^(131)Ⅰ-17-丙烯胺基-17-去甲氧基格尔德霉素对人乳腺癌细胞生长的抑制作用

Inhibitory effects of ^(131)I labeled 17-allylamino-17-demethoxygeldanamycin on breast cancer cell line

目的探讨^(131)I-17-丙烯胺基-17-去甲氧基格尔德霉素(17-AAG)对人乳腺癌细胞生长的影响及其相关机制。方法采用过氧化氢标记法制备^(131)I-17-AAG。细胞杀伤实验分为5组:二甲基亚砜(DMSO)对照(A)组,Na^(131)I 370 kBq(B)组,17-AAG 2.5 mg/L(C)组,^(131)I-17-AAG 370 kBq(D)组,^(131)I-17-AAG 370 kBq+17-AAG 2.5 mg/L(E)组。用四甲基偶氮唑蓝(MTT)法检测各种药物对人乳腺癌细胞 MCF-7的生长抑制作用,流式细胞术分析细胞凋亡及细胞周期变化,RT-PCR 检测药物处理前后 MCF-7细胞中 Akt2基因的 mRNA 表达情况。结果 ^(131)I-17-AAG 的标记率为83%,放化纯为96.6%,比活度为1.48×10~5MBq/μmol。各组药物对细胞的杀伤呈时间效应,随着时间的延长,细胞的抑制率都呈明显上升趋势,尤以 E 组趋势明显。A～E组药物作用48 h 后,通过亚 G1峰检测 MCF-7细胞凋亡率分别为(1.54±0.13)%,(5.72±1.05)%,(12.97±1.44)%,(20.65±1.36)%,(35.39±4.15)%,各组细胞凋亡率差异有统计学意义(P 均<0.05)。C 组,D 组及 E 组Akt2基因的 mRNA 表达均比 A 组降低,其中 E 组降低尤为明显。结论 ^(131)I-17AAG 能够抑制 MCF-7细胞的生长并促进其凋亡,且能有效抑制 Akt2基因的 mRNA 表达,和17-AAG 联合应用能够增强肿瘤细胞对放疗的敏感性。

Objective 17-allylamino-17-demethoxygeldanamycin（17-AAG） is a less toxic analogue of geldanamycin （GA） that retains the tumoricidal features of GA. Same as its parent compound, 17-AAG inhibits several signaling pathways through binding to heat shock protein （HSP） 90, which results in destabilization of signaling complexes and degradation of client proteins in a variety of tumor cell growth. Treatment with 17-AAG was effective to inhibit tumor growth and induce apoptosis in colon cancer, glioblastoma, and breast cancer cell lines. This study aimed at exploring the anti-proliferation effects and mechanism of ^131I labeled 17-AAG on human breast cancer cell line MCF-7. Methods ^1311-17-AAG was prepared by the reaction of 17-AAG with Na ^131I in the presence of hydrogen peroxide. The MCF-7 cells were divided into 5 groups with different additional drugs： group A, dimethyl sulfoxide （DMSO） ; group B, 370 kBq Na ^131I; group C, 2.5 mg/L 17-AAG; group D, 370 kBq ^131I-17-AAG; group E, 370 kBq 131I-17-AAG ＋ 2.5 mg/L 17-AAG. 3- （ 4,5-dimethyhhiazol-2-yl ） -2,5, diphenylte-trazolium bromide （MTT） assay was used to evaluate the effect of growth inhibition of MCF-7 cells. Cell cycle and apoptosis were analyzed by flow cytometry. The change of the expression of Akt2 mRNA in MCF-7 cells was examined by RT-PCR. Results The labeling yield of ^131I-17-AAG was 83%. The radiochemical purity of ^131I-17-AAG after purification was 96.6%. The specific activity was 1.48 × 10^5 MBq/μmol. All drugs could significantly inhibit the growth of MCF-7 cells in vitro as the duration lasts longer, especially for group E. After 48 h, sub-G1 peaks detected by flow cytometry were（1.54 ±0.13）% ,（5.72 ± 1.05）% ,（12.97 ± 1.44）%, （20.65 ± 1.36）%, （35.39 ±4.15）% for group A, B, C, D and E, respectively. The experimental groups （ B - E） were all significantly higher than the control group（A, all P 〈0.05）. The expression of Akt2 mRNA in treated MCF-7 cells（groups C -E）were all lower than that of the control group （ A）, especially for group E. Conclusions ^131I-17-AAG could suppress the growth of human breast cancer cell line MCF-7 and hasten the apoptosis. It could significantly suppress the expression of Akt2 mRNA. Combined with 17-AAG, ^131I-17-AAG could improve the effect of radiotherapy.