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通过CRISPRMAX递送蛋白激酶A及其对乳腺癌干细胞样特性的影响。

Delivery of Protein Kinase A by CRISPRMAX and Its Effects on Breast Cancer Stem-Like Properties.

作者信息

Zhou Jun-Nian, Rautio Tzu-Chen, Liu Chang, Xu Xiao-Yu, Wang Dong-Qing, Guo Yong, Eriksson John, Zhang Hongbo

机构信息

Experimental Hematology and Biochemistry Lab, Beijing Institute of Radiation Medicine, Beijing 100850, China.

Stem Cell and Regenerative Medicine Lab, Institute of Health Service and Transfusion Medicine, Beijing 100850, China.

出版信息

Pharmaceutics. 2020 Dec 23;13(1):11. doi: 10.3390/pharmaceutics13010011.

DOI:10.3390/pharmaceutics13010011
PMID:33374889
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7824330/
Abstract

Protein kinase A (PKA) activation has recently been reported to inhibit epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) ability, which is considered to be responsible for chemoresistance and tumor recurrence in patients. While current studies mainly focus on gene manipulation of the EMT process, the direct delivery of PKA enzymes to cancer cells has never been investigated. Here, we utilize the commercial Lipofectamine CRISPRMAX reagent to directly deliver PKAs to breast cancer cells and evaluate its effects on EMT regulation. We optimized the delivery parameters with fluorescent-labeled bovine serum albumin, and successfully delivered fluorescent PKAs through CRISPRMAX into breast cancer cells. Then, we evaluated the biological effects by immunofluorescence, flow cytometry, mammosphere assay, and chemoresistance assay. Our data showed the expression of EMT-related markers, α-smooth muscle actin and N-cadherin, was downregulated after CRISPRMAX-PKA treatment. Although the CD44/CD24 population did not change considerably, the size of mammospheres significantly decreased. In paclitaxel and doxorubicin chemoresistance assays, we noticed PKA delivery significantly inhibited paclitaxel resistance rather than doxorubicin resistance. Taken together, these results suggest our direct enzyme delivery can be a potential strategy for inhibiting EMT/CSC-associated traits, providing a safer approach and having more clinical translational efficacy than gene manipulation. This strategy will also facilitate the direct testing of other target enzymes/proteins on their biological functions.

摘要

最近有报道称蛋白激酶A（PKA）的激活可抑制上皮-间质转化（EMT）和癌症干细胞（CSC）的能力，而EMT和CSC被认为与患者的化疗耐药性和肿瘤复发有关。虽然目前的研究主要集中在EMT过程的基因操作上，但PKA酶直接递送至癌细胞的研究从未开展过。在此，我们利用商业化的Lipofectamine CRISPRMAX试剂将PKA直接递送至乳腺癌细胞，并评估其对EMT调控的影响。我们用荧光标记的牛血清白蛋白优化了递送参数，并通过CRISPRMAX成功地将荧光PKA递送至乳腺癌细胞中。然后，我们通过免疫荧光、流式细胞术、乳腺球形成实验和化疗耐药性实验评估了其生物学效应。我们的数据显示，CRISPRMAX-PKA处理后，EMT相关标志物α-平滑肌肌动蛋白和N-钙黏蛋白的表达下调。虽然CD44/CD24细胞群没有显著变化，但乳腺球的大小显著减小。在紫杉醇和阿霉素化疗耐药性实验中，我们发现PKA递送显著抑制了紫杉醇耐药性，而非阿霉素耐药性。综上所述，这些结果表明我们的直接酶递送可能是一种抑制与EMT/CSC相关特性的潜在策略，与基因操作相比，它提供了一种更安全的方法且具有更高的临床转化效力。该策略还将有助于直接测试其他靶标酶/蛋白的生物学功能。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/80c4/7824330/d61b74368afe/pharmaceutics-13-00011-g010.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/80c4/7824330/0b4a68224080/pharmaceutics-13-00011-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/80c4/7824330/825712aa6705/pharmaceutics-13-00011-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/80c4/7824330/097ffc651a93/pharmaceutics-13-00011-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/80c4/7824330/86dead7a1d1d/pharmaceutics-13-00011-g004.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/80c4/7824330/14df133d5ebf/pharmaceutics-13-00011-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/80c4/7824330/147ad004170a/pharmaceutics-13-00011-g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/80c4/7824330/2577c893b4c9/pharmaceutics-13-00011-g008.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/80c4/7824330/89a7a7fb18bf/pharmaceutics-13-00011-g009.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/80c4/7824330/d61b74368afe/pharmaceutics-13-00011-g010.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/80c4/7824330/0b4a68224080/pharmaceutics-13-00011-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/80c4/7824330/825712aa6705/pharmaceutics-13-00011-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/80c4/7824330/097ffc651a93/pharmaceutics-13-00011-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/80c4/7824330/86dead7a1d1d/pharmaceutics-13-00011-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/80c4/7824330/923861047ea0/pharmaceutics-13-00011-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/80c4/7824330/14df133d5ebf/pharmaceutics-13-00011-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/80c4/7824330/147ad004170a/pharmaceutics-13-00011-g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/80c4/7824330/2577c893b4c9/pharmaceutics-13-00011-g008.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/80c4/7824330/89a7a7fb18bf/pharmaceutics-13-00011-g009.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/80c4/7824330/d61b74368afe/pharmaceutics-13-00011-g010.jpg

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