Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, 30 S 2000 E Rm 301, Salt Lake City, UT, 84112, USA.
Philipps-Universitat Marburg, Biegenstraße 10, Marburg, 35037, Germany.
J Ovarian Res. 2019 May 15;12(1):45. doi: 10.1186/s13048-019-0516-2.
While tumor suppressor p53 functions primarily as a transcription factor in the nucleus, cellular stress can cause p53 to translocate to the mitochondria and directly trigger a rapid apoptotic response. We have previously shown that fusing p53 (or its DNA binding domain, DBD, alone) to the mitochondrial targeting signal (MTS) from Bak or Bax can target p53 to the mitochondria and induce apoptosis in gynecological cancer cell lines including cervical cancer cells (HeLa; wt p53), ovarian cancer cells (SKOV-3; p53 267del non-expressing), and breast cancer cells (T47D; L194F p53 mutation). However, p53 with Bak or Bax MTSs have not been previously tested in cancers with strong dominant negative (DN) mutant p53 which are capable of inactivating wt p53 by homo-oligomerization. Since p53-Bak or Bax MTS constructs act as monomers, they are not subject to DN inhibition. For this study, the utility of p53-Bak or p53-Bax MTS constructs was tested for ovarian cancers which are known to have varying p53 statuses, including a strong DN contact mutant p53 (Ovcar-3 cells), a p53 DN structural mutant (Kuramochi cells), and a p53 wild type, low expressing cells (ID8).
Our mitochondrial p53 constructs were tested for their ability to localize to the mitochondria in both mutant non-expressing p53 (Skov-3) and p53 structural mutant (Kuramochi) cell lines using fluorescence microscopy and a nuclear transcriptional activity assay. The apoptotic activity of these mitochondrial constructs was determined using a mitochondrial outer membrane depolarization assay (TMRE), caspase assay, and a late stage cell death assay (7-AAD). We also tested the possibility of using our constructs with paclitaxel, the current standard of care in ovarian cancer treatment. Our data indicates that our mitochondrial p53 constructs are able to effectively localize to the mitochondria in cancer cells with structural mutant p53 and induce apoptosis in many ovarian cancer cell lines with different p53 statuses. These constructs can also be used in combination with paclitaxel for an increased apoptotic effect.
The results suggest that targeting p53 to mitochondria can be a new strategy for ovarian cancer treatment.
尽管肿瘤抑制因子 p53 主要在核内作为转录因子发挥作用，但细胞应激会导致 p53 易位到线粒体，并直接引发快速凋亡反应。我们之前曾表明，将 p53（或其 DNA 结合域 DBD 单独）与 Bak 或 Bax 的线粒体靶向信号（MTS）融合，可将 p53 靶向到线粒体，并在包括宫颈癌细胞（HeLa；wt p53）、卵巢癌细胞（SKOV-3；p53 267del 不表达）和乳腺癌细胞（T47D；L194F p53 突变）在内的妇科癌症细胞系中诱导凋亡。然而，带有 Bak 或 Bax MTS 的 p53 以前尚未在具有强显性负（DN）突变 p53 的癌症中进行过测试，这些癌症能够通过同源寡聚化使 wt p53 失活。由于 p53-Bak 或 p53-Bax MTS 构建体作为单体起作用，因此它们不受 DN 抑制的影响。在这项研究中，我们测试了 p53-Bak 或 p53-Bax MTS 构建体在卵巢癌中的效用，已知卵巢癌具有不同的 p53 状态，包括强 DN 接触突变 p53（Ovcar-3 细胞）、p53 DN 结构突变（Kuramochi 细胞）和 p53 野生型、低表达细胞（ID8）。
我们使用荧光显微镜和核转录活性测定法，在突变型非表达 p53（Skov-3）和 p53 结构突变（Kuramochi）细胞系中，测试了这些线粒体 p53 构建体将 p53 定位到线粒体的能力。我们使用线粒体膜去极化测定法（TMRE）、半胱天冬酶测定法和晚期细胞死亡测定法（7-AAD）来确定这些线粒体构建体的凋亡活性。我们还测试了将我们的构建体与紫杉醇联合使用的可能性，紫杉醇是卵巢癌治疗的当前标准。我们的数据表明，我们的线粒体 p53 构建体能够有效地将 p53 定位到具有结构突变 p53 的癌细胞中，并在具有不同 p53 状态的许多卵巢癌细胞系中诱导凋亡。这些构建体也可以与紫杉醇联合使用以增强凋亡效果。
结果表明，将 p53 靶向到线粒体可能是治疗卵巢癌的一种新策略。
