Rodriguez R, Lim H Y, Bartkowski L M, Simons J W
Department of Urology, Brady Urological Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA.
Prostate. 1998 Mar 1;34(4):259-69. doi: 10.1002/(sici)1097-0045(19980301)34:4<259::aid-pros3>3.0.co;2-c.
Metastatic human prostate cancer requires novel therapeutic strategies in order to overcome its low proliferative rate and its resistance to conventional chemotherapeutic agents. To identify potential cytotoxin gene products for use in experimental therapeutics such as in vivo gene therapy, an in vitro screen was designed.
Eight recombinant cellular toxins were tested for activity against a spectrum of metastatic human prostate cancer cell phenotypes. Pseudomonas exotoxin A, ricin, tumor necrosis factor alpha (TNF-alpha), diphtheria toxin (DT), Crotalus durissus terrificus toxin, crotalus adamenteus toxin, Naja naja toxin, and Naja mocambique toxin were evaluated. Comparative survival distinguished the relative potencies of these cytotoxins for irreparable prostate cancer cell death.
Of the phospholipase A2 toxins, Crotalus durissus terrificus and Naja mocambique are active against the PSA secreting LNCaP cell line; however, the effect is reversible, and no other hormone refractory prostate cell line tested is sensitive. Screening identified toxin-specific differences: dose-dependent cytotoxic activity against all human prostate cancer cell lines tested was only identified for ricin and diphtheria toxin (DT) as highly potent. DT has an IC50 in the range of 20-00 pM by clonogenic survival and kills irreversibly by both apoptosis as well as nonapototic pathways. Acquisition of p53 mutant status conferred no reduction in sensitivity to DT cytotoxicity. Cell cycle arrest by aphidicolin did not protect human prostate cells from irreversible DT-induced cell death. TNF-alpha had modest cytostatic activity in the screen; however, the combination of TNF-alpha and DT resulted in marked acceleration of the time to prostate cancer cell death.
The rational screening of cytotoxins allows the identification of cell cycle-independent agents of variable potency against human prostate cancer. DT-mediated cell death is cell cycle independent, and p53 independent, making it particularly attractive for application to cytoreductive gene therapy, targeted monoclonal antibodies, and prodrug delivery of toxins applied to human prostate cancer therapeutics.
转移性人类前列腺癌需要新的治疗策略，以克服其低增殖率以及对传统化疗药物的耐药性。为了鉴定可用于实验性治疗（如体内基因治疗）的潜在细胞毒素基因产物，设计了一项体外筛选。
测试了八种重组细胞毒素对一系列转移性人类前列腺癌细胞表型的活性。评估了绿脓杆菌外毒素A、蓖麻毒素、肿瘤坏死因子α（TNF-α）、白喉毒素（DT）、南美响尾蛇毒素、中美珊瑚蛇毒素、眼镜蛇毒素和莫桑比克眼镜蛇毒素。通过比较存活率来区分这些细胞毒素对不可修复的前列腺癌细胞死亡的相对效力。
在磷脂酶A2毒素中，南美响尾蛇毒素和莫桑比克眼镜蛇毒素对分泌前列腺特异性抗原（PSA）的LNCaP细胞系有活性；然而，这种作用是可逆的，且测试的其他激素难治性前列腺细胞系均不敏感。筛选确定了毒素特异性差异：仅鉴定出蓖麻毒素和白喉毒素（DT）对所有测试的人类前列腺癌细胞系具有剂量依赖性细胞毒性活性，且效力很强。通过克隆形成存活率测定，DT的半数抑制浓度（IC50）在20-200 pM范围内，并且通过凋亡以及非凋亡途径不可逆地杀伤细胞。获得p53突变状态并未降低对DT细胞毒性的敏感性。阿非迪霉素导致的细胞周期停滞并不能保护人类前列腺细胞免受DT诱导的不可逆细胞死亡。在筛选中，TNF-α具有适度的细胞生长抑制活性；然而，TNF-α与DT联合使用导致前列腺癌细胞死亡时间显著加快。
对细胞毒素进行合理筛选可鉴定出对人类前列腺癌具有不同效力的细胞周期非依赖性药物。DT介导的细胞死亡与细胞周期无关且与p53无关，这使其在应用于减瘤基因治疗、靶向单克隆抗体以及用于人类前列腺癌治疗的毒素前药递送方面特别具有吸引力。
