Inhibition of human lung adenocarcinoma cells by 5F11-doxorubicin immunoconjugate in vitro and in vivo

Abstract

随着抗体技术的发展,越来越多的抗体复合物用于肿瘤的治疗。本研究试图探讨单克隆抗体化疗药物结合物5F11-DOX在体内外对人肺腺癌A2细胞的抑制作用及其可能的作用机制。方法 戊二醛法制备5F11-DOX结合物,集落形成实验观察结合物对肿瘤细胞的体外杀伤作用,荧光显微镜观察DOX在肿瘤细胞中的分布,免疫组化检测抗体与肿瘤细胞的结合部位及分布。裸鼠皮下或腹腔内接种A2细胞异种移植瘤,比较5F11-DOX与游离DOX的疗效。结果 浓度为0.04mg/L的5F11-DOX即可在体外杀死全部人肺腺癌A2细胞,5F11-DOX是游离DOX作用的10倍。荧光镜检发现在DOX浓度为3mg/L时作用3h,去除药物,继续培养24h后,5F11-DOX组的DOX荧光较游离DOX组强。免疫组化检测发现5F11定位于细胞膜与胞浆中,而荧光镜检发现DOX分布于细胞内。裸鼠皮下及腹腔内异种移植瘤治疗实验显示5F11DOX组裸鼠的移植瘤明显小于对照组和同剂量游离DOX组(P < 0.05);5F11-DOX疗效相当于4～8倍的游离DOX。移植瘤的病理切片HE染色显示,5F11-DOX结合物组癌巢中央和周围均有大片的瘤组织坏死。结论 5F11-DOX结合物对人肺腺癌A2细胞的杀伤作用显著高于游离的DOX。

Background and objective With the development of antibody technology, more and more immunoconjugates are used in clinical treatment for different cancers. The aim of this study is to investigate the inhibitive effects of 5F11-DOX immunoconjugate on human lung adenocarcinoma cell line LTEP-A2 in vitro and in vivo and to explore the potential mechanism. Methods The 5F11-DOX immunoconjugate was produced by diluted glutaraldehyde crosslinking. The killing efficiency of 5F11-DOX was detected by clonogenic assay. The distribution of DOX was observed under fluorescence microscope and the 5F11 location was determined by immunohistochemistry. The therapeutic efficacy of 5F11-DOX and free DOX was detected on subcutaneous or intraperitoneal exnogenic transplanted tumors of human lung adenocarcinoma A2 cells in nude mice. Results 5F11-DOX of 0.04mg/L could kill all the A2 cells in vitro and the killing efficiency was 10 times as that of the free DOX. Fluorescence microscopy showed that fluorescence of DOX in 3mg/L 5F11-DOX group was much stronger than that in 3mg/L free DOX group after treating A2 cells with 3mg/L 5F11-DOX or DOX for 3h, then incubating the cells with fresh medium for another 24 hours. Immunohistochemistry showed that 5F11 located in cell membrane and cytoplasm and fluorescence microscopy proved that DOX located inside the cells. The average sizes of subcutaneous or intraperitoneal exnogenic transplanted tumors in 5F11-DOX group were obviously smaller than those of the control group and free DOX group at the same dosage (P < 0.05), and the anti-tumorogenicity efficacy of 5F11-DOX was 4--8 times as that of free DOX. The HE staining showed that extensive necrosis occurred in the center of tumors and around cancer nests in 5F11-DOX group. Conclusion The killing efficacy of 5F11-DOX on human lung adenocarcinoma cell line A2 is obviously higher than that of the free DOX.