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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.


mRNA levels of macrophage surface markers. THP-1 cells (100,000) were treated with either 1 or 3 ng/ml PMA for 48 h, 72 h or 72 h + rest (72 h PMA incubation followed by 48 h resting in fresh medium). Expression of surface markers CD14 (a) and Mcl-1 (b) was determined by qRT-PCR and the results expressed relative to untreated THP-1 cells. Data represent the mean ± SE of at least three experiments (*p < 0.05, **p < 0.01, ***p < 0.005)
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Fig2: mRNA levels of macrophage surface markers. THP-1 cells (100,000) were treated with either 1 or 3 ng/ml PMA for 48 h, 72 h or 72 h + rest (72 h PMA incubation followed by 48 h resting in fresh medium). Expression of surface markers CD14 (a) and Mcl-1 (b) was determined by qRT-PCR and the results expressed relative to untreated THP-1 cells. Data represent the mean ± SE of at least three experiments (*p < 0.05, **p < 0.01, ***p < 0.005)

Mentions: PMA treatment of THP-1 monocytes has been commonly used to study macrophages in vitro and different differentiation protocols including variable concentration of PMA for different time periods have been reported so far [18]. We analyzed the effect of either 1 or 3 ng/ml PMA in THP-1 cells for 48 h, 72 h or 72 h + rest (72 h followed by 48 h resting in fresh medium). Cell morphology after PMA treatment was examined under the microscope for all conditions. Cell adhesion and spreading, which are hallmarks of macrophages, were observed at all indicated time points with both 1 and 3 ng/ml PMA. CD14 [19, 20, 23–25] and Mcl-1 [26] up-regulation have been reported to be differentiation markers of macrophages upon incubation of THP-1 cells with PMA. Therefore, to determine the concentration and incubation time of PMA required to differentiate THP-1 monocytes to macrophages, mRNA levels of CD14 and Mcl-1 were determined by qRT-PCR (Fig. 2). CD14 expression was highly enhanced using 3 ng/ml PMA for 72 h + rest (Fig. 2a) and Mcl-1 expression was increased at 48 h and its elevated level maintained at 72 h + rest for both concentrations of PMA (Fig. 2b).Fig. 2


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
mRNA levels of macrophage surface markers. THP-1 cells (100,000) were treated with either 1 or 3 ng/ml PMA for 48 h, 72 h or 72 h + rest (72 h PMA incubation followed by 48 h resting in fresh medium). Expression of surface markers CD14 (a) and Mcl-1 (b) was determined by qRT-PCR and the results expressed relative to untreated THP-1 cells. Data represent the mean ± SE of at least three experiments (*p < 0.05, **p < 0.01, ***p < 0.005)
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Related In: Results  -  Collection

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Fig2: mRNA levels of macrophage surface markers. THP-1 cells (100,000) were treated with either 1 or 3 ng/ml PMA for 48 h, 72 h or 72 h + rest (72 h PMA incubation followed by 48 h resting in fresh medium). Expression of surface markers CD14 (a) and Mcl-1 (b) was determined by qRT-PCR and the results expressed relative to untreated THP-1 cells. Data represent the mean ± SE of at least three experiments (*p < 0.05, **p < 0.01, ***p < 0.005)
Mentions: PMA treatment of THP-1 monocytes has been commonly used to study macrophages in vitro and different differentiation protocols including variable concentration of PMA for different time periods have been reported so far [18]. We analyzed the effect of either 1 or 3 ng/ml PMA in THP-1 cells for 48 h, 72 h or 72 h + rest (72 h followed by 48 h resting in fresh medium). Cell morphology after PMA treatment was examined under the microscope for all conditions. Cell adhesion and spreading, which are hallmarks of macrophages, were observed at all indicated time points with both 1 and 3 ng/ml PMA. CD14 [19, 20, 23–25] and Mcl-1 [26] up-regulation have been reported to be differentiation markers of macrophages upon incubation of THP-1 cells with PMA. Therefore, to determine the concentration and incubation time of PMA required to differentiate THP-1 monocytes to macrophages, mRNA levels of CD14 and Mcl-1 were determined by qRT-PCR (Fig. 2). CD14 expression was highly enhanced using 3 ng/ml PMA for 72 h + rest (Fig. 2a) and Mcl-1 expression was increased at 48 h and its elevated level maintained at 72 h + rest for both concentrations of PMA (Fig. 2b).Fig. 2

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 &alpha; SIRP&alpha;.

Background: In the present work, we designed Polypurine reverse Hoogsteen hairpins, PPRHs, to silence the expression of CD47 in tumor cells and SIRP&alpha; 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-1&beta;, IL-18, IL-6, IL-8 and TNF-&alpha;) were measured by qRT-PCR. The ability of PPRHs to silence CD47 and SIRP&alpha; 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 SIRP&alpha;. 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-1&beta;, IL-18, IL-6, IL-8 and TNF-&alpha;). PPRHs were able to decrease both CD47 expression in MCF-7 cell line and SIRP&alpha; 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/SIRP&alpha; 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.