铁线莲皂苷对人胃癌SGC-7901细胞增殖、凋亡及NF-κB表达的影响
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摘要
背景和目的:胃癌是我国最常见的恶性肿瘤之一,其发病率和死亡率位居各恶性肿瘤前列。虽然近年来多学科综合治疗模式的进展使胃癌的疗效有了很大的提高,但其5年生存率依然较低。在对胃癌患者进行化疗过程中,往往会产生肿瘤细胞耐药和严重不良反应从而导致治疗失败。因此,寻找高效、低毒的新型药物或治疗方法应用于肿瘤的治疗具有重要的临床意义。中医药是我国医学珍贵的宝库,近年来随着成分提取技术和药理研究的发展,由中药中提取的多种成分如姜黄素、苦参碱、白藜芦醇等被证实对多种肿瘤细胞具有抑制增殖、促进凋亡的作用,部分已应用于临床。其中植物来源的紫杉类药物在临床应用取得了较好的效果。铁线莲属(Clematis L.)系毛茛科(Ranunculacea)植物,在我国分布广泛,铁线莲的根茎在中国被称为威灵仙,通常被用于止痛、镇静、抗菌、消炎、抗肿瘤及利尿。已有研究发现,铁线莲属植物中的一些成分具有抑制肿瘤细胞增殖的活性,但研究尚不够深入。本研究以胃癌SGC-7901细胞为对象,观察扬子铁线莲中提取的三萜类皂苷成分对人胃癌SGC-7901细胞株生长及凋亡的作用,研究其对核转录因子NF-κB活性的影响,初步探讨其可能的作用机理,为进一步深入研究奠定基础。
方法:1. MTT法检测铁线莲皂苷单独应用或联合化疗药物5-FU对胃癌SGC-7901细胞增殖的影响。2.流式细胞仪检测铁线莲皂苷单独应用或与化疗药物5-FU联合应用对肿瘤细胞凋亡的影响。3.应用Western Blot方法检测各实验组胞核NF-κB表达情况。4.应用胞核NF-κB活性检测手段(ELISA法)进一步确定胞核内NF-κB的表达情况。
结果: 1.不同浓度铁线莲皂苷作用于胃癌SGC-7901细胞后,对细胞的增殖抑制率均明显高于对照组(P<0.05),且呈现浓度依赖性。铁线莲皂苷浓度越高,细胞增殖抑制率则越高。联合用药比单独用药细胞抑制率明显上升,铁线莲皂苷10μg/ml培养48小时细胞抑制率23%,5-FU 5μg/ml细胞抑制率为28%。而两者联合应用细胞抑制率为48%。2.流式细胞术检测结果显示铁线莲皂苷对5-FU诱导的肿瘤细胞凋亡有明显的促进作用,对照组细胞凋亡率约为1.9%,单用5-FU时为8.3%,单用铁线莲皂苷为12.3%,联合用药组凋亡率为29.1%(P<0.01)。3. Western Blot及ELISA法均证实铁线莲皂苷对胞核NF-κB表达及活性有一定的抑制作用,并且这种抑制作用存在着剂量依赖现象。
结论:铁线莲皂苷对胃癌SGC-7901细胞具有抑制增殖、诱导凋亡的作用,其诱导细胞凋亡的机制部分可能与抑制胞核NF-κB表达有关,提示铁线莲皂苷作为一种作用于NF-κB的抑制剂可能会在诱导肿瘤凋亡的治疗方面具有临床应用前景。
Objective:Gastric cancer is one of the most common malignacies in our country. The incidence and fatality rate is in the forefront of malignancies. Although recent progress of multidisciplinary therapy has improved the curative effect, the 5 year overall survival of gastric cancer patients is still unsatisfactory. It is realized that the failure of treatment can be attributed to multi-drug resistance and severe toxic response. Investigation of efficient, low-toxic drugs or treatments has important clinical significance. Traditional Chinese medicine is a treasure trove of our country in aspect of medicine. In the recent years, as the development of technology of elements extracting and pharmacological research, many kinds of elements such as Curcumin and Resveratrol extracted from the Chinese medicine were confirmed to have an effect to inhibit proliferation and induce apoptosis of tumor cells. And some have been applied to clinical, such as paclitaxel. Clematis is a large genus in the Ranunculaceae family, which is widely distributed in China and has many pharmacological actions. The roots and rhizomes of Clematis are called weilingxian in Chinese, which is commonly used as an analgesic, abirritative, antibacterial, antiphlogistic, anticancer and diuretic agent. It is investigated that some elements from Clematis has cytotoxic activity and inhibit proliferation in vitro. But the research is not deeply enough .We investigate in vitro the effect of a new triterpene saponins extracted from Clematis ganpiniana on growth and apoptosis of human gastric carcinoma cell line SGC-7901, and on NF-κB activation. Furthermore, we preliminarily explore its possible mechanism of NF-κB inhibition, which is helpful for our further study.
Methods: 1.Human gastric carcinoma cell line SGC-7901 cells were treated with Clematis saponins and/or with 5-FU. MTT assay was used to evaluate the growth inhibition rates .Negative control and blank control were set. 2. Apoptosis rate of SGC- 7901 cells treated with Clematis saponins and 5 - fluorouracil (5-FU) was determined by Annexin V /PI double staining flow cytometry (FCM). 3. Western Blot was used to test the expression of NF-κB in nuclear. 4. The intranuclear activity of NF-κB was confirmed by ELISA assay.
Results: 1.The inhibition rates on SGC-7901 cells in each Clematis saponins treatment group were significantly higher than those in control group (P < 0. 05), and the inhibition rates increased with the concentration increase of Clematis saponins. The growth inhibitory effect of 5-FU on SGC-7901 cells was significantly enhanced by Clematis saponins. 10μg/ml of Clematis saponins caused 23% inhibitory rate. The inhibitory rate of 5-FU was 28%. Combined treatment of Clematis saponins and 5-FU resulted in 48% inhibitory rate. The Clematis saponins could obviously promote on cell apoptosis induced by 5-FU. 2. Western Blot and ELISA confirmed that the Clematis saponins could significantly inhibit the expression and activity of NF-κB in nuclei. And the inhibition pattern showed in dose- dependent manners. 3. Clematis saponins had a promotion of 5-FU induced cell apoptosis. The apoptosis rate of SGC-7901 cells was of 8.3% in 5-FU treated group and of 12.3% in Clematis saponins treated group, and of 29.1% in combined treatment of the two agents as well (P<0.01).
Conclusion: Clematis saponins can significantly inhibit proliferation and induce apoptosis of gastric carcinoma cell SGC-7901, which might be partly related to the inhibition of the expression of NF-κB. These data suggests that Clematis saponins, as a novel candidate of NF-κB inhibitor, might be of clinical significance in apoptosis-inducing cancer therapy.
方法:1. MTT法检测铁线莲皂苷单独应用或联合化疗药物5-FU对胃癌SGC-7901细胞增殖的影响。2.流式细胞仪检测铁线莲皂苷单独应用或与化疗药物5-FU联合应用对肿瘤细胞凋亡的影响。3.应用Western Blot方法检测各实验组胞核NF-κB表达情况。4.应用胞核NF-κB活性检测手段(ELISA法)进一步确定胞核内NF-κB的表达情况。
结果: 1.不同浓度铁线莲皂苷作用于胃癌SGC-7901细胞后,对细胞的增殖抑制率均明显高于对照组(P<0.05),且呈现浓度依赖性。铁线莲皂苷浓度越高,细胞增殖抑制率则越高。联合用药比单独用药细胞抑制率明显上升,铁线莲皂苷10μg/ml培养48小时细胞抑制率23%,5-FU 5μg/ml细胞抑制率为28%。而两者联合应用细胞抑制率为48%。2.流式细胞术检测结果显示铁线莲皂苷对5-FU诱导的肿瘤细胞凋亡有明显的促进作用,对照组细胞凋亡率约为1.9%,单用5-FU时为8.3%,单用铁线莲皂苷为12.3%,联合用药组凋亡率为29.1%(P<0.01)。3. Western Blot及ELISA法均证实铁线莲皂苷对胞核NF-κB表达及活性有一定的抑制作用,并且这种抑制作用存在着剂量依赖现象。
结论:铁线莲皂苷对胃癌SGC-7901细胞具有抑制增殖、诱导凋亡的作用,其诱导细胞凋亡的机制部分可能与抑制胞核NF-κB表达有关,提示铁线莲皂苷作为一种作用于NF-κB的抑制剂可能会在诱导肿瘤凋亡的治疗方面具有临床应用前景。
Objective:Gastric cancer is one of the most common malignacies in our country. The incidence and fatality rate is in the forefront of malignancies. Although recent progress of multidisciplinary therapy has improved the curative effect, the 5 year overall survival of gastric cancer patients is still unsatisfactory. It is realized that the failure of treatment can be attributed to multi-drug resistance and severe toxic response. Investigation of efficient, low-toxic drugs or treatments has important clinical significance. Traditional Chinese medicine is a treasure trove of our country in aspect of medicine. In the recent years, as the development of technology of elements extracting and pharmacological research, many kinds of elements such as Curcumin and Resveratrol extracted from the Chinese medicine were confirmed to have an effect to inhibit proliferation and induce apoptosis of tumor cells. And some have been applied to clinical, such as paclitaxel. Clematis is a large genus in the Ranunculaceae family, which is widely distributed in China and has many pharmacological actions. The roots and rhizomes of Clematis are called weilingxian in Chinese, which is commonly used as an analgesic, abirritative, antibacterial, antiphlogistic, anticancer and diuretic agent. It is investigated that some elements from Clematis has cytotoxic activity and inhibit proliferation in vitro. But the research is not deeply enough .We investigate in vitro the effect of a new triterpene saponins extracted from Clematis ganpiniana on growth and apoptosis of human gastric carcinoma cell line SGC-7901, and on NF-κB activation. Furthermore, we preliminarily explore its possible mechanism of NF-κB inhibition, which is helpful for our further study.
Methods: 1.Human gastric carcinoma cell line SGC-7901 cells were treated with Clematis saponins and/or with 5-FU. MTT assay was used to evaluate the growth inhibition rates .Negative control and blank control were set. 2. Apoptosis rate of SGC- 7901 cells treated with Clematis saponins and 5 - fluorouracil (5-FU) was determined by Annexin V /PI double staining flow cytometry (FCM). 3. Western Blot was used to test the expression of NF-κB in nuclear. 4. The intranuclear activity of NF-κB was confirmed by ELISA assay.
Results: 1.The inhibition rates on SGC-7901 cells in each Clematis saponins treatment group were significantly higher than those in control group (P < 0. 05), and the inhibition rates increased with the concentration increase of Clematis saponins. The growth inhibitory effect of 5-FU on SGC-7901 cells was significantly enhanced by Clematis saponins. 10μg/ml of Clematis saponins caused 23% inhibitory rate. The inhibitory rate of 5-FU was 28%. Combined treatment of Clematis saponins and 5-FU resulted in 48% inhibitory rate. The Clematis saponins could obviously promote on cell apoptosis induced by 5-FU. 2. Western Blot and ELISA confirmed that the Clematis saponins could significantly inhibit the expression and activity of NF-κB in nuclei. And the inhibition pattern showed in dose- dependent manners. 3. Clematis saponins had a promotion of 5-FU induced cell apoptosis. The apoptosis rate of SGC-7901 cells was of 8.3% in 5-FU treated group and of 12.3% in Clematis saponins treated group, and of 29.1% in combined treatment of the two agents as well (P<0.01).
Conclusion: Clematis saponins can significantly inhibit proliferation and induce apoptosis of gastric carcinoma cell SGC-7901, which might be partly related to the inhibition of the expression of NF-κB. These data suggests that Clematis saponins, as a novel candidate of NF-κB inhibitor, might be of clinical significance in apoptosis-inducing cancer therapy.
引文
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17 J ing - Pin Lin, Jai - Sing Yang, Jau - Hong Lee, et al. Berberine induces cell cycle arrest and apop tosis in human gastric carcinoma SNU - - 5 cell line [ J ]. World Journal of Gastroenterology, 2006, 12 (1) : 21 - 28.
18孙璟,涂水平,吴裕炘,等.天花粉蛋白诱导胃癌细胞MKN - 45凋亡中P53、Bcl- 2、c - myc蛋白表达变化[ J ].上海医学, 2001, 24 (5) : 292 - 294.
19董晓荣,伍钢,董继华,等.丹参酮ⅡA对MKN - 45细胞生长的影响[ J ].肿瘤防治杂志, 2005, 12 (19) : 1465 - 1468.
20陈坚,钟良,钱立平,等.丹参酮ⅡA诱导SGC - 7901胃癌细胞凋亡及机制[ J ] .复旦学报(医学版) , 2007, 34 ( 1) : 57- 61, 65.
21李晓光,谢锦,李文梅,等.大蒜油诱导人胃癌细胞分化和凋亡的机制研究[ J ] .中华肿瘤杂志, 1998, 20 ( 5 ) : 325 -327.
22 Benjamin CL, Melnikova VO, Ananthas -wamy HN. P53 p rotein and pathogenesis of melanoma and nonmelanoma skin cancer[ J ]. Advances in Experimental Medicine and Biology, 2008, 624: 265 - 282.
23姚保泰,吴敏,王博,等.盐酸小檗碱对人胃癌细胞凋亡与P53、Bcl - 2基因表达水平的影响[ J ].中华临床医学杂志,2006, 7 (1) : 1 - 4.
24戴亚红,刘淑梅,孟昕,等.三氧化二砷对人胃癌细胞株nm23、P53基因表达的影响[ J ] .中国康复理论与实践, 2005, 11(8) : 626 - 627.
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26徐立,王明艳,许冬青,等.康尔爱片诱导人胃腺癌SGC - 7901细胞凋亡及对p53基因表达影响的研究[ J ].南京中医药大学学报, 1999, 15 (6) : 347 - 349.
27 Manicassamy S, Sun Z. The critical role of protein kinase C - theta in Fas/Fas ligand -mediated apoptosis [ J ]. Journal of immunology, 2007, 178 (1) : 312 - 319.
28许相范,孙抒,孙百研,等.抗癌口服液体外对人胃癌MGC - 803细胞株凋亡相关基因作用的研究[ J ].中华综合临床医学杂志, 2003, 5 (1) : 14 - 15.
29苏勉诫,戴萍,吕晓英,等.癌宁对人胃腺癌细胞SGC - 7901细胞因子及Fas基因表达的影响[ J ] .现代预防医学, 2001,28 (1) : 6 - 7.
30吕晓英,刘新华,李由,等.红毛五加多糖对胃癌细胞的诱导凋亡作用[ J ] .中国癌症杂志, 2001, 11 (2) : 137 - 139.
31郑舜华,崔儒涛,蔡淦,等.乐胃煎对大鼠胃黏膜上皮异型增生细胞凋亡及调控基因(Bcl-2、Fas、ICE)蛋白表达的影响[ J ] .浙江中医学院学报, 1998, 23(6) : 45 - 48.
32 JunttilaMR, Westermarck J. Mechanisms of MYC stabilization in human malignancies[ J ]. Cell Cycle, 2008, 7 ( 5 ) : 592 -596.
33 HU Mei - jie, ZHANG Shu, WU Yu - xin,et al. Apop tosis in gastric cancer induced by Trichosanthinand relationship od this apoptosis with expression of C - myc [ J ]. Journal of Shanghai Second Medical University, 2002,14 (1) : 43 - 46.
34邓友平,林晨,张雪,等.三氧化二砷(As2O3 )诱导人胃腺癌SGC7901细胞程序化死亡并降低c - myc基因的表达[ J ].药学学报, 1999, 34 (5) : 333 - 337.
35张俊文,王丕龙.三氧化二砷诱导胃癌细胞凋亡及其对C - myc和TGF -β1表达的影响[ J ] .胃肠病学和肝病学杂志,2006, 15 (1) : 7 - 9.
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