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乳腺癌是女性最常见的恶性肿瘤之一,严重危害女性健康和生命[1-2],向临床提供安全、有效的抗肿瘤药物是当今医药工作者的使命。热休克蛋白90(heat shock protein, Hsp90)在肿瘤细胞中的表达比正常细胞高2~10倍,并可调控众多信号蛋白的构象成熟和稳定性,在肿瘤细胞生长、分化、凋亡等方面发挥重要作用,已成为抗肿瘤药物研究的重要靶点之一[3-5]。格尔德霉素(geldanamycin, GA)是第一个鉴定出的Hsp90抑制剂,虽然其抗肿瘤活性很强,但是由于具有肝毒性强、水溶性差等缺点,严重限制了临床上的应用[6-7]。糖基化修饰是提高化合物水溶性的最常用方法之一,不仅可以增加化合物的水溶性和稳定性,丰富结构多样性,并可改善药物在体内的药代动力学性质,有利于药物的药效发挥[8]。最近,本课题组通过体外酶法糖基化反应制备了若干个GA衍生物糖基化产物,但是对低毒性的非苯醌GA衍生物17-demethoxy-reblastatin(17-DR)糖基化产物的准确结构及其抗肿瘤活性未进行研究报道[9-10]。因此,本研究中将通过体外酶法糖基化法制备新型非苯醌GA糖基化衍生物,并利用ESI-MS和NMR波谱解析鉴定其结构,同时探讨和阐明新型非苯醌GA糖基化衍生物对Hsp90活性、人乳腺癌MCF-7细胞增殖、凋亡的影响,为今后的持续研究提供科学依据。
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HPLC分析结果(见图 1)显示,非苯醌GA衍生物17-DR的体外酶法糖基化反应液中可观察到其糖基化产物峰的存在。17-DR峰(底物)的保留时间(tR)为8.8 min,与此对应的糖基化产物峰tR为7.2 min。
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化合物1为白色粉末,其分子式为C34H52N2O12,ESI-MS [M-H]-m/z 679;1H-NMR(700 MHz, DMSO-d6)数据为:δH 9.45(-NH, s), 7.0(1H, brd, H-19), 6.54 (1H, s, H-17), 6.49 (1H, s, H-21), 5.80 (1H, brs, H-3), 5.27 (1H, d, J=9.1 Hz), 4.85 (1H, d, J=7.7 Hz, H-7), 4.74 (1H, d, J=7.0 Hz, H-1′), 3.69 (1H, d, J=10.5 Hz, H-6′), 3.50 (1H, dd, J=5.6, 10.5 Hz, H-6′), 3.32 (3H, s, 6-OCH3), 3.22 (3H, s, 12-OCH3), 3.20 (1H, m, H-11), 3.18-3.25 (overlap, H-6, H-3′, H-4′, and H-5′), 3.0 (2H, m, H-12 and H-2′), 2.34-2.60 (overlap, H-13 and H-15), 2.33 (1H, m, H-10), 2.14 (2H, m, H-4), 1.75 (1H, m, H-14), 1.71 (3H, s, H-22), 1.39 (3H, s, H-23), 1.23 (2H, m, H-5), 0.90 (3H, d, J=6.3 Hz, H-24), 0.78 (3H, d, J=5.6 Hz, H-25);13C-NMR (175 MHz, DMSO-d6)数据为:δC 170.7(C-1), 157.6 (C-18), 156.1(7-OCONH2), 141.5 (C-16), 139.9 (C-20), 134.7 (C-3), 133.3 (C-9), 131.9 (C-2), 129.8 (C-8), 117.0 (C-21), 112.9 (C-17), 106.7 (C-19), 100.7 (C-1′), 80.7 (C-7, C-12, and C-2′), 79.4 (C-6), 77.1 (C-5′), 76.7 (C-3′), 73.2 (C-11), 69.6 (C-4′), 60.6 (C-6′), 58.3 (6-OCH3), 56.4 (12-OCH3), 42.7 (C-13 and C-15), 33.9 (C-10), 30.9 (C-14), 29.7 (C-5), 23.4 (C-4), 19.1 (C-25), 16.3 (C-24), 13.1 (C-22), 11.8 (C-23)。化合物1除了具有17-DR类似的波谱特征之外,还具有1分子葡萄糖单元信号[δH 4.74 (H-1′), 3.0 (H-2′), 3.18-3.25 (H-3′, H-4′, H-5′), 3.69 (H-6′), 3.50 (H-6′), 和δC 100.7 (C-1′), 80.7 (C-2′), 76.7 (C-3′), 69.6 (C-4′), 77.1 (C-5′), 60.6 (C-6′)][11]。进一步,根据HMBC确认了化合物1中的糖分子、甲氧基、7-氨基甲酰基等主要官能团的连接位置(H-1′/C-18, 6-OCH3/C-6, 12-OCH3/C-12, H-7/C-6, 7-OCONH2, C-8等)(见图 2)。因此,新化合物1的结构鉴定为17-demethoxy-reblastatin-18-O-β-D-glucopyranoside。
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本研究通过体外酶法糖基化反应制备了化合物1,并发现化合物1具有较强的特异性地抑制酵母Hsp90蛋白ATPase活性。结果显示,化合物1显著地抑制Hsp90活性,其IC50值为8.98 μmol/L,而经典的Hsp90抑制剂GA的IC50值为3.06 μmol/L (见表 1)。
分组 IC50/ (μmol/L) F P MS组内 化合物1 8.98±0.56 — — — 17-DR 1.82±0.13** 347.78 < 0.01 0.126 GA 3.06±0.22△△ 859.83 < 0.01 0.244 q检验:与化合物1比较**P < 0.01;与17-DR比较△△P < 0.01 表 1 化合物1抑制Hsp90 ATPase活性(ni=3;x±s)
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结果表明,化合物1具有一定的抑制乳腺癌MCF-7细胞增殖的作用,并呈现时间与浓度依赖性,作用于MCF-7细胞72 h的IC50值为81.84 μmol/L (见图 3)。
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PI染色/流式细胞术结果显示,化合物1具有一定的诱导MCF-7细胞凋亡的能力,并呈现浓度依赖性,不同浓度化合物1(50、100、200 μmol/L)其诱导肿瘤细胞凋亡率分别为5.4%、6.1%、14.3%,用200 μmol/L的化合物1处理细胞组诱导细胞凋亡与阴性对照组2.4%比较差异有统计学意义(P < 0.05) (见图 4)。
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结果显示,Hsp90顾客蛋白Akt和Bcl-2随着化合物浓度的增加,其表达逐渐下调,呈现浓度依赖关系,而对Hsp90蛋白表达基本不产生影响(见图 5)。与阴性对照组相比,不同浓度的化合物1诱导Akt和Bcl-2蛋白下调率分别为5%、20%、51%和10%、29%、51%(见表 2)。
化合物/(μmol/L) Akt Bcl-2 Hsp90 0 1.00±0.02 1.00±0.15 1.00±0.11 50 0.95±0.03 0.90±0.01 0.86±0.04* 100 0.80±0.03* 0.71±0.05* 0.88±0.03* 200 0.49±0.07** 0.49±0.04** 0.87±0.04* F 25.20 15.81 7.09 P < 0.01 < 0.01 < 0.05 MS组内 0.005 0.008 0.010 与0 μmol/L组比较**P < 0.01或*P < 0.05 表 2 Akt、Bcl-2和Hsp90蛋白表达灰度值的定量分析结果(ni=3;x±s)
新型格尔德霉素葡萄糖苷的制备及其诱导人乳腺癌MCF-7细胞凋亡的作用
Preparation of a novel geldanamycin glucoside and its induction effects on the apoptosis of human breast cancer MCF-7 cells
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摘要:
目的对非苯醌格尔德霉素进行酶法糖基化修饰,并探讨其糖基化产物对乳腺癌细胞增殖、凋亡的影响。 方法利用体外酶法糖基化反应制备新型非苯醌格尔德霉素糖基化产物,并经质谱和核磁共振解析鉴定其结构。采用孔雀绿-钼酸铵显色反应、MTT法、PI单染/流式细胞术检测化合物的体外抗肿瘤活性。免疫印迹法检测化合物对Akt、Bcl-2及热休克蛋白90(Hsp90)表达的影响。 结果新型非苯醌格尔德霉素糖基化产物鉴别为17-demethoxy-reblastatin-18-O-β-D-glucopyranoside(1)。化合物1具有显著抑制Hsp90 ATPase活性,其IC50值为8.98 μmol/L。化合物1对人乳腺癌MCF-7细胞表现出一定的抑制增殖、诱导凋亡的作用,且呈现浓度依赖性。免疫印迹法结果显示,随着化合物1浓度的增加,诱导Akt和Bcl-2蛋白的降解越明显,而对Hsp90蛋白的表达没有影响。 结论新型Hsp90抑制剂化合物1具有抑制MCF-7细胞增殖、诱导凋亡能力,其机制可能是通过抑制Hsp90 ATPase活性以及诱导降解Hsp90顾客蛋白Akt和Bcl-2有关。 Abstract:ObjectiveTo explore the effects of the enzymatic glycosylation product modified by enzymatically non-benzoquinone geldanamycin(GA) on the proliferation and apoptosis of breast cancer cells. MethodsA new non-benzoquinone GA glucoside was prepared using enzymatic glycosylation in vitro.The product was characterized by ESI-MS and nuclear magnetic resonance(NMR) analysis.The anti-cancer activities of the compound were evaluated using malachite green-molybda colour reaction, MTT assay and flow cytometry with PI staining in vitro.The effects of the compound on expression levels of Akt, Bcl-2 and heat shock protein 90(Hsp90) were detected using Western blot. ResultsThe structure of novel non-benzoquinone GA glucoside was characterized as 17-demethoxy-reblastatin-18-O-β-D-glucopyranoside(1).Compound 1 could significantly inhibit the activity of Hsp90 ATPase, the IC50 value of which was 8.98 μmol/L.Compound 1 could inhibit the proliferation, and induce apoptosis in MCF-7 cells of human breast cancer in a concentration-dependent manner.The results of Western blot showed that, with the increasing of compound 1 concentration, the induced degradation of Akt and bcl-2 protein was more obvious, while the expression of Hsp90 protein was not affected. ConclusionsThe new Hsp90 inhibitor, compound 1, can inhibit the proliferation, and induce apoptosis of MCF-7 cells, the mechanism of which may be by inhibiting the activity of Hsp90 ATPase, and inducing the degradation of Hsp90 client protein Akt and Bcl-2. -
Key words:
- breast neoplasms /
- geldanamycin /
- heat shock protein 90 /
- glycosylation /
- apoptosis
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表 1 化合物1抑制Hsp90 ATPase活性(ni=3;x±s)
分组 IC50/ (μmol/L) F P MS组内 化合物1 8.98±0.56 — — — 17-DR 1.82±0.13** 347.78 < 0.01 0.126 GA 3.06±0.22△△ 859.83 < 0.01 0.244 q检验:与化合物1比较**P < 0.01;与17-DR比较△△P < 0.01 表 2 Akt、Bcl-2和Hsp90蛋白表达灰度值的定量分析结果(ni=3;x±s)
化合物/(μmol/L) Akt Bcl-2 Hsp90 0 1.00±0.02 1.00±0.15 1.00±0.11 50 0.95±0.03 0.90±0.01 0.86±0.04* 100 0.80±0.03* 0.71±0.05* 0.88±0.03* 200 0.49±0.07** 0.49±0.04** 0.87±0.04* F 25.20 15.81 7.09 P < 0.01 < 0.01 < 0.05 MS组内 0.005 0.008 0.010 与0 μmol/L组比较**P < 0.01或*P < 0.05 -
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