Dean N, McKay R, Miraglia L, Howard R, Cooper S, Giddings J, Nicklin P, Meister L, Ziel R, Geiger T, Muller M, Fabbro D
Department of Molecular Pharmacology, ISIS Pharmaceuticals, Carlsbad, California 92008, USA.
Cancer Res. 1996 Aug 1;56(15):3499-507.
A 20-mer phosphorothioate oligodeoxynucleotide (ISIS 3521) designed to hybridize sequences in the 3'-untranslated region of human protein kinase C-alpha (PKC-alpha) mRNA has been shown to inhibit the expression of PKC-alpha in multiple human cell lines. In human bladder carcinoma (T-24) cells, inhibition of PKC-alpha was both concentration dependent and oligonucleotide sequence specific. ISIS 3521 had a IC50 of 50-100 nM for PKC-alpha mRNA reduction and was without effect on the expression of other members of the PKC family of genes (PKC-eta and zeta). Toxicity studies in mice revealed that the oligodeoxynucleotide was well tolerated at repeat doses of 100 mg/kg i.v. for up to 14 days, with no acute toxicity apparent. The oligodeoxynucleotide was found to also inhibit the growth of three different human tumor cell lines, the T-24 bladder, human lung carcinoma (A549), and Colo 205 colon carcinoma grown in nude mice. The inhibition was dose dependent with ID50 values for the growth inhibition between 0.06 and 0.6 mg/kg daily when given i.v., depending on the cell line examined. Three control phosphorothioate oligodeoxynucleotides not targeting human PKC-alpha were without effect on the growth of the tumors at doses as high as 6 mg/kg. Recovery of ISIS 3521 from tumor tissue and resolution by capillary gel electrophoresis revealed that 24 It after the final dose of oligodeoxynucleotide, intact, full-length 20-mer material was present as well as some apparent exonuclease degradation products (e.g., n-1 and n-2 mers). These studies demonstrate the in vivo antitumor effects of an antisense oligodeoxynucleotide targeting PKC-alpha and suggest that this compound may be of value as a chemotherapeutic agent in the treatment of human cancers.
一种设计用于与人蛋白激酶C-α（PKC-α）mRNA的3'-非翻译区序列杂交的20聚体硫代磷酸酯寡脱氧核苷酸（ISIS 3521）已被证明能抑制多种人类细胞系中PKC-α的表达。在人膀胱癌细胞（T-24）中，PKC-α的抑制作用既是浓度依赖性的，也是寡核苷酸序列特异性的。ISIS 3521对PKC-α mRNA减少的IC50为50 - 100 nM，对PKC基因家族的其他成员（PKC-η和ζ）的表达没有影响。对小鼠的毒性研究表明，该寡脱氧核苷酸以100 mg/kg静脉重复给药长达14天，耐受性良好，没有明显的急性毒性。发现该寡脱氧核苷酸还能抑制三种不同的人类肿瘤细胞系在裸鼠体内的生长，即T-24膀胱癌细胞、人肺癌细胞（A549）和结肠癌细胞（Colo 205）。抑制作用是剂量依赖性的，静脉给药时，根据所检测的细胞系不同，生长抑制的ID50值在每天0.06至0.6 mg/kg之间。三种不靶向人PKC-α的对照硫代磷酸酯寡脱氧核苷酸在高达6 mg/kg的剂量下对肿瘤生长没有影响。通过毛细管凝胶电泳从肿瘤组织中回收ISIS 3521并进行分析，结果显示在最后一剂寡脱氧核苷酸24小时后，完整的全长20聚体物质以及一些明显的核酸外切酶降解产物（如n - 1和n - 2聚体）都存在。这些研究证明了靶向PKC-α的反义寡脱氧核苷酸的体内抗肿瘤作用，并表明该化合物作为治疗人类癌症的化疗药物可能具有价值。
