Olaparib nanoformulation in H-ferritin as a promising option for both BRCA-mutated and sporadic triple negative breast cancer: An in vitro study
Olaparib nanoformulation in H-ferritin as a promising option for both BRCA-mutated and sporadic triple negative breast cancer: An in vitro study
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H-铁蛋白中的奥拉帕尼纳米制剂是治疗 BRCA 突变和散发性三阴性乳腺癌的有前景的选择：一项体外研究
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发表时间：
2018
期刊：
影响因子：
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通讯作者：
F. Corsi
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作者：
S. Mazzucchelli;M. Truffi;L. Sorrentino;M. Bellini;M. Rizzuto;R. Ottria;P. Ciuffreda;D. Prosperi;F. Corsi
背景资料：聚ADP-核糖聚合酶（PARP）抑制剂是治疗三阴性乳腺癌（TNBC）的一种新的有前途的策略，TNBC通常显示基因组不稳定或BRCA突变。然而，临床结果是有争议的，在野生型BRCA的情况下没有表现出任何益处，可能是由于生物利用度差和核递送不足。纳米技术可以克服这些主要限制。本研究的目的是评估H-铁蛋白纳米制剂奥拉帕尼（HOla）与游离奥拉帕尼（奥拉）对BRCA突变和非突变TNBC细胞的抗癌功效。方法：用HOla或游离奥拉体外处理BRCA突变的HCC 1937细胞和BRCA野生型的MDA MB-231和MDA MB-468细胞。通过流式细胞术评估HOla对TNBC细胞上过表达的转铁蛋白受体I（TfR-1）的主动靶向和结合能力。通过共聚焦显微镜评估奥拉和HOla的内化和细胞内定位。通过施用增加剂量的HOla或奥拉，比较细胞活力、细胞周期、细胞死亡、PARP-1切割和DNA损伤来评估抗癌功效。最后，比较了两种治疗的抗PARP疗效和核隔室中药物的比例。结果：TNBC细胞均过表达TfR-1，并能被HOla识别。共聚焦显微镜显示纳米颗粒快速内化到细胞中，细胞内持续时间长达48小时。与奥拉相比，用HOla观察到药物的核浓度显著增加，这是由于通过自触发机制介导的H-铁蛋白的核递送的强烈改善。使用10 nM、50 nM或100 nM的奥拉没有显示出显著的抗增殖作用。相反，50 nM和100 nM的HOla在所有TNBC细胞系中显示出高1000倍的抗癌活性。排除了H-铁蛋白纳米载体本身对细胞毒性的可能贡献，用空隙纳米颗粒处理细胞。HOla处理的样品中G2/M期细胞周期停滞、细胞死亡、裂解的PARP-1和磷酸化组蛋白H2 A. X方面的DNA损伤的比例高于用游离奥拉处理的样品。结论：我们的研究结果表明，奥拉的纳米制剂作为独立疗法强烈增强了PARP抑制对BRCA突变型和野生型TNBC的细胞毒性功效，从而允许靶向递送至TNBC细胞中并迅速归巢至核隔室中。Citation Format：Mazzucchelli S，Truffi M，Sorrentino L，Bellini M，Rizzuto MA，Ottria R，Ciuffreda P，Prosperi D，Corsi F.奥拉帕尼纳米制剂在H-铁蛋白中作为BRCA突变和散发性三阴性乳腺癌的有希望的选择：体外研究[摘要]。In：Proceedings of the 2017圣安东尼奥Breast Cancer Symposium; 2017 Dec 5-9;圣安东尼奥，TX. Philadelphia（PA）：AACR; Cancer Res 2018;78（4 Suppl）：Abstract nr P1-10-13.
Background: PolyADP-ribose polymerase (PARP) inhibitors are a novel promising strategy toward triple-negative breast cancer (TNBC), which often shows genomic instability or BRCA mutations. However, clinical results are controversial, and no benefits were demonstrated in case of wild type BRCA, possibly due to poor bioavailability, and inadequate nuclear delivery. Nanotechnology could overcome these major limitations. The aim of this study was to assess the anticancer efficacy of H-Ferritin nanoformulated Olaparib (HOla) vs. free Olaparib (Ola) on BRCA-mutated and non mutated TNBC cells. Methods: BRCA-mutated HCC1937 cells and BRCA-wild type MDA MB-231 and MDA MB-468 cells were treated with HOla or free Ola in vitro. Active targeting and binding capability of HOla toward transferrin receptor 1 (TfR-1), over-expressed on TNBC cells, was assessed by flow cytometry. Internalization and intracellular localization of Ola and HOla was assessed by confocal microscopy. Anticancer efficacy was assessed by administration of increasing doses of HOla or Ola, comparing cell viability, cell cycle, cell death, PARP-1 cleavage and DNA damage. Finally, anti-PARP efficacy and proportion of drug in the nuclear compartment were compared between treatments. Results: All TNBC cell lines over-expressed TfR-1 and were succesfully recognized by HOla. Confocal microscopy showed a fast internalization of nanoparticles into cells, with intracellular persistence up to 48h. A marked increase in nuclear concentration of drug was observed with HOla compared to Ola, due to a strongly improved nuclear delivery by H-Ferritin mediated by a self-triggered mechanism. No significant antiproliferative effect was demonstrated with Ola at 10 nM, 50 nM or 100 nM. Conversely, HOla at 50 nM and 100 nM showed a 1000-fold higher anticancer activity in all TNBC cell lines. A possible contribution in cytotoxicity by H-Ferritin nanovector itself was excluded treating cells with void nanoparticles. Proportions of cell cycle arrest in G2/M, cell death, cleaved PARP-1 and DNA damage in terms of phosphorylated histone H2A.X were higher in HOla treated samples than in ones treated with free Ola. Conclusions:Our findings suggest that nanoformulation of Ola strongly enhances cytotoxic efficacy of PARP inhibition as a stand-alone therapy, on both BRCA-mutated and wild type TNBCs allowing a targeted delivery into TNBC cells and a prompt homing into the nuclear compartment. Citation Format: Mazzucchelli S, Truffi M, Sorrentino L, Bellini M, Rizzuto MA, Ottria R, Ciuffreda P, Prosperi D, Corsi F. Olaparib nanoformulation in H-ferritin as a promising option for both BRCA-mutated and sporadic triple negative breast cancer: An in vitro study [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-10-13.