Belenkov A I, Alakhov V Y, Kabanov A V, Vinogradov S V, Panasci L C, Monia B P, Chow T Y K
Department of Oncology, Division of Radiation Oncology, Montreal General Hospital, McGill University, Montreal, Quebec, Canada.
Gene Ther. 2004 Nov;11(22):1665-72. doi: 10.1038/sj.gt.3302355.
In an effort to improve the efficacy of antisense delivery, we evaluated polyethyleneimine (PEI, 2 kDa) alone or grafted with nonionic amphiphilic block copolymer Pluronic (P85) as a carrier for Ku86 antisense oligonucleotide (ASO) delivery. Ku86 is an abundant nuclear protein that plays an important role in nonhomologous DNA end joining and has implications in tumorigenesis and acquired drug resistance. Transfection of adherent and suspension cell lines with Ku86 ASOs complexed with P85-g-PEI (2 kDa) conjugates was associated with a specific decrease in Ku86 mRNA levels (EC50<75 nM and EC50<250 nM, respectively, n=3). More importantly, no requirement for reduced serum conditions was necessary during transfection. In contrast, whereas Ku86 ASOs complexed with PEI (2 kDa) alone were effective in decreasing Ku86 mRNA levels in adherent cell lines (EC50<75 nM, n=3), the formulation did not produce any detectable decrease in Ku86 mRNA levels in suspension cell lines. Transfection of adherent cell lines with 500 nM Ku86 ASOs formulated with P85-g-PEI (2 kDa) was associated with a specific decrease (<10% remaining of control) in Ku86 protein expression and a two-fold increased cell death after treatment with ionizing radiation (IR). In athymic nude mice bearing subcutaneous human HT29 colon adenocarcinoma xenografts, Ku86 ASO-P85-g-PEI (2 kDa) administration (15 mg/kg, subcutaneously) with a Q1D x 7 treatment schedule, when combined with a single dose of IR (6 Gy), caused a significant inhibition of HT29 tumor growth compared with mismatch- and naked antisense-pretreated control groups (time from 200 to 1000 mm3, 126.9 versus 84.18 and 87.76 days, P<0.005). A potentiation of the antitumor activity was observed in all mice treated with Ku86 ASO-P85-g-PEI (2 kDa) formulation; however, tumor growth inhibition was reversible upon treatment cessation. No morbidity/mortality or changes in histopathology were observed under this treatment regiment. Our results indicate that P85-g-PEI (2 kDa) conjugates may increase the efficacy of Ku86 ASO delivery in management of resistant malignancies, thus providing a rationale for their evaluation in cancer patients in combination with conventional anticancer therapies.
为提高反义寡核苷酸传递的效率，我们评估了单独的聚乙烯亚胺（PEI，2 kDa）或接枝了非离子两亲性嵌段共聚物普朗尼克（P85）的聚乙烯亚胺作为Ku86反义寡核苷酸（ASO）传递载体的效果。Ku86是一种丰富的核蛋白，在非同源DNA末端连接中起重要作用，并且与肿瘤发生和获得性耐药有关。用与P85-g-PEI（2 kDa）缀合物复合的Ku86 ASO转染贴壁和悬浮细胞系，与Ku86 mRNA水平的特异性降低相关（EC50分别<75 nM和<250 nM，n = 3）。更重要的是，转染过程中无需降低血清条件。相比之下，虽然单独与PEI（2 kDa）复合的Ku86 ASO在贴壁细胞系中能有效降低Ku86 mRNA水平（EC50<75 nM，n = 3），但该制剂在悬浮细胞系中未使Ku86 mRNA水平出现任何可检测到的降低。用500 nM与P85-g-PEI（2 kDa）配制的Ku86 ASO转染贴壁细胞系，与Ku86蛋白表达的特异性降低（<对照的10%）以及电离辐射（IR）处理后细胞死亡增加两倍相关。在携带人HT29结肠腺癌皮下异种移植物的无胸腺裸鼠中，按照Q1D×7治疗方案皮下给予Ku86 ASO-P85-g-PEI（2 kDa）（15 mg/kg），当与单剂量IR（6 Gy）联合使用时，与错配和裸反义预处理对照组相比，显著抑制了HT29肿瘤生长（从200到1000 mm3的时间，分别为126.9天、84.18天和87.76天，P<0.005）。在用Ku86 ASO-P85-g-PEI（2 kDa）制剂治疗的所有小鼠中均观察到抗肿瘤活性增强；然而，停止治疗后肿瘤生长抑制是可逆的。在此治疗方案下未观察到发病率/死亡率或组织病理学变化。我们的结果表明，P85-g-PEI（2 kDa）缀合物可能会提高Ku86 ASO在耐药恶性肿瘤治疗中的传递效率，从而为其与传统抗癌疗法联合用于癌症患者的评估提供了理论依据。
