Novel vitamin E analogue and 9-nitro-camptothecin administered as liposome aerosols decrease syngeneic mouse mammary tumor burden and inhibit metastasis
Novel vitamin E analogue and 9-nitro-camptothecin administered as liposome aerosols decrease syngeneic mouse mammary tumor burden and inhibit metastasis
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DOI：
10.1007/s00280-004-0817-y
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发表时间：
2004-11-01
影响因子：
3
通讯作者：
Kline, K
中科院分区：
文献类型：
--
作者：
Lawson, KA;Anderson, K;Kline, K
目的：为了测试维生素E的不可水解的醚连接乙酸类似物2，5，7，8-四甲基-2R-（4 R，8 R，12-三甲基十三烷基）色满-6-基氧基乙酸（α-TEA）和喜树碱衍生物9-硝基喜树碱（9-NC）单独和组合抗小鼠乳腺肿瘤细胞（用绿色荧光蛋白稳定转染的系66克隆4; 66 c1 -4-GFP）体外培养或皮下移植到雌性BALB/c小鼠的腹股沟区以形成建立的肿瘤。研究方法：在用α-TEA和次优浓度的9-NC单独或组合体外处理66 cl-4-GFP细胞后，通过用4 ′，6-二脒基-2-苯基吲哚（DAPI）染色的细胞核的形态学评价来测量细胞凋亡，并通过氚化胸苷摄取来测量DNA合成停滞。对于体内分析，使用二月桂酰磷脂酰胆碱将α-TEA和9-NC（均为水不溶性化合物）配制成脂质体，并通过气雾剂给药，计算出的剂量分别为每天36和0.4 μ g/小鼠（单独给药或单独给药）联合治疗）每周7天。结果如下：用α-TEA（10 μ g/ml;单独产生38%凋亡）和次优浓度的9-NC（15.6、31.3、62.5或125 ng/ml;单独产生2 - 7%凋亡）的组合处理培养物中的66 cl-4-GFP细胞3天，产生47%、58%、64%和69%凋亡。同样地，α-TEA + 9-NC的组合比单独施用的任一种药剂更多地抑制DNA合成。用脂质体配制和雾化递送α-TEA + 9-NC至BALB/c小鼠，观察到皮下移植肿瘤生长的显著减少（P< 0.001）。肉眼可见的肺转移的发生率在对照组中为83%，而在α-TEA-、9-NC-或组合处理的小鼠中为8%。肺以及腋窝和臂淋巴结的荧光显微镜检查显示，与对照组相比，在α-TEA-、9-NC-和组合治疗的动物中观察到的转移统计学显著降低。原发性肿瘤组织的增殖和凋亡分析显示，治疗组分别具有较低的Ki-67和较高的末端脱氧核苷酸转移酶介导的缺口末端标记。治疗显示对两个血管生成参数没有可测量的影响，即通过血红蛋白含量评估的肿瘤内血容量和通过CD 31染色评估的肿瘤内血管密度。结论：联合治疗增强了细胞培养物中的抗增殖和促凋亡活性，并且当在脂质体中配制并通过雾化递送以治疗侵袭性和转移性同基因鼠乳腺肿瘤时，与单独的任一种治疗相比，联合治疗显示肿瘤体积显著减小。从机制上讲，似乎增强的细胞凋亡、降低的细胞增殖和降低的血管密度都不能完全解释组合治疗的增强效果。
Purpose: To test the anticancer properties of a nonhydrolyzable ether-linked acetic acid analogue of vitamin E, 2,5,7,8-tetramethyl-2R-(4R, 8R, 12-trimethyltridecyl) chroman-6-yloxyacetic acid (alpha-TEA), and a derivative of camptothecin, 9-nitrocamptothecin (9-NC) singly and in combination against mouse mammary tumor cells ( line 66 clone 4 stably transfected with green fluorescent protein; 66c1-4-GFP) cultured in vitro or transplanted subcutaneously into the inguinal region of female BALB/c mice to form established tumors. Methods: Following in vitro treatment of 66cl-4-GFP cells with alpha-TEA and suboptimal concentrations of 9-NC, singly or in combination, apoptosis was measured by morphological evaluation of nuclei stained with 4', 6-diamidino- 2-phenylindole ( DAPI), and DNA synthesis arrest was measured by tritiated thymidine uptake. For in vivo analyses alpha-TEA and 9-NC, both water-insoluble compounds, were formulated into liposomes using dilauroylphosphatidylcholine and administered by aerosol to deliver doses calculated to be 36 and 0.4 mug/mouse per day, respectively, ( singly or each separately for combined treatments) 7 days per week. Results: Treatment of 66cl-4-GFP cells in culture for 3 days with a combination of alpha-TEA ( 10 mug/ml; singly produces 38% apoptosis), and suboptimal concentrations of 9-NC (15.6, 31.3, 62.5, or 125 ng/ml; singly produce 2 - 7% apoptosis), produced 47%, 58%, 64%, and 69% apoptosis. Likewise, combinations of alpha-TEA + 9-NC inhibited DNA synthesis more than either agent administered singly. A significant reduction (P< 0.001) in growth of subcutaneous transplanted tumors was observed with liposome-formulated and aerosolized delivery of alpha-TEA+ 9-NC to BALB/c mice. The incidence of macroscopic lung metastasis was 83% in control vs 8% in alpha-TEA-, 9-NC-, or combination-treated mice. Fluorescence microscopic examination of lungs and axillary and brachial lymph nodes showed a statistically significant decrease in metastasis observed in alpha-TEA-, 9-NC-, and combination- vs control-treated animals. Analyses of primary tumor tissue for proliferation and apoptosis showed treatment groups to have lower Ki-67 and higher terminal deoxynucleotidyl transferase-mediated nick end labeling, respectively. Treatments showed no measurable effects on two angiogenesis parameters, namely intratumoral blood volume as assessed by hemoglobin content and intratumoral blood vessel density as assessed with CD31 staining. Conclusions: Combination treatments enhanced antiproliferative and proapoptotic activities in cell culture, and when formulated in liposomes and delivered via aerosolization to treat an aggressive and metastatic syngeneic murine mammary tumor, the combination treatment showed a significant reduction in tumor volume in comparison to either treatment alone. Mechanistically, it appears that neither enhanced apoptosis, reduced cell proliferation, nor reduced blood vessel density can fully account for the enhanced effects of the combination treatment.