Unidad de Transferencia Genética, Instituto de Oncología Ángel H. Roffo, Universidad de Buenos Aires, Avenida San Martin 5481, 1417 Buenos Aires, Argentina.
Biomed Pharmacother. 2013 May;67(4):269-75. doi: 10.1016/j.biopha.2013.01.002. Epub 2013 Feb 6.
Bleomycin is a chemotherapeutic agent barely diffusible through the plasmatic membrane. We evaluated DNA/cationic lipids complexes (lipoplexes) as mediators of its uptake in four spontaneous canine melanoma derived cell lines (Ak, Bk, Br and Rkb). Cell survival after lipofection plus or minus bleomycin was determined by the acid phosphatase method and the cellular uptake of lipoplexes, carrying the E. coli β-galactosidase gene, was evidenced by SYBR Green I staining. The four cell lines resulted sensitive to the bleomycin/lipoplexes system in both spatial configurations. Survival rates values were lower than 20% in monolayers of the four tested lines and lower than 30% in three lines (Ak, Bk and Rkb) when grown as spheroids. The sensitization to bleomycin depended on lipoplexes in Ak and Rkb while Bk (in both spatial configurations) and Br (as monolayers) were sensitive to bleomycin alone. Although some degree of sensitivity to bleomycin was induced by cationic lipids alone in Ak and Rkb monolayers, the maximal bleomycin effects appeared in the presence of lipoplexes. The sensitization was independent of transcriptional activity. The co-administration of lipoplexes diminished bleomycin IC50: 10-fold in Ak and Rkb monolayers; and sensitized the Ak and Rkb resistant spheroids. The bleomycin cytotoxic effects depended on lipoplexes concentration and diminished when cells were incubated at 8°C. Our results suggest that lipoplexes sensitize cells to bleomycin, increasing its uptake by an active transport mechanism, such as endocytosis. The bleomycin/lipoplexes system appears as a promising combination of chemotherapy and non-viral cancer gene therapy.
博莱霉素是一种几乎不能透过质膜扩散的化疗药物。我们评估了 DNA/阳离子脂质体复合物（脂质体）作为四种自发性犬黑色素瘤衍生细胞系（Ak、Bk、Br 和 Rkb）摄取博莱霉素的介质。转染脂质体后加或不加博莱霉素对细胞存活的影响通过酸性磷酸酶法测定，携带大肠埃希菌β-半乳糖苷酶基因的脂质体的细胞摄取通过 SYBR Green I 染色证明。四种细胞系在两种空间构型下均对博莱霉素/脂质体系统敏感。在四种测试细胞系的单层培养物中，存活率值低于 20%，在作为球体生长的三种细胞系（Ak、Bk 和 Rkb）中，存活率值低于 30%。博莱霉素的敏感性取决于 Ak 和 Rkb 中的脂质体，而 Bk（在两种空间构型下）和 Br（作为单层）则对博莱霉素单独敏感。尽管在 Ak 和 Rkb 单层培养物中，阳离子脂质体本身在一定程度上诱导了对博莱霉素的敏感性，但在存在脂质体的情况下，博莱霉素的最大作用才显现出来。这种敏感性与转录活性无关。脂质体的共同给药使 Ak 和 Rkb 单层中的博莱霉素 IC50 降低了 10 倍；并使 Ak 和 Rkb 耐药球体敏感化。博莱霉素的细胞毒性作用取决于脂质体的浓度，当细胞在 8°C 下孵育时，其作用减弱。我们的结果表明，脂质体通过主动运输机制（如内吞作用）使细胞对博莱霉素敏感，增加其摄取。博莱霉素/脂质体系统似乎是化疗和非病毒癌症基因治疗的有前途的组合。
