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.
Highlights
CRISPRMAX was shown as a light gray outer outline of the complexes, while the encapsulated bovine serum albumin (BSA) or Protein kinase A (PKA) proteins were presented in black (Figure 1a)
For null CRISPRMAX, there was only a light gray outline. These results showed that the proteins were successfully coated by CRISPRMAX
We previously demonstrated that transfection with CRISPRMAX-PKAs can initiate an inhibition process that represses epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) in breast cancer cells, thereby further weakening chemoresistance in breast cancer cells
Summary
The cancer stem cell (CSC) theory has been proposed as one of the viable explanations for the resistance of cancer cells to conventional cancer therapies, including chemotherapy and radiotherapy in the clinic [1]. Emerging studies demonstrate that epithelial-tomesenchymal transition (EMT) plays a crucial role in the dedifferentiation of differentiated cancer cells into CSCs [2,3]. Snail family transcriptional repressor 1 (SNAIL1), Twist family BHLH transcription factor. 1 (TWIST), zinc finger E-Box binding homeobox 1 (ZEB1), and microRNAs (miRNAs), such as miR-200 family, let-7, have been revealed and have shown promise in inhibition of the EMT process in studies [4]. Targeting EMT-induced CSCs or reversing EMT has become an attractive strategy for inhibiting CSCs and CSC-associated chemoresistance
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