5935-5950.
3.Yilmaz M,Christofori G.EMT,the cytoskeleton,and cancer cell invasion.Cancer Metastasis Rev 2009;28:15-33.
4.Tu T,Budzinska MA,Maczurek AE,Cheng R,Di Bartolomeo A,Warner FJ,et al.Novel aspects of the liver microenvironment in hepatocellular carcinoma pathogenesis and development.Int J MolSci 2014;6:9422-9458.
5.Murielle M,Surinder K Batra.Hypoxia-inducing factors as master regulators of stemness properties and altered metabolism of cancer-and metastasis-initiating cells.J Cell Mol Med 2013;1:30-54.
6.Zhang L,Huang G,Li X,Zhang Y,Jiang Y,Shen J,et al.Hypoxia induces epithelial-mesenchymal transition via activation of SNAI1 by hypoxia-inducible factor-1αin hepatocellular carcinoma.BMC Cancer 2013;13:108.
7.Pallier K,Cessot A,C?téJF,Just PA,Cazes A,Fabre E,et al.TWIST1 a new determinant of epithelial to mesenchymal transition in EGFR mutated lung adenocarcinoma.PLoSOne 2012;1:e29954.
8.Gajula RP,Chettiar ST,Williams RD,Thiyagarajan S,Kato Y,Aziz K,et al.The Twist box domain is required for Twist1-induced prostate cancer metastasis.Mol Cancer Res 2013;11:1541-7786.
9.Li G,Liu D,Zhang Y,Qian Y,Zhang H,Guo S,et al.Celastrol inhibits lipopolysaccharide stimulated rheumatoid fibroblast-like synoviocyte invasion through suppression of TLR4/NF-κB-mediated matrix metalloproteinase-9expression.PLoS One 2013;7:e68905.
10.Qian YY,Zhang H,Hou Y,Yuan L,Li GQ,Guo SY,et al.Celastrus orbiculatus extract inhibits tumor angiogenesis by targeting vascular endothelial growth factor signaling pathway and shows potent antitumor activity in hepatocarcinomas in vitro and in vivo.Chin J Integr Med 2012;10:752-760.
11.Zhang H,Qian YY,Liu YQ,Li GQ,Cui PF,Zhu YD,et al.Celastrus orbiculatus extract induces mitochondrialmediated apoptosis in human hepatocellular carcinoma cells.J Tradit Chin Med 2012;32:621-626.
12.Wang MR,Zhang X,Liu YQ.Acetoacetate extract from Celastrus orbiculatus Thunb inhibits growth of RFP-xenografted human liver carcinoma.Chin J Hepatol(Chin)2012;20:377-380.
13.Zhu Y,Liu Y,Qian Y,Dai X,Yang L,Chen J,et al.Research on the efficacy of Celastrus Orbiculatus in suppressing TGF-β1-induced epithelial-mesenchymal transition by inhibiting HSP27 and TNF-α-induced NF-κB/Snail signaling pathway in human gastric adenocarcinoma.BMC Complement Altern Med 2014;14:433.
14.Zhou G,Dada LA,Wu M,Kelly A,Trejo H,Zhou Q,et al.Hypoxia-induced alveolar epithelial-mesenchymal transition requires mitochondrial ROS and hypoxia-inducible factor 1.Am J Physiol Lung Cell Mol Physiol 2009;6:L1120-L1130.
15.Tsai JH,Yang J.Epithelial-mesenchymal plasticity in carcinoma metastasis.Genes Dev 2013;20:2192-2206.
16.Yeo EJ,Chun YS,Park JW.New anticancer strategies targeting HIF-1.Biochem Pharmacol 2004;68:1061-1069.
17.Kakodkar R,Soin AS.Liver transplantation for HCC:a review.Indian J Surg 2012;1:100-117.
18.Vil anueva DR,Llovet JM.Targeted therapies for hepatocel ular carcinoma.Gastroenterology 2011;5:1410-1426.
19.Hurst DR,Welch DR.Metastasis suppressor genes:at the interface between the environment and tumor cell growth.Int Rev Cell Mol Biol 2011;286:107-180.
20.Wang YF,Zhou BH.Epithelial-mesenchymal transition in breast cancer progression and metastasis.Chin J Cancer2011;9:603-611.
21.Tsuji T,Ibaragi S,Hu GF.Epithelial-mesenchymal transition and cell cooperativity in metastasis.Cancer Res2009;18:7135-7139.
22.Zhao Z,Cheng X,Wang Y,Han R,Li L,Xiang T,et al.Metformin inhibits the IL-6-induced epithelial-mesenchymal transition and lung adenocarcinoma growth and metastasis.PLoS One 2014;4:e95884.
23.Ponnusamy MP,Seshacharyulu P,Lakshmanan I,Vaz AP,Chugh S,Batra SK.Emerging role of mucins in epithelial to mesenchymal transition.Curr Cancer Drug Targets2013;9:945-956.
24.Lamouille S,Subramanyam D,Blelloch R,Derynck R.Regulation of epithelial-mesenchymal and mesenchymalepithelial transitions by microRNAs.Curr Opin Cell Biol2013;2:200-207.
25.Gonzalez DM,Medici M.Signaling mechanisms of the epithelial-mesenchymal transition.Sci Signal 2014;344:re8.
26.Huang SG,Zhang LL,Niu Q,Xiang GM,Liu LL,Jiang DN,et al.Hypoxia promotes epithelial-mesenchymal transition of hepatocellular carcinoma cells via inducing GLIPR-2expression.PLoS One 2013;10:e77497.
27.Huang SG,Zhang LL,Niu Q,Xiang GM,Liu LL,Jiang DN.Hypoxia-inducible factor prolyl-hydroxylase-2 mediates transforming growth factor beta 1-induced epithelialmesenchymal transition in renal tubular cells.Biochim Biophys Acta 2013;6:1454-1462.
28.Wu CH,Tang SC,Wang PH,Lee H,KoJ L.Nickelinduced epithelial-mesenchymal transition by reactive oxygen species generation and E-cadherin promoter hypermethylation.J Biol Chem 2012;30:25292-25302.
29.Oyanagi J,Ogawa T,Sato H,Higashi S,Miyazaki K.Epithelial-mesenchymal transition stimulates human cancer cells to extend microtubule-based invasive protrusions and suppresses cell growth in collagen gel.PLoS One2012;12:e53209.
30.Kang Y,Luczaj L,Kang J,Zhang S.Wild food plants and wild edible fungi in two valleys of the Qinling Mountains(Shaanxi,central China).J Ethnobiol Ethnomed 2013;9:26.
31.Li G,Liu D,Guo S,Sunagawa M,Hisamitsu T,Liu Y.Antiinvasive effects of Celastrus Orbiculatus extract on interleukin-1beta and tumour necrosis factor-alpha combination-stimulated fibroblast-like synoviocytes.Lement Altern Med 2014;14:62.
32.Zhu YD,Liu YQ,Qian YY,Zhang H,Li GQ,Yang L.Extracts of Celastrus Orbiculatus exhibit anti-proliferative and anti-invasive effects on human gastric adenocarcinoma cells.Chin J Integr Med 2014;11:1-8.