人参皂苷Rg3体外通过抑制Wnt/β联蛋白通路阻止胃癌SGC7901细胞血管生成拟态的形成
|
摘要
目的:探索人参皂苷Rg3体外对胃癌SGC7901细胞血管生成拟态(VM)形成的影响及其分子机制。方法:MTT法检测不同浓度Rg3对SGC7901细胞增殖的影响;SGC7901细胞分为BML-284组、XAV-939组、Rg3组、Rg3+BML-284组和空白组,Transwell实验检测细胞的侵袭和迁移,成管实验观察细胞管样结构的形成,ELISA检测细胞MMP-9和MMP2分泌变化,qPCR检测细胞中GSK-3β、Wnt2B m RNA表达水平,WB检测细胞中β联蛋白表达水平,免疫荧光检测β联蛋白进入细胞核情况。结果:人参皂苷Rg3可以时间-浓度依赖的方式抑制SGC7901细胞增殖。与空白组相比,40 mg/L Rg3显著抑制SGC7901细胞侵袭和迁移(均P<0.05)、VM的形成(P<0.05),同时细胞中GSK-3β、Wnt2B mRNA和β联蛋白的表达及其进核行为均受到显著抑制(均P<0.05);Rg3+BML-284组细胞的侵袭、迁移以及VM的形成情况与空白组无显著差异(均P>0.05)。结论:Rg3通过抑制SGC7901细胞中Wnt/β联蛋白通路激活从而抑制细胞的侵袭、迁移以及VM的形成。
Objective: To investigate the effects of ginsenoside Rg3 on the formation of vasculogenic mimicry(VM) in gastric cancer cell line SGC7901 and its molecular mechanism. Methods: MTT assay was used to detect the effect of different concentrations of Rg3 on the proliferation of SGC7901 cells. SGC7901 cells were grouped as follows: BML-284 group, XAV-939 group, Rg3 group, Rg3+BML-284 group and blank group. Transwell chamber assay was used to detect cell invasion and migration; the formation of VM was observed by tube formation assay; the secretion of MMP-9 and MMP2 was detected by ELISA; the m RNA expressions of GSK-3β and Wnt2 B were detected by qPCR; the expression of β-Catenin protein in cells was analyzed by WB; and nuclear entry of β-Catenin was examined by Immunofluorescence. Results: Ginsenoside Rg3 inhibited the proliferation of SGC7901 cells in a time-and concentrationdependent manner; compared with the blank group, 40 mg/L Rg3 significantly inhibited the invasion and migration of SGC7901 cells(both P<0.05) and VM formation(P<0.05); in the meanwhile, the expressions of intracellular GSK-3β, Wnt2 B mRNA and β-catenin protein, as well as the nuclear entry of β-catenin were significantly inhibited(all P<0.05). The invasion, migration and VM formation of SGC7901 cells in Rg3+BML-284 group were not significantly different from those in the blank group(all P>0.05). Conclusion: Rg3 can inhibit cell invasion, migration and VM formation in SGC7901 cells by inhibiting the activation of Wnt/β-Catenin pathway.
Objective: To investigate the effects of ginsenoside Rg3 on the formation of vasculogenic mimicry(VM) in gastric cancer cell line SGC7901 and its molecular mechanism. Methods: MTT assay was used to detect the effect of different concentrations of Rg3 on the proliferation of SGC7901 cells. SGC7901 cells were grouped as follows: BML-284 group, XAV-939 group, Rg3 group, Rg3+BML-284 group and blank group. Transwell chamber assay was used to detect cell invasion and migration; the formation of VM was observed by tube formation assay; the secretion of MMP-9 and MMP2 was detected by ELISA; the m RNA expressions of GSK-3β and Wnt2 B were detected by qPCR; the expression of β-Catenin protein in cells was analyzed by WB; and nuclear entry of β-Catenin was examined by Immunofluorescence. Results: Ginsenoside Rg3 inhibited the proliferation of SGC7901 cells in a time-and concentrationdependent manner; compared with the blank group, 40 mg/L Rg3 significantly inhibited the invasion and migration of SGC7901 cells(both P<0.05) and VM formation(P<0.05); in the meanwhile, the expressions of intracellular GSK-3β, Wnt2 B mRNA and β-catenin protein, as well as the nuclear entry of β-catenin were significantly inhibited(all P<0.05). The invasion, migration and VM formation of SGC7901 cells in Rg3+BML-284 group were not significantly different from those in the blank group(all P>0.05). Conclusion: Rg3 can inhibit cell invasion, migration and VM formation in SGC7901 cells by inhibiting the activation of Wnt/β-Catenin pathway.
引文
[1] LAHMIDANI N, EL YOUSF M, AQODAD N, et al. Update on gastric cancer epidemiology and risk factors[J]. JCT, 2018, 9(3):242-254. DOI:10.4236/jct.2018.93021.
[2] RACORDON D, VALDIVIA A, MINGO G, et al. Structural and functional identification of vasculogenic mimicry in vitro[J/OL]. Sci Rep, 2017, 7(1):6985[2018-12-20]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5539303/. DOI:10.1038/s41598-017-07622-w.
[3] ZHANG J, QIAO L, LIANG N, et al. Vasculogenic mimicry and tumor metastasis[J/OL]. J Buon, 2016, 21(3):533-541[2018-12-20].http://jbuon.com/pdfs/JBUON-21-3-1.pdf.
[4]陈冠男,余飞,盛冠男,等.肿瘤血管生成拟态研究的希望与挑战[J].现代肿瘤医学, 2017, 25(1):138-141. DOI:10.3969/j.issn.1672-4992.2017.01.038.
[5] GUO Q J, YUAN Y, JIN Z C, et al. Association between tumor vasculogenic mimicry and the poor prognosis of gastric cancer in China:an updated systematic review and meta-analysis[J/OL]. Biomed Res Int, 2016, 2016:2408645[2018-12-20]. https://www. ncbi. nlm.nih.gov/pmc/articles/PMC5080470/. DOI:10.1155/2016/2408645.
[6] YAO L L, ZHANG D F, ZHAO X L, et al. Dickkopf-1-promoted vasculogenic mimicry in non-small cell lung cancer is associated with EMT and development of a cancer stem-like cell phenotype[J/OL]. J Cell Mol Med, 2016, 20(9):1673-1685[2018-12-20]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988283/. DOI:10.1111/jcmm.12862.
[7] GONG W C, SUN B C, ZHAO X L, et al. Nodal signaling promotes vasculogenic mimicry formation in breast cancer via the Smad2/3pathway[J/OL]. Oncotarget, 2016, 7(43):70152-70167[2018-12-20]. https://www. ncbi. nlm. nih. gov/pmc/articles/PMC5342542/.DOI:10.18632/oncotarget.12161.
[8] QI L S, SONG W Z, LIU Z Y, et al. Wnt3a promotes the vasculogenic mimicry formation of colon cancer via wnt/β-catenin signaling[J]. Int J Mol Sci, 2015, 16(8):18564-18579. DOI:10.3390/ijms160818564.
[9]汪丛丛,庄静,冯福彬,等.姜黄素抑制肺癌细胞血管拟态形成机制探讨[J].中华肿瘤防治杂志, 2015, 22(4):243-246. DOI:10.16073/j.cnki.cjcpt.2015.04.001.
[10]黄月云,夏婷,赵成国,等.人参皂苷Rh2和Rg3抗肿瘤作用研究进展[J].实用中医药杂志, 2016, 32(8):846-847. DOI:10.3969/j.issn.1004-2814.2016.08.084.
[11] KIM J W, JUNG S Y, KWON Y H, et al. Ginsenoside Rg3 attenuates tumor angiogenesis via inhibiting bioactivities of endothelial progenitor cells[J]. Cancer Biol Ther, 2012, 13(7):504-515. DOI:10.4161/cbt.19599.
[12] TANG M Y, WANG W B, CHENG L Y, et al. The inhibitory effects of 20(R)-ginsenoside Rg3 on the proliferation, angiogenesis, and collagen synthesis of hypertrophic scar derived fibroblasts in vitro[J]. Iran J Basic Med Sci, 2018, 21(3):309-317. DOI:10.22038/ijbms.2018.19451.5153.
[13] ZHOU Y Y, ZHENG X, LU J J, et al. Ginsenoside 20(S)-rg3 inhibits the warburg effect via modulating DNMT3A/miR-532-3p/HK2pathway in ovarian cancer cells[J]. Cell Physiol Biochem, 2018, 45(6):2548-2559. DOI:10.1159/000488273.
[14]杨茂鹏,王艳,王晓红,等.人参皂苷Rg3对大肠癌细胞系Lovo/5-Fu侵袭性及耐药影响的相关研究[J].哈尔滨医科大学学报,2018, 52(1):24-28.
[15] LU M X, FEI Z H, ZHANG G L. Synergistic anticancer activity of20(S)-Ginsenoside Rg3 and Sorafenib in hepatocellular carcinoma by modulating PTEN/Akt signaling pathway[J]. Biomed Pharmacother, 2018, 97:1282-1288. DOI:10.1016/j.biopha.2017.11.006.
[16] MA J L, HAN S X, ZHU Q, et al. Role of Twist in vasculogenic mimicry formation in hypoxic hepatocellular carcinoma cells in vitro[J]. Biochem Biophys Res Commun, 2011, 408(4):686-691.DOI:10.1016/j.bbrc.2011.04.089.
[17]林俊豪,冯思同,栗枭杰,等.基质金属蛋白酶和肿瘤血管生成拟态的相关研究[J].现代肿瘤医学, 2017, 25(3):470-473. DOI:10.3969/j.issn.1672-4992.2017.03.036.
[18]顾晓萌,于涛.恶性胃间质瘤的血管生成拟态与恶性程度的关系[J].山东大学学报(医学版), 2009, 47(5):117-119+130.
[19]朱磊,薛英威.胃癌血管生成拟态与胃癌分期及预后的研究[C].北京:阳光长城肿瘤学术会议, 2013.
[20] LI Y L, SUN B C, ZHAO X L, et al. MMP-2 and MMP-13 affect vasculogenic mimicry formation in large cell lung cancer[J]. J Cell Mol Med, 2017, 21(12):3741-3751. DOI:10.1111/jcmm.13283.
[21]李国栋,姚碧辉,金文海,等. Wnt/β-catenin的表达与肿瘤关系研究进展[J].世界最新医学信息文摘, 2018,(7):30-31.
[22] CLEVERS H, NUSSE R. Wnt/β-catenin signaling and disease[J].Cell, 2012, 149(6):1192-1205. DOI:10.1016/j.cell.2012.05.012.
[2] RACORDON D, VALDIVIA A, MINGO G, et al. Structural and functional identification of vasculogenic mimicry in vitro[J/OL]. Sci Rep, 2017, 7(1):6985[2018-12-20]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5539303/. DOI:10.1038/s41598-017-07622-w.
[3] ZHANG J, QIAO L, LIANG N, et al. Vasculogenic mimicry and tumor metastasis[J/OL]. J Buon, 2016, 21(3):533-541[2018-12-20].http://jbuon.com/pdfs/JBUON-21-3-1.pdf.
[4]陈冠男,余飞,盛冠男,等.肿瘤血管生成拟态研究的希望与挑战[J].现代肿瘤医学, 2017, 25(1):138-141. DOI:10.3969/j.issn.1672-4992.2017.01.038.
[5] GUO Q J, YUAN Y, JIN Z C, et al. Association between tumor vasculogenic mimicry and the poor prognosis of gastric cancer in China:an updated systematic review and meta-analysis[J/OL]. Biomed Res Int, 2016, 2016:2408645[2018-12-20]. https://www. ncbi. nlm.nih.gov/pmc/articles/PMC5080470/. DOI:10.1155/2016/2408645.
[6] YAO L L, ZHANG D F, ZHAO X L, et al. Dickkopf-1-promoted vasculogenic mimicry in non-small cell lung cancer is associated with EMT and development of a cancer stem-like cell phenotype[J/OL]. J Cell Mol Med, 2016, 20(9):1673-1685[2018-12-20]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988283/. DOI:10.1111/jcmm.12862.
[7] GONG W C, SUN B C, ZHAO X L, et al. Nodal signaling promotes vasculogenic mimicry formation in breast cancer via the Smad2/3pathway[J/OL]. Oncotarget, 2016, 7(43):70152-70167[2018-12-20]. https://www. ncbi. nlm. nih. gov/pmc/articles/PMC5342542/.DOI:10.18632/oncotarget.12161.
[8] QI L S, SONG W Z, LIU Z Y, et al. Wnt3a promotes the vasculogenic mimicry formation of colon cancer via wnt/β-catenin signaling[J]. Int J Mol Sci, 2015, 16(8):18564-18579. DOI:10.3390/ijms160818564.
[9]汪丛丛,庄静,冯福彬,等.姜黄素抑制肺癌细胞血管拟态形成机制探讨[J].中华肿瘤防治杂志, 2015, 22(4):243-246. DOI:10.16073/j.cnki.cjcpt.2015.04.001.
[10]黄月云,夏婷,赵成国,等.人参皂苷Rh2和Rg3抗肿瘤作用研究进展[J].实用中医药杂志, 2016, 32(8):846-847. DOI:10.3969/j.issn.1004-2814.2016.08.084.
[11] KIM J W, JUNG S Y, KWON Y H, et al. Ginsenoside Rg3 attenuates tumor angiogenesis via inhibiting bioactivities of endothelial progenitor cells[J]. Cancer Biol Ther, 2012, 13(7):504-515. DOI:10.4161/cbt.19599.
[12] TANG M Y, WANG W B, CHENG L Y, et al. The inhibitory effects of 20(R)-ginsenoside Rg3 on the proliferation, angiogenesis, and collagen synthesis of hypertrophic scar derived fibroblasts in vitro[J]. Iran J Basic Med Sci, 2018, 21(3):309-317. DOI:10.22038/ijbms.2018.19451.5153.
[13] ZHOU Y Y, ZHENG X, LU J J, et al. Ginsenoside 20(S)-rg3 inhibits the warburg effect via modulating DNMT3A/miR-532-3p/HK2pathway in ovarian cancer cells[J]. Cell Physiol Biochem, 2018, 45(6):2548-2559. DOI:10.1159/000488273.
[14]杨茂鹏,王艳,王晓红,等.人参皂苷Rg3对大肠癌细胞系Lovo/5-Fu侵袭性及耐药影响的相关研究[J].哈尔滨医科大学学报,2018, 52(1):24-28.
[15] LU M X, FEI Z H, ZHANG G L. Synergistic anticancer activity of20(S)-Ginsenoside Rg3 and Sorafenib in hepatocellular carcinoma by modulating PTEN/Akt signaling pathway[J]. Biomed Pharmacother, 2018, 97:1282-1288. DOI:10.1016/j.biopha.2017.11.006.
[16] MA J L, HAN S X, ZHU Q, et al. Role of Twist in vasculogenic mimicry formation in hypoxic hepatocellular carcinoma cells in vitro[J]. Biochem Biophys Res Commun, 2011, 408(4):686-691.DOI:10.1016/j.bbrc.2011.04.089.
[17]林俊豪,冯思同,栗枭杰,等.基质金属蛋白酶和肿瘤血管生成拟态的相关研究[J].现代肿瘤医学, 2017, 25(3):470-473. DOI:10.3969/j.issn.1672-4992.2017.03.036.
[18]顾晓萌,于涛.恶性胃间质瘤的血管生成拟态与恶性程度的关系[J].山东大学学报(医学版), 2009, 47(5):117-119+130.
[19]朱磊,薛英威.胃癌血管生成拟态与胃癌分期及预后的研究[C].北京:阳光长城肿瘤学术会议, 2013.
[20] LI Y L, SUN B C, ZHAO X L, et al. MMP-2 and MMP-13 affect vasculogenic mimicry formation in large cell lung cancer[J]. J Cell Mol Med, 2017, 21(12):3741-3751. DOI:10.1111/jcmm.13283.
[21]李国栋,姚碧辉,金文海,等. Wnt/β-catenin的表达与肿瘤关系研究进展[J].世界最新医学信息文摘, 2018,(7):30-31.
[22] CLEVERS H, NUSSE R. Wnt/β-catenin signaling and disease[J].Cell, 2012, 149(6):1192-1205. DOI:10.1016/j.cell.2012.05.012.