蟾毒灵对MHCC97H迁移运动的影响及Rac1蛋白表达的实验研究
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摘要
背景及目的：肝细胞癌(HCC)是我国最常见的恶性肿瘤之一,恶性程度高。而手术或介入治疗术后复发率较高,原因在于肝癌容易发生侵袭转移,因此明确其侵袭转移的发生机制和探索有效的治疗方法仍然是科研和临床上面临的严峻的挑战。蟾毒灵(Bufalin)是从中药蟾酥中提取的蟾毒配基之一。有研究表明蟾毒灵具有抗肝癌作用,但是目前关于蟾毒灵对肝癌转移的作用和相关机制研究还不清楚。本研究的目的是观察蟾毒灵对不同转移潜能肝癌细胞系迁移运动的影响以及蟾毒灵作用下MHCC97H细胞中Rac1蛋白的表达情况并探讨其机制。
方法：以高转移潜能肝癌细胞系(MHCC97H)、低转移潜能肝癌细胞系(MHCC97L)、正常肝细胞(L02)为基础来评价蟾毒灵在抗肝癌细胞迁移运动中的作用和相关机制的初步研究。采用XTT、划痕实验和Transwell chamber实验分别评价蟾毒灵对高转移潜能肝癌细胞(MHCC97H)增殖活性及对细胞迁移运动的抑制作用；采用Western blot检测Rac1蛋白在MHCC97H、MHCC97L、L02三种细胞系及经蟾毒灵处理的MHCC97H(MHCC97HI)中的表达情况。
结果：XTT实验结果显示：MHCC97H与MHCC97L和L02细胞增殖活性相比,有差异有统计学意义(P<0.05)；L02细胞增殖活性和MHCC97L细胞增殖活性相比,差异无统计学意义(P>0.05)。Transwell小室实验结果显示：不同浓度蟾毒灵作用下MHCC97H细胞迁移数目,组间比较,差异有统计学意义(P<0.05)。Western blot检测结果显示：Rac1蛋白在三种细胞系中都有表达,在MHCC97H中高表达。MHCC97H(Ⅰ)与MHCC97H、MHCC97L和L02这三种细胞系中的Rac1蛋白表达情况相比较,其Rac1的表达量明显降低,差异具有统计学意义(P<0.05)。
结论：蟾毒灵可抑制MHCC97L、MHCC97L、L02细胞增殖活性,且对MHCC97H增殖抑制较明显,并呈现出时间剂量依赖效应。蟾毒灵可抑制高转移潜能肝癌细胞系(MHCC97H)的迁移运动。蟾毒灵可降低Rac1在高转移潜能的肝癌细胞系中表达,提示可能通过Rac1依赖的SAPK/JNK通路抑制细胞迁移运动进而抑制MHCC97H细胞转移特性。
BACKGORND & AIMS:Hepatocellular carcinoma (HCC) is one of the most aggressive malignant tumors highly prevalent in China. It transfer easily, there is high recurrence rate after surgical or inerventional therapy. Therefor, to study on the mechanism of invasion and metastasis, and to explore effective treatment of HCC is the challenge in science and clinic. Bufalin is extracted from toxin of Bufo bufo gargarizans Cantor, was reported to have inhibitory effects on HCC. However, the role of anti-metastasis and the underlying mechanism remain elusive. The purpose of this study was to observe influence of bufalin in high potential metastatic HCC cell lines and expression of Racl Protein.
METHOD:To study the role of bufalin and relationship in anti-metastasis and the underlying mechanism, we used HCC cell lines (MHCC97H, MHCC97L, LO2) as a based assay system. The inhibitory effect on bufalin of HCC cell's viability and migration was determined by XTT cytotoxicity assay, Scrape migration assay and Cell migration assay. Cell migration and invasion assays were performed by transwell chambers. The expressed of Racl protein in HCC lines, were determined by Western blot.
RESULTS:The result of XTT showed that the cell proliferation of MHCC97H was a statistically significant difference compared with MHCC97L/LO2 (P< 0.05). MHCC97L compared with LO2 was showed no significant differece (P< 0.05). The result of Transwell Chamber showed that there was a statistically significant among the three cell lines. The result of Western blot showed that Racl protein did express in the three cell lines (MHCC97H, MHCC97L, LO2). The difference was statistically significant among the three cell lines on expression of Rac1 (P<0.5). Rac1 is highly expressed in aggressive HCC cell lines. Bufalin would reduce the expressed of Racl in aggressive HCC cell lines.
CONCLUSIONS:Bufalin could inhibit the proliferation of HCC cell lines (MHCC97H, MHCC97L, LO2), and there showed a time and dose dependent effect. Bufalin could inhibit the migration of HCC lines. And Rac1 was highly expressed in aggressive HCC cell lines. Bufalin could reduce the expressed of Racl in aggressive HCC cell lines. Bufalin may be inhibit MHCC97H cell lines through Rac1 dependent SAPK/JNK pathway.
方法：以高转移潜能肝癌细胞系(MHCC97H)、低转移潜能肝癌细胞系(MHCC97L)、正常肝细胞(L02)为基础来评价蟾毒灵在抗肝癌细胞迁移运动中的作用和相关机制的初步研究。采用XTT、划痕实验和Transwell chamber实验分别评价蟾毒灵对高转移潜能肝癌细胞(MHCC97H)增殖活性及对细胞迁移运动的抑制作用；采用Western blot检测Rac1蛋白在MHCC97H、MHCC97L、L02三种细胞系及经蟾毒灵处理的MHCC97H(MHCC97HI)中的表达情况。
结果：XTT实验结果显示：MHCC97H与MHCC97L和L02细胞增殖活性相比,有差异有统计学意义(P<0.05)；L02细胞增殖活性和MHCC97L细胞增殖活性相比,差异无统计学意义(P>0.05)。Transwell小室实验结果显示：不同浓度蟾毒灵作用下MHCC97H细胞迁移数目,组间比较,差异有统计学意义(P<0.05)。Western blot检测结果显示：Rac1蛋白在三种细胞系中都有表达,在MHCC97H中高表达。MHCC97H(Ⅰ)与MHCC97H、MHCC97L和L02这三种细胞系中的Rac1蛋白表达情况相比较,其Rac1的表达量明显降低,差异具有统计学意义(P<0.05)。
结论：蟾毒灵可抑制MHCC97L、MHCC97L、L02细胞增殖活性,且对MHCC97H增殖抑制较明显,并呈现出时间剂量依赖效应。蟾毒灵可抑制高转移潜能肝癌细胞系(MHCC97H)的迁移运动。蟾毒灵可降低Rac1在高转移潜能的肝癌细胞系中表达,提示可能通过Rac1依赖的SAPK/JNK通路抑制细胞迁移运动进而抑制MHCC97H细胞转移特性。
BACKGORND & AIMS:Hepatocellular carcinoma (HCC) is one of the most aggressive malignant tumors highly prevalent in China. It transfer easily, there is high recurrence rate after surgical or inerventional therapy. Therefor, to study on the mechanism of invasion and metastasis, and to explore effective treatment of HCC is the challenge in science and clinic. Bufalin is extracted from toxin of Bufo bufo gargarizans Cantor, was reported to have inhibitory effects on HCC. However, the role of anti-metastasis and the underlying mechanism remain elusive. The purpose of this study was to observe influence of bufalin in high potential metastatic HCC cell lines and expression of Racl Protein.
METHOD:To study the role of bufalin and relationship in anti-metastasis and the underlying mechanism, we used HCC cell lines (MHCC97H, MHCC97L, LO2) as a based assay system. The inhibitory effect on bufalin of HCC cell's viability and migration was determined by XTT cytotoxicity assay, Scrape migration assay and Cell migration assay. Cell migration and invasion assays were performed by transwell chambers. The expressed of Racl protein in HCC lines, were determined by Western blot.
RESULTS:The result of XTT showed that the cell proliferation of MHCC97H was a statistically significant difference compared with MHCC97L/LO2 (P< 0.05). MHCC97L compared with LO2 was showed no significant differece (P< 0.05). The result of Transwell Chamber showed that there was a statistically significant among the three cell lines. The result of Western blot showed that Racl protein did express in the three cell lines (MHCC97H, MHCC97L, LO2). The difference was statistically significant among the three cell lines on expression of Rac1 (P<0.5). Rac1 is highly expressed in aggressive HCC cell lines. Bufalin would reduce the expressed of Racl in aggressive HCC cell lines.
CONCLUSIONS:Bufalin could inhibit the proliferation of HCC cell lines (MHCC97H, MHCC97L, LO2), and there showed a time and dose dependent effect. Bufalin could inhibit the migration of HCC lines. And Rac1 was highly expressed in aggressive HCC cell lines. Bufalin could reduce the expressed of Racl in aggressive HCC cell lines. Bufalin may be inhibit MHCC97H cell lines through Rac1 dependent SAPK/JNK pathway.
引文
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17 Kurosawa M, Tani Y, Nishimura S, et al. Distinct PKC isozymes regulate bufalin-induced differentiation and apoptosis in human monocytic cells. Am J Physiol Cell Physiol 200.1;280(3):C459-64.
18,Hashimoto S, Jing YK, Kawazoe N, et al. Bufalin reduces the level of topoisomerase II in human leukemia cells and affects the cytotoxicity of anticancer drugs. Leukemia Research 1997; 21 (9):875-88
19 Numazawa S, Inoue N, Nakura H, et al. A cardiotonic steroid bufal in-induced differentiation of THP-1 cells:Involvement of NA+, K+-ATPase inhibition in the early changes in proto-oncogene expression. Biochemical Pharmacology 1996;52 (2):321-9.
20Watabe M, Kawazoe N, Masuda Y, et al. Bcl-2 Protein Inhibits Bufalin-induced Apoptosis through Inhibition of Mitogen-activated Protein Kinase Activation in human leukemia U937 Cells. Cancer Res 1997;57(15):3097-100.
21刘云鹏,曲秀娟,王萍萍,等.蟾蜍灵诱导K562细胞分化和凋亡过程中WT1表达的下调.中华血液学杂志.2002;23(7)：356-9.
22 Masuda Y, Kawazoe N, Nakajo S, et al. Bufalin induces apoptosis and influences the expression of apoptosis-related genes in human leukemia cells. Leuk Res 1995;19(8):549-56.
23殷佩浩,李琦,秦爱军等.蟾毒灵诱导人胰腺癌细胞凋亡及其作用机制研究.上海中医药杂志.2008：42(6)：78-81.
24胡强,李琦,殷佩浩,等1蟾毒灵诱导人胰腺癌细胞凋亡及其对Survivin基因表达的影响.上海中医药大学学报.2008;22(1)：55-58.
25徐永中,范钰,张尤历,等.蟾蜍灵对结肠癌SW620细胞凋亡的影响.江 苏大学学报(医学版).2007;17(06)：515-7.
26 De Witt N. Introduction Angiogenesis. Nature.2005.483:931
27翟笑枫,吕祥,顾伟.等.蟾毒灵抑制血管生成作用的初步观察.中国中医药信息杂志.2010;8：29-31.
28 Lee DY, Yasuda M, Yamamoto T, et al. Bufalin inhibits endothelial cell proliferation and angiogenesis in vitro. Life Sci 1997;60(2): 127-34.
29王南瑶,李苏宜,赵伟,等.华蟾素联合三氧化二砷抑制鸡胚尿囊膜血管生成的实验研究.临床肿瘤学杂志.2006;11(7)：494-6.
30方凡夫,张亚妮,顾伟.蟾毒灵抑制人白血病多药耐药K562/VCR细胞株增殖的实验研究.中药新药与临床药理.2010;21(3)：213-5.
31顾伟,韩克起,苏永华等.蟾毒灵对裸鼠人肝癌移植瘤的抑制作用及对Bcl-2和Bax蛋白表达的影响.中西医结合学报.2007;5(2)：155-9.
32 Lee DY, Yasuda M, Yamamoto T, et al. Bufalin inhibits endothelial cell proliferation and angiogenesis in vitro. Life Sci. 1997; 60 (2):127-34.
33 Yamada, K., Honma, Y., Asahi, K. I., et al. Differentiation of human acute myeloid leukaemia cells in primary culture in response to cotylenin A, a plant growth regulator. Br J Haematol.2001; 114:814-21
34李二妮,刘云鹏,侯科佐,等.蟾毒灵对人鼻咽癌细胞CNE-1的放射增敏作用.中华肿瘤防治杂志.2006;13(13)：978-80.
35 Pastor N., Felipe. Bufalin influences the repair of X-ray-induced DNA breaks in Chinese hamster cells.2003. DNA Repair (Amst) 2(12)：1353-60.
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27 Keely PJ, Westwick JK, Whitchead IP, et al. Cdc42 and Raclinduce integrin-mediated cell motility and invasiveness through PI (3) K. Nature 1997,390:632-6.
28 Liu SJ, Yu M, He Y, et. al. Melittin Prevents Liver Cancer Cell Metastasis Through Inhibition of the Rac1-Dependent Pathway. Hepatology 2008; 47(6);1964-73.
1 Rohrer DC. Bufalin. Acta Cryst.1982. B38,1865-8.
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6 Takai N, Ueda T, Nishida M, et al. Bufalin induces growth inhibition、cell cycle arrest and apoptosis in human endometrial and ovarian cancer cells. Int J Mol Med 2008;21 (5):637-43.
7孟晓燕,方凡夫,顾伟,等.蟾毒灵对人肝癌多药耐药BEL-7402/5-FU细胞增殖活性的影响.山东医药.2009;49(5)：37-9.
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13 Liu YP, QU XJ, Wang PPL, et al. WT1 downregulation during K562 cell differentiation and apop tosis induced by bufalin. Chin J Hem atol 2002;23 (7):356-9.
14 Numazawa S, Inoue N, Nukura H, et al. A cardiotonic steroid bufalin induced differentiation of THP-1 cells Involvement of Na+-K+-ATPase inhibition in the early changes in proto on cogene expression. Bio Chem Pharm acol 1996;52 (2):321-9.
15 Yamada K, Hino K, Tomoyasu S, et all Enhancement by bufalin of retinoic acid2induced differentiation of acute promyelocytic leukemia cells in p rimary culture. Leuk Res 1998;22(7):589-595.
16 Kerr JF, Wyllie AH, Currie AR. Apoptosis:a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972;26(4):239-57.
17 Kurosawa M, Tani Y, Nishimura S, et al. Distinct PKC isozymes regulate bufalin-induced differentiation and apoptosis in human monocytic cells. Am J Physiol Cell Physiol 200.1;280(3):C459-64.
18,Hashimoto S, Jing YK, Kawazoe N, et al. Bufalin reduces the level of topoisomerase II in human leukemia cells and affects the cytotoxicity of anticancer drugs. Leukemia Research 1997; 21 (9):875-88
19 Numazawa S, Inoue N, Nakura H, et al. A cardiotonic steroid bufal in-induced differentiation of THP-1 cells:Involvement of NA+, K+-ATPase inhibition in the early changes in proto-oncogene expression. Biochemical Pharmacology 1996;52 (2):321-9.
20Watabe M, Kawazoe N, Masuda Y, et al. Bcl-2 Protein Inhibits Bufalin-induced Apoptosis through Inhibition of Mitogen-activated Protein Kinase Activation in human leukemia U937 Cells. Cancer Res 1997;57(15):3097-100.
21刘云鹏,曲秀娟,王萍萍,等.蟾蜍灵诱导K562细胞分化和凋亡过程中WT1表达的下调.中华血液学杂志.2002;23(7)：356-9.
22 Masuda Y, Kawazoe N, Nakajo S, et al. Bufalin induces apoptosis and influences the expression of apoptosis-related genes in human leukemia cells. Leuk Res 1995;19(8):549-56.
23殷佩浩,李琦,秦爱军等.蟾毒灵诱导人胰腺癌细胞凋亡及其作用机制研究.上海中医药杂志.2008：42(6)：78-81.
24胡强,李琦,殷佩浩,等1蟾毒灵诱导人胰腺癌细胞凋亡及其对Survivin基因表达的影响.上海中医药大学学报.2008;22(1)：55-58.
25徐永中,范钰,张尤历,等.蟾蜍灵对结肠癌SW620细胞凋亡的影响.江 苏大学学报(医学版).2007;17(06)：515-7.
26 De Witt N. Introduction Angiogenesis. Nature.2005.483:931
27翟笑枫,吕祥,顾伟.等.蟾毒灵抑制血管生成作用的初步观察.中国中医药信息杂志.2010;8：29-31.
28 Lee DY, Yasuda M, Yamamoto T, et al. Bufalin inhibits endothelial cell proliferation and angiogenesis in vitro. Life Sci 1997;60(2): 127-34.
29王南瑶,李苏宜,赵伟,等.华蟾素联合三氧化二砷抑制鸡胚尿囊膜血管生成的实验研究.临床肿瘤学杂志.2006;11(7)：494-6.
30方凡夫,张亚妮,顾伟.蟾毒灵抑制人白血病多药耐药K562/VCR细胞株增殖的实验研究.中药新药与临床药理.2010;21(3)：213-5.
31顾伟,韩克起,苏永华等.蟾毒灵对裸鼠人肝癌移植瘤的抑制作用及对Bcl-2和Bax蛋白表达的影响.中西医结合学报.2007;5(2)：155-9.
32 Lee DY, Yasuda M, Yamamoto T, et al. Bufalin inhibits endothelial cell proliferation and angiogenesis in vitro. Life Sci. 1997; 60 (2):127-34.
33 Yamada, K., Honma, Y., Asahi, K. I., et al. Differentiation of human acute myeloid leukaemia cells in primary culture in response to cotylenin A, a plant growth regulator. Br J Haematol.2001; 114:814-21
34李二妮,刘云鹏,侯科佐,等.蟾毒灵对人鼻咽癌细胞CNE-1的放射增敏作用.中华肿瘤防治杂志.2006;13(13)：978-80.
35 Pastor N., Felipe. Bufalin influences the repair of X-ray-induced DNA breaks in Chinese hamster cells.2003. DNA Repair (Amst) 2(12)：1353-60.