凌涛,王委,胡琛,严鑫,许雨婷,唐淳,寇玉,刘量
LING Tao,WANG Wei,HU Chen,YAN Xin,XU Yu-ting,TANG Chun,KOU Yu,LIU Liang

• 1:扬州大学医学院(转化医学研究院)江苏省中西医结合老年病防治重点实验室

摘要(Abstract)：

研究中药绶草中分离得到的主要的9,10-二氢菲类单体化合物orchinol对人胃癌SGC-7901细胞侵袭和迁移的抑制作用及初步的分子机制。体外培养人胃癌SGC-7901细胞,分别加入终浓度为5,10,20,40,80μmol·L~(-1)的orchinol处理细胞24,48,72 h,采用MTT法检测orchinol对SGC-7901细胞活力的影响;利用伤口愈合实验、Transwell实验分别分析5,10,20μmol·L~(-1)orchinol作用48 h后对SGC-7901细胞的迁移和侵袭能力的影响;Western blot法检测orchinol对SGC-7901细胞β-catenin,Wnt-3a,DvL2,cyclinD1,GSK-3β蛋白表达水平的影响。结果显示,orchinol在5～80μmol·L~(-1)能以剂量和时间依赖性的方式抑制SGC-7901细胞的活力,orchinol分别处理细胞24,48,72 h后,其对SGC-7901细胞的半数抑制浓度(IC_(50))分别为77.79,42.96,7.85μmol·L~(-1);与对照组相比,5,10,20μmol·L~(-1)orchinol作用48 h,可明显抑制SGC-7901细胞侵袭和迁移的能力(P<0.05,P<0.01),且呈剂量依赖关系;Western blot结果显示,orchinol可显著下调β-catenin,Wnt3a,DvL2,cyclinD1蛋白、显著上调GSK-3β蛋白表达水平(P<0.05,P<0.01,P<0.001)。以上结果提示orchinol能显著抑制SGC-7901细胞侵袭和迁移能力,其机制可能是与orchinol抑制Wnt3a/β-catenin信号通路及下游基因蛋白的表达有关。
The purpose of this study was to investigate the inhibitory effect of the main 9,10-dihydrophenanthrene orchinol isolated from Spiranthes sinensis Radix et Herba on the invasion and migration of human gastric cancer SGC-7901 cells and its preliminary molecular mechanism. SGC-7901 cells were cultured in vitro, after the cells were treated with different final concentrations(5, 10, 20, 40, 80 μmol·L~(-1)) of orchinol for 24, 48 or 72 hours, the effect of orchinol on cell viability was measured by MTT assay. Wound healing and Transwell assays were performed to determine the effects of different final concentrations(5, 10, 20, 40 μmol·L~(-1)) of orchinol for 48 hour on invasion and migration abilities of SGC-7901 cells, respectively. The protein expression levels of β-catenin, Wnt-3α, DvL2, cyclinD1 and GSK-3β were detected by Western blot. The results showed that 5-80 μmol·L~(-1)orchinol inhibited the viability of SGC-7901 cells in a dose-dependent and time-dependent manner, and the IC_(50)values of 24, 48 and 72 hours were 77.79, 42.96 and 7.85 μmol·L~(-1), respectively. Compared with the control group, the ability of invasion and migration of SGC-7901 cells was significantly inhibited after treated with 5, 10 and 20 μmol·L~(-1) orchinol for 48 hours(P<0.05, P<0.01), and the dose-effect relationship was observed. The results of Western blot showed that orchinol could significantly down-regulate the protein expression levels of β-catenin, Wnt3 a, DvL2 and cyclinD1, and up-regulate the protein expression level of GSK-3β(P<0.05, P<0.01, P<0.001). The above results suggest that orchinol can obviously inhibit the invasion and migration of SGC-7901 cells, which may be related to its inhibition of Wnt3 a/β-catenin signaling pathway and the proteins expression of downstream genes.

关键词(KeyWords)： 绶草;orchinol;人胃癌SGC-7901细胞;迁移;侵袭;Wnt3a/β-catenin信号通路
Spiranthes sinensis;orchinol;human gastric cancer SGC-7901 cells;migration;invasion;Wnt3a/β-catenin signaling pathway

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金青年基金项目(81703812);; 中国博士后基金面上项目(2016M601865);; 扬州大学大学生学术科技创新基金项目(x20170849,x20180734)

作者(Author): 凌涛,王委,胡琛,严鑫,许雨婷,唐淳,寇玉,刘量
LING Tao,WANG Wei,HU Chen,YAN Xin,XU Yu-ting,TANG Chun,KOU Yu,LIU Liang

参考文献(References)：

• [1] 张雯, 王金万. 胃癌的流行病学及其分型[J]. 中国全科医学, 2010, 13(11): 16.
• [2] 周际昌. 实用肿瘤内科学[M]. 北京:人民卫生出版社, 2010: 589.
• [3] 董瑞祥, 李刚, 孔庆霞. 奥沙利铂联合替吉奥一线治疗晚期胃癌效果[J]. 中国医药导报, 2014, 11(14): 56.
• [4] Liu J, Li C Y, Zhong Y J, et al. Chemical constituents from Spiranthes sinensis[J]. Biochem Syst Ecol, 2013, 47(8): 108.
• [5] 李建晨, 冯丽, 野原稔弘. 细叶石仙桃的化学成分[J]. 中国中药杂志, 2008, 33(14): 1691.
• [6] Auberon F, Olatunji O J, Krisa S, et al. Two new stilbenoids from the aerial parts of Arundina graminifolia (Orchidaceae)[J]. Molecules, 2016, 21(11): 1430.
• [7] Zhao G Y, Deng B W, Zhang C Y, et al. New phenanthrene and 9,10-dihydrophenanthrene derivatives from the stems of Dendrobium officinale with their cytotoxic activities[J]. J Nat Med, 2018, 72(1): 1.
• [8] Kovács A, Vasas A, Hohmann J. Natural phenanthrenes and their biological activity[J]. Phytochemistry, 2008, 69(5):1084.
• [9] 帝尔玛·丹增彭措. 晶珠本草[M]. 上海: 上海科学技术出版社, 1986: 360.
• [10] 李文丽. 盘龙参S-(180)肉瘤的实验观察[J]. 数理医药学杂志, 2005, 18(3): 193.
• [11] Liu J, Su X H, Li C Y, et al. Two new dihydrophenanthrenes from Spiranthes sinensis (Pers.) Ames[J]. Nat Prod Res Dev, 2012, 24: 866.
• [12] Li C Y, Liu J, Su X H, et al. New dimeric phenanthrene and flavone from Spiranthes sinensis[J]. J Asian Nat Prod Res, 2013, 15(4): 417.
• [13] Lin Y L, Huang R L, Don M J, et al. Dihydrophenanthrenes from Spiranthes sinensis[J]. J Nat Prod, 2000, 63(12): 1608.
• [14] Yasuhiro T, Midori U, Tohru K. Studies on the constituents of orchidaceous plant Ⅷ. Constituents of Spiranthe sinensis (Pers.) Ames var. amoena (M. BIEBERSON) HARA. (1). Isolation and structure elucidation of spiranthol-A, spiranthol-B, and spirasineol-A, new isopentenyldihydrophenanthrenes[J]. Chem Pharm Bull, 1989, 37(12): 3195.
• [15] Yasuhiro T, Li J, Hiroyuki H, et al. Studies on the constituents of orchidaceous plant Ⅷ. Constituents of Spiranthe sinensis (Pers.) AMES var. amoena (M. BIEBERSON) HARA. (2). Structures of spiranthesol, spiranthoquinone, spiranthol-C, and spirasineol-B, new isopentenyldihydrophenanthrenes[J]. Chem Pharm Bull, 1990, 38(2): 629.
• [16] Yasuhiro T, Keiko N, Hiroyuki H, et al. Spiranthesol, a dimeric dihydrophenanthrene from the roots of Spiranthes sinensis(Pers.) Ames(M. Bieberson) Hara[J]. Chem Pharm Bull, 1989, 37(6): 1667.
• [17] Lin Y L, Wang W Y, Kuo Y H, et al. Homocyclotirucallane and two dihydrophenanthrenes from Spiranthes sinensis[J]. Chem Pharm Bull, 2001, 49(9): 1098.
• [18] 彭亮. 胃癌侵袭转移相关功能基因的研究[D]. 北京:中国协和医科大学, 2009.
• [19] 高启龙,朱亚楠,石变,等.青龙衣含药血清抑制胃癌SGC-7901细胞生长和诱导凋亡[J].中国实验方剂学杂志,2017,23(13):111.
• [20] Oshima H, Matsunaga A, Fujimura T, et al. Carcinogenesis in mouse stomach by simultaneous activation of the Wnt signaling and prostaglandin E2 pathway[J]. Gastroenterology, 2006, 131(4): 1086.
• [21] Abeer E W, Enzo L. The Wnt/beta-catenin pathway in adrenocortical development and cancer [J]. Mol Cell Endocrinol, 2011, 332 (1/2): 32.
• [22] Berthon A, Martinez A, Bertherat J, et al. Wnt/β-catenin signalling in adrenal physiology and tumour development [J]. Mol Cell Endocrinol, 2012, 351(1): 87.
• [23] Lalefar N R,Witkowski A,Simonsen J B, et al. Wnt3α nanodisks promote ex vivo expansion bof hematopoietic stem and progenitor cells[J]. J Nanobiotechnol, 2016, 14(1): 66.
• [24] Zhao X, Lu Y, Nie Y, et al. MicroRNAs as critical regulators involved in regulating epithelial-mesenchymal transition[J].Curr Cancer Drug Tar, 2013(13): 1.
• [25] Laya M S, Azócar J A, Vera W, et al. Total synthesis of orchinol[J]. J Chem Res, 2000, 2000(7):324.