Silencing of CD47 and SIRPα by Polypurine reverse Hoogsteen hairpins to promote MCF-7 breast cancer cells death by PMA-differentiated THP-1 cells.
BACKGROUND
In the context of tumor immunology, tumor cells have been shown to overexpress CD47, an anti-phagocytic signal directed to macrophages to escape from phagocytosis by interacting with Signal Regulatory Protein α SIRPα. In the present work, we designed Polypurine reverse Hoogsteen hairpins, PPRHs, to silence the expression of CD47 in tumor cells and SIRPα in macrophages with the aim to eliminate tumor cells by macrophages in co-culture experiments.


METHODS
THP-1 cells were differentiated to macrophages with PMA. The mRNA levels of differentiation markers CD14 and Mcl-1 mRNA and pro-inflammatory cytokines (IL-1β, IL-18, IL-6, IL-8 and TNF-α) were measured by qRT-PCR. The ability of PPRHs to silence CD47 and SIRPα was evaluated at the mRNA level by qRT-PCR and at the protein level by Western Blot. Macrophages were co-cultured with tumor cells in the presence of PPRHs to silence CD47 and/or SIRPα. Cell viability was assessed by MTT assays.


RESULTS
THP-1 cells differentiated to macrophages with PMA showed an increase in macrophage surface markers (CD14, Mcl-1) and pro-inflammatory cytokines (IL-1β, IL-18, IL-6, IL-8 and TNF-α). PPRHs were able to decrease both CD47 expression in MCF-7 cell line and SIRPα expression in macrophages at the mRNA and protein levels. In the presence of PPRHs, MCF-7 cells were eliminated by macrophages in co-culture experiments, whereas they survived in the absence of PPRHs.


CONCLUSIONS
Our data support the usage of PPRHs to diminish CD47/SIRPα interaction by decreasing the expression of both molecules thus resulting in an enhanced killing of MCF-7 cells by macrophages, which might translate into beneficial effects in cancer therapy. These results indicate that PPRHs could represent a new approach with immunotherapeutic applications.
Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, IN2UB, Barcelona, Spain.
Gizem
G
Bener
Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, IN2UB, Barcelona, Spain.
Alex
A
J Félix
Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, IN2UB, Barcelona, Spain.
Cristina
C
Sánchez de Diego
Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, IN2UB, Barcelona, Spain.
Isabel
I
Pascual Fabregat
Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, IN2UB, Barcelona, Spain.
Carlos J
CJ
Ciudad
Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, IN2UB, Barcelona, Spain. vnoe@ub.edu.
Véronique
http://orcid.org/0000-0001-7937-8038
V
Noé
CD47; Immunotherapy; Macrophage; PPRH; SIRPα
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