PO.CL08.02 · 临床研究
[123I]CC1，一种用于靶向放射性核素治疗（TRT）的放射性药物，通过PARP结合与捕获来放大人类癌细胞系中的DNA损伤和细胞毒性。
[ 123 I]CC1, a radiopharmaceutical for Targeted radionuclide therapy (TRT), exploits PARP binding and trapping to amplify DNA damage and cytotoxicity in human cancer cell lines.
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摘要 Abstract
中文摘要
我们研究了[123I]CC1的抗肿瘤作用是否由PARP捕获所驱动。[123I]CC1是一种PARP结合型放射性药物，可发射极短射程的电离俄歇（Auger）电子，具有用于靶向放射性核素治疗（TRT）的潜力。捕获作用会延长其在DNA上的驻留时间，并触发更多的DNA损伤。我们在暴露于[123I]CC1的PSN1和U87人类癌细胞系中对此进行了检测，通过western blot评估染色质组分中的PARP水平，并使用伽马计数器定量核组分和胞质组分中的123I计数。下游效应通过gammaH2AX免疫荧光（作为DNA损伤标志物）以及DNA纤维实验（测量复制速度）进行评估。此外，我们通过使用甲基甲磺酸（MMS）增加被捕获的PARP以及使用PARG抑制剂JA2131减少被捕获的PARP来调节PARP与DNA的结合，并利用western blot和伽马计数器分析[123I]CC1如何追踪PARP从染色质到核可溶性组分的移动。最后，采用gammaH2AX免疫荧光评估与MMS或PARGi的联合处理是否影响[123I]CC1产生DNA损伤的能力。[123I]CC1提高了PSN1和U87细胞中DNA上被捕获的PARP水平，随后gammaH2AX焦点迅速增加。有趣的是，DNA纤维实验显示，在30分钟和24小时时PSN1中的复制受到明显阻碍，表明存在大量DNA损伤。在加入[123I]CC1之前用0.01% MMS预处理，与单用[123I]CC1相比进一步提高了PARP水平，导致染色质中123I的更大积累。相反，PARG抑制剂处理减少了PARP在DNA上的驻留时间，与[123I]CC1联用时降低了DNA中123I的递送。最后，在30分钟和24小时时对gammaH2AX的评估显示，MMS预处理增强了[123I]CC1诱导的DNA损伤，其损伤程度大于单用MMS，表现出相加效应，而PARGi预处理无显著影响。这些结果表明，[123I]CC1的细胞毒性作用由PARP捕获所介导，这是我们团队此前观察到的其强效抗肿瘤活性的基础。此外，对PARylation循环的调节改变了放射性向DNA的递送以及DNA损伤的程度，提示了一种增强[123I]CC1治疗获益及其临床转化潜力的策略。
查看英文原文 English abstract
We investigated whether the antitumor effects of [ 123 I]CC1, a PARP-binding radiopharmaceutical emitting very short-range ionising Auger electrons with potential use in Targeted Radionuclide Therapy (TRT), are driven by PARP trapping. Trapping will prolong its residence time on DNA and triggers more DNA damage. We tested this in PSN1 and U87 human cancer cell lines exposed to [ 123 I]CC1, by assessing PARP levels in the chromatin fraction via western blot and quantifying 123 I counts in nuclear and cytoplasmic fractions using a gamma counter. Downstream effects were evaluated using gammaH2AX immunofluorescence as a marker of DNA damage and DNA fiber assays to measure replication speed. Furthermore, we modulated PARP association with DNA by using methyl methanesulfonate (MMS) to increase and the PARG inhibitor JA2131 to decrease trapped PARP, using western blot and gamma counter to analyze how [ 123 I]CC1 tracks PARP movements from chromatin to nuclear soluble fraction. Finally, gammaH2AX immunofluorescence was used to assess whether combinatory treatments with MMS or PARGi affect the ability of [ 123 I]CC1 in producing DNA damage. [ 123 I]CC1 elevated trapped PARP levels on DNA in PSN1 and U87 cells, followed by a rapid increase in gammaH2AX foci. Interestingly, DNA fiber assays showed that replication was markedly hindered in PSN1 at 30 min and 24 h, indicating substantial DNA damage. Pretreatment with 0.01% MMS before [ 123 I]CC1
addition further elevated PARP levels compared to [ 123 I]CC1 alone, resulting in greater chromatin 123 I accumulation. In contrast, PARG inhibitor treatment reduced PARP residence time on DNA, and in combination with [ 123 I]CC1 decreased DNA 123 I delivery. Finally, assessment of gammaH2AX at 30 min and 24 hours showed that MMS pretreatment enhanced DNA damage induced by [ 123 I]CC1 greater damage than MMS alone, demonstrating an additive effect, whereas PARGI pretreatment had no significant effect.The results indicate that the cytotoxic effect of [ 123 I]CC1 is mediated by PARP trapping, which underlies its potent antitumoral activity previously observed by our group. Furthermore, modulation of the PARylation cycle alters delivery of radioactivity to DNA and the extent of DNA damage, suggesting a strategy to enhance the therapeutic benefit of [ 123 I]CC1 and its potential for clinical translation.
利益披露 Disclosure
L. Hernandez Cano, None..
N. Delgado Mayenco, None..
H. Kramer, None..
E. Diekstra, None..
F. Amoroso, None..
S. Unnikrishnan, None..
G. Alachouzos, None..
W. Szymanski, None..
F. A. Kruyt, None..
B. Cornelissen, None.