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Silencing of CD47 and SIRP α by Polypurine reverse Hoogsteen hairpins to promote MCF-7 breast cancer cells death by PMA-differentiated THP-1 cells

View Article: PubMed Central - PubMed

ABSTRACT

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.

Background: 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.

No MeSH data available.


Related in: MedlinePlus

Schematic representation of co-culture experiments. MCF-7 and THP-1 cells were plated in separate dishes and THP-1 cells were immediately transfected with PPRHs against SIRPα. After 24 h, transfected THP-1 cells were added to MCF-7 cells and differentiated to macrophages with PMA for 3 days. After that period of time, the medium was replaced with fresh F12 medium and PPRHs against CD47 were transfected. Cell viability (MTT) assay was assessed 5 days after the last transfection
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Fig1: Schematic representation of co-culture experiments. MCF-7 and THP-1 cells were plated in separate dishes and THP-1 cells were immediately transfected with PPRHs against SIRPα. After 24 h, transfected THP-1 cells were added to MCF-7 cells and differentiated to macrophages with PMA for 3 days. After that period of time, the medium was replaced with fresh F12 medium and PPRHs against CD47 were transfected. Cell viability (MTT) assay was assessed 5 days after the last transfection

Mentions: MCF-7 (60,000) and THP-1 cells (1000) were plated in 6-well dishes separately and THP-1 cells were immediately transfected with PPRHs against SIRPα and the negative control, as explained in the corresponding section. After 24 h, transfected THP-1 cells were added to MCF-7 cells and treated with 3 ng/ml PMA for differentiation (Fig. 1). Three days after PMA treatment, the PMA-containing medium was aspirated and PPRHs against CD47 and the negative control were transfected in fresh F12 medium. At the same time, antibody against CD47 was added as positive control. Cell viability was assessed 5 days after transfection by MTT assays.Fig. 1


Silencing of CD47 and SIRP α by Polypurine reverse Hoogsteen hairpins to promote MCF-7 breast cancer cells death by PMA-differentiated THP-1 cells
Schematic representation of co-culture experiments. MCF-7 and THP-1 cells were plated in separate dishes and THP-1 cells were immediately transfected with PPRHs against SIRPα. After 24 h, transfected THP-1 cells were added to MCF-7 cells and differentiated to macrophages with PMA for 3 days. After that period of time, the medium was replaced with fresh F12 medium and PPRHs against CD47 were transfected. Cell viability (MTT) assay was assessed 5 days after the last transfection
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5037635&req=5

Fig1: Schematic representation of co-culture experiments. MCF-7 and THP-1 cells were plated in separate dishes and THP-1 cells were immediately transfected with PPRHs against SIRPα. After 24 h, transfected THP-1 cells were added to MCF-7 cells and differentiated to macrophages with PMA for 3 days. After that period of time, the medium was replaced with fresh F12 medium and PPRHs against CD47 were transfected. Cell viability (MTT) assay was assessed 5 days after the last transfection
Mentions: MCF-7 (60,000) and THP-1 cells (1000) were plated in 6-well dishes separately and THP-1 cells were immediately transfected with PPRHs against SIRPα and the negative control, as explained in the corresponding section. After 24 h, transfected THP-1 cells were added to MCF-7 cells and treated with 3 ng/ml PMA for differentiation (Fig. 1). Three days after PMA treatment, the PMA-containing medium was aspirated and PPRHs against CD47 and the negative control were transfected in fresh F12 medium. At the same time, antibody against CD47 was added as positive control. Cell viability was assessed 5 days after transfection by MTT assays.Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

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.

Background: 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.

No MeSH data available.


Related in: MedlinePlus